Title of Invention

ERYTHROPOIETIN: REMODELING AND GLYCOCONJUGATION OF ERYTHROPOIETIN .

Abstract A glycoPEGylated EPO peptide comprising an recombinant EPO peptide and atleast one glycan and atleast one poly(ethylene glycol) molecule covalently attached to said glycan, wherein said poIy(ethylene glycol) molecule is added to said EPO peptide using a glycosyltransferace.
Full Text TITLE OF THE INVENTION
ERYTHROPOIETIN: REMODELING AND GLYCOCONJUGATION OF
ERYTHROPOIETIN
BACKGROUND OF THE INVENTION
Most naturally occurring peptides contain carbohydrate moieties attached to the
peptide via specific linkages to a select number of amino acids along the length of the
primary peptide chain. Thus, many naturally occurring peptides are termed "glycopeptides.
The variability of the glycosylation pattern on any given peptide has enormous implications
for the function of that peptide. For example, the structure of the N-linked glycans on a
peptide can impact various characteristics of the peptide, including the protease
susceptibility, intracellular trafficking, secretion, tissue targeting, biological half-life and
antigenicity of the peptide in a cell or organism. The alteration of one or more of these
characteristics greatly affects the efficacy of a peptide in its natural setting, and also affects
the efficacy of the peptide as a therapeutic agent in situations where the peptide has been
generated for that purpose.
The carbohydrate structure attached to the peptide chain is known as a "glycan"
molecule. The specific glycan structure present on a peptide affects the solubility and
aggregation characteristics of the peptide, the folding of the primary peptide chain and
therefore its functional or enzymatic activity, the resistance of the peptide to proteolytic
attack and the control of proteolysis leading to the conversion of inactive forms of the peptide
to active forms. Importantly, terminal sialic acid residues present on the glycan molecule
affect the length of the half life of the peptide in the mammalian circulatory system. Peptides
whose glycans do not contain terminal sialic acid residues are rapidly removed from the
circulation by the liver, an event which negates any potential therapeutic benefit of the
peptide.
The glycan structures found in naturally occurring glycopeptides are typically divided
into two classes, N-linked and O-linked glycans.
Peptides expressed in eukaryotic cells are typically N-glycosylated oh asparagine
residues at sites in the peptide primary structure containing the sequence asparagine-X-
serine/threonine where X can be any amino acid except proline and aspartic acid. The
carbohydrate portion of such peptides is known as an N-linked glycan. The early events of
N-glycosylation occur in the endoplasmic reticulum (ER) and are identical in mammals,
plants, insects and other higher eukaryotes. First, an oligosaccharide chain comprising
fourteen sugar residues is constructed on a lipid carrier molecule. As the nascent peptide is
translated and translocated into the ER. the entire oligosaccharide chain is transferred to the
amide group of the asparagine residue in a reaction catalyzed by a membrane bound
glyeosyitransferase enzyme. The N-linked glycan is further processed both in the ER and in
the Golgi apparatus. The further processing generally entails removal of some of the sugar
residues and addition of other sugar residues in reactions catalyzed by glycosidases and
glycosyltransferascs specific for the sugar residues removed and added.
Typically, the final structures of the N-linked glycans are dependent upon the
organism in which the peptide is produced. For example, in general, peptides produced in
bacteria are completely unglycosylated. Peptides expressed in insect cells contain high
mannose and paunci-mannose N-linked oligosaccharide chains, among others. Peptides
produced in mammalian cell culture are usually glycosylated differently depending, e.g..
upon the species and cell culture conditions. Even in the same species and under the same
conditions, a certain amount of heterogeneity in the glycosyl chains is sometimes
encountered. Further, peptides produced in plant cells comprise glycan structures that differ
significantly from those produced in animal cells. The dilemma in the art of the production
of recombinant peptides, particularly when the peptides are to be used as therapeutic agents,
is to be able to generate peptides that are correctly glycosylated, i.e., to be able to generate a
peptide having a glycan structure that resembles, or is identical to that present on the
naturally occurring form of the peptide. Most peptides produced by recombinant means
comprise glycan structures that are different from the naturally occurring glycans.
A variety of methods have been proposed in the art to customize the glycosylation
pattern of a peptide including those described in WO 99/22764, WO 98/58964, WO 99/54342
and U.S. Patent No. 5,047,335, among others. Essentially, many of the enzymes required for
the in vitro glycosylation of peptides have been cloned and sequenced. In some instances,
these enzymes have been used in vitro to add specific sugars to an incomplete glycan
molecule on a peptide. In other instances, cells have been genetically engineered to express a
combination of enzymes and desired peptides such that addition of a desired sugar moiety to
an expressed peptide occurs within the cell.
Peptides may also be modified by addition of O-linked glycans, also called mucin-
type glycans because of their prevalence on mucinous glycopeptide. Unlike "N-glycans that
are linked to asparagine residues and are formed by en bloc transfer of oligosaccharide from
lipid-bound intermediates, O-glycans are linked primarily to serine and threonine residues
and are formed by the stepwise addition of sugars from nucleotide sugars (Tanner et al,
Biochim. Biophys. Acta. 906:81-91 (1987); and Hounsell el al., Glycoconj. J. 13:19-26
(1996)). Peptide function can be affected by the structure of the O-linked glycans present
thereon. For example, the activity of P-selectin ligand is affected by the O-linked glycan
structure present thereon. For a review of O-linked glycan structures, see Schachter and
Brockhausen. The Biosynthesis of Branched O-Linked Glycans. 1989, Society for
Experimental Biology, pp. 1-26 (Great Britain). Other glycosylation patterns are formed by
linking glycosylphosphatidylinositol to the carboxyl-terminal carboxyl group of the protein
(Takeda et al.. Trends Biochem. Sci. 20:367-371 (1995); and Udenfriend et al.Ann. Rev.
Biochem. 64:593-591 (1995).
Although various techniques currently exist to modify the N-linked glycans of
peptides, there exists in the art the need for a generally applicable method of producing
peptides having a desired, i.e., a customized glycosylation pattern. There is a particular need
in the art for the customized in vitro glycosylation of peptides, where the resulting peptide
can be produced at industrial scale. This and other needs are met by the present invention.
The administration of glycosylated and non-glycosylated peptides for engendering a
particular physiological response is well known in the medicinal arts. Among the best known
peptides utilized for this purpose is insulin, which is used to treat diabetes. Enzymes have
also been used for their therapeutic benefits. A major factor, which has limited the use of
therapeutic peptides is the immunogenic nature of most peptides. In a patient, an
immunogenic response to an administered peptide can neutralize the peptide and/or lead to
the development of an allergic response in the patient. Other deficiencies of therapeutic
peptides include suboptimal potency and rapid clearance rates. The problems inherent in
peptide therapeutics are recognized in the art, and various methods of eliminating the
problems have been investigated. To provide soluble peptide therapeutics, synthetic
polymers have been attached to the peptide backbone.
Poly(ethylene glycol) ("PEG") is an exemplary polymer that has been conjugated to
peptides. The use of PEG to derivatize peptide therapeutics has been demonstrated to reduce
the immunogenicity of the peptides and prolong the clearance time from the circulation. For
example. U.S. Pat. No. 4,179,337 (Davis et al.) concerns non-immunogenic peptides, such as
enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene
glycol. Between 10 and 100 moles of polymer are used per mole peptide and at least 15% of
the physiological activity is maintained.
WO 93/15189 (Veronese et al.) concerns a method to maintain the activity of
polyethylene glycol-modified proteolytic enzymes by linking the proteolytic enzyme to a
macromolecularized inhibitor. The conjugates are intended for medical applications.
The principal mode of attachment of PEG, and its derivatives, to peptides is a non-
specific bonding through a peptide amino acid residue. For example, U.S. Patent No.
4,088,538 discloses an enzymatically active polymer-enzyme conjugate of an enzyme
covalently linked to PEG. Similarly, U.S. Patent No. 4,496,689 discloses a covalently
attached complex of a-1 protease inhibitor with a polymer such as PEG or
methoxypoly(ethylene glycol) ("mPEG"). Abuchowski et al. (./. Biol. Chem. 252: 3578
(1977) discloses the covalent attachment of mPEG to an amine group of bovine serum
albumin. U.S. Patent No. 4,414,147 discloses a method of rendering interferon less
hydrophobic by conjugating it to an anhydride of a dicarboxylic acid, such as poly(ethy!ene
succinic anhydride). PCT WO 87/00056 discloses conjugation of PEG and
poly(oxyethylated) polyols to such proteins as interferon-P, interleukin-2 and immunotoxins.
EP 154.316 discloses and claims chemically modified lymphokines, such as IL-2 containing
PEG bonded directly to at least one primary amino group of the lymphokine. U.S. Patent No.
4.055,635 discloses pharmaceutical compositions of a water-soluble complex of a proteolytic
enzyme linked covalently to a polymeric substance such as a polysaccharide.
Another mode of attaching PEG to peptides is through the non-specific oxidation of
glycosyl residues on a peptide. The oxidized sugar is utilized as a locus for attaching a PEG
moiety to the peptide. For example, MTimkulu (WO 94/05332) discloses the use of a
hydrazine- or amino-PEG to add PEG to a glycoprotein. The glycosyl moieties are randomly
oxidized to the corresponding aldehydes, which are subsequently coupled to the armno-PEG.
See also. Bona et al. (WO 96/40731), where a PEG is added to an immunoglobulin molecule
by enzymatically oxidizing a glycan on the immunoglobulin and then contacting the glycan
with an amino-PEG molecule.
In each of the methods described above. poly(ethylene glycol) is added in a random,
non-specific manner to reactive residues on a peptide backbone. For the production of
therapeutic peptides, it is clearly desirable to utilize a derivatization strategy that results in the
formation of a specifically labeled, readily characterizable, essentially homogeneous product.
Two principal classes of enzymes are used in the synthesis of carbohydrates,
glycosyltransferases (e.g., sialyltransferases, oligosaccharyltransferases. N-
acetylglucosaminyltransferases), and glycosidases. The glycosidases are further classified as
exoglycosidases {e.g., P-mannosidase, (3-glucosidase), and endoglycosidases (e.g., Endo-A.
Endo-M). Each of these classes of enzymes has been successfully used synthetically to
prepare carbohydrates. For a general review, see, Crout et al., Curr. Opin. Chem. Biol. 2: 98-
111 (1998).
Glycosyltransferases modify the oligosaccharide structures on peptides.
Glycosyltransferases are effective for producing specific products with good stereochemical
and regiochemical control. Glycosyltransferases have been used to prepare oligosaccharides
and to modify terminal N- and O-linked carbohydrate structures, particularly on peptides
produced in mammalian cells. For example, the terminal oligosaccharides of glycopeptides
have been completely sialylated and/or fucosylated to provide more consistent sugar
structures, which improves glycopeptide pharmacodynamics and a variety of other biological
properties. For example, p-l,4-galactosyltransferase is used to synthesize lactosamine. an
illustration of the utility of glycosyltransferases in the synthesis of carbohydrates (see, e.g.,
Wong et al., J. Org. Chem. 47: 5416-5418 (1982)). Moreover, numerous synthetic
procedures have made use of ß-sialyltransferases to transfer sialic acid from cytidine-5'-
monophospho-N-acetylneuraminic acid to the 3-OH or 6-OH of galactose (see, e.g., Kevin et
al.. Chem. Eur. J. 2: 1359-1362 (1996)). Fucosyltransferases are used in synthetic pathways
to transfer a fucose unit from guanosine-5'-diphosphofucose to a specific hydroxyl of a
saccharide acceptor. For example, Ichikawa prepared sialyl Lewis-X by a method that
involves the fucosylation of sialylated lactosamine with a cloned fucosyltransferase
(Ichikawa et al, J. Am. Cham. Soc. 114: 9283-9298 (1992)). For a discussion of recent
advances in glycoconjugate synthesis tor therapeutic use see, Koeller et al. Nature
Biotechnology 18: 835-841 (2000). See also, U.S. Patent No. 5,876,980; 6,030,815;
5,728.554; 5.922,577: and WO/9831826.
Glycosidases can also be used to prepare saccharides. Glycosidases normally catalyze
the hydrolysis of a glycosidic bond. However, under appropriate conditions, they can be used
to form this linkage. Most glycosidases used for carbohydrate synthesis are exoglycosidases;
the glycosyl transfer occurs at the non-reducing terminus of the substrate. The glycosidase
binds a glycosyl donor in a glycosyl-enzyme intermediate that is either intercepted by water
to yield the hydrolysis product, or by an acceptor, to generate a new glycoside or
oligosaccharide. An exemplary pathway using an exoglycosidase is the synthesis of the core
trisaccharide of all N-linked glycopeptides, including the ß-mannoside linkage, which is
formed by the action of P-mannosidase (Singh et al., Chem. Commun. 993-994 (1996)).
Tn another exemplary application of the use of a glycosidase to form a glycosidic
linkage, a mutant glycosidase has been prepared in which the normal nucleophilic amino acid
within the active site is changed to a non-nucleophilic amino acid. The mutant enzyme does
not hydrolyze glycosidic linkages, but can still form them. Such a mutant glycosidase is used
to prepare oligosaccharides using an a-glycosyl fluoride donor and a glycoside acceptor
molecule (Withers et al., U.S. Patent No. 5,716,812).
Although their use is less common than that of the exoglycosidases, endoglycosidases
are also utilized to prepare carbohydrates. Methods based on the use of endoglycosidases
have the advantage that an oligosaccharide, rather than a monosaccharide, is transferred.
Oligosaccharide fragments have been added to substrates using end-P-N-acetylglucosamines
such as endo-F, endo-M (Wang et al., Tetrahedron Lett. 37: 1975-1978); and Haneda et al.,
Carbohydr. Res. 292: 61-70 (1996)).
In addition to their use in preparing carbohydrates, the enzymes discussed above arc
applied to the synthesis of glycopeptides as well. The synthesis of a homogenous glycoform
of ribonuclease B has been published (Witte K.. et al., J. Am. Chem. Soc. 119: 2114-2118
(1997)). The high mannose core of ribonuclease B was cleaved by treating the glycopeptide
with endoglycosidase H. The cleavage occurred specifically between the two core GlcNAc
residues. The tetrasaccharide sialyl Lewis X was then enzymatically rebuilt on the remaining
GlcNAc anchor site on the now homogenous protein by the sequential use of p-1,4-
galactosyltransferase. a-2,3-sialyltransferase and a-1,3-fucosyltransferase V. However,
while each enzymatically catalyzed step proceeded in excellent yield, such procedures have
not been adapted for the generation of glycopeptides on an industrial scale.
Methods combining both chemical and enzymatic synthetic elements are also known
in the art. For example. Yamamoto and coworkers (Carbohydr. Res. 305: 415-422 (1998))
reported the chemoenzymatic synthesis of the glycopeptide, glycosylated Peptide T, using an
endoglycosidase. The N-acetylglucosaminyl peptide was synthesized by purely chemical
means. The peptide was subsequently enzymatically elaborated with the oligosaccharide of
human transferrin peptide. The saccharide portion was added to the peptide by treating it
with an endo-P-N-acetylglucosaminidase. The resulting glycosylated peptide was highly
stable and resistant to proteolysis when compared to the peptide T and N-acetylglucosaminyl
peptide T.
The use of glycosyltransferases to modify peptide structure with reporter groups has
been explored. For example, Brossmer et al. (U.S. Patent No. 5,405,753) discloses the
formation of a fluorescent-labeled cytidine monophosphate ("CMP") derivative of sialic acid
and the use of the fluorescent glycoside in an assay for sialyl transferase activity and for the
fluorescent-labeling of cell surfaces, glycoproteins and peptides. Gross et al. {Analyt.
Biochem. 186: 127 (1990)) describe a similar assay. Bean et al. (U.S. Patent No. 5.432.059)
discloses an assay for glycosylation deficiency disorders utilizing reglycosylation of a
deficiently glycosylated protein. The deficient protein is reglycosylated with a fluorescent-
labeled CMP glycoside. Each of the fluorescent sialic acid derivatives is substituted with the
fluorescent moiety at either the 9-position or at the amine that is normally acetylated in sialic
acid. The methods using the fluorescent sialic acid derivatives are assays for the presence of"
glycosyltransferases or for non-glycosylated or improperly glycosylated glycoproteins. The
assays are conducted on small amounts of enzyme or glycoprotein in a sample of biological
origin. The enzymatic derivatization of a glycosylated or non-glycosylated peptide on a
preparative or industrial scale using a modified sialic acid has not been disclosed or suggested
in the prior art.
Considerable effort has also been directed towards the modification of cell surfaces by
altering glycosyl residues presented by those surfaces. For example. Fukuda and coworkers
have developed a method for attaching glycosides of defined structure onto cell surfaces.
The method exploits the relaxed substrate specificity of a fucosyltransferase that can transfer
fucose and fucose analogs bearing diverse glycosyl substrates (Tsuboi et ah../. Biol. Cheni.
271:27213(1996)).
Enzymatic methods have also been used to activate glycosyl residues on a
glycopeptide towards subsequent chemical elaboration. The glycosyl residues are typically
activated using galactose oxidase, which converts a terminal galactose residue to the
corresponding aldehyde. The aldehyde is subsequently coupled to an amine-containing
modifying group. For example, Casares et al. {Nature Biotech. 19: 142 (2001)) have attached
doxorubicin to the oxidized galactose residues of a recombinant MHCH-peptide chimera.
Glycosyl residues have also been modified to contain ketone groups. For example,
Mahal and co-workers {Science 276: 1125 (1997)) have prepared N-levulinoyl mannosamine
("ManLey"). which has a ketone functionality at the position normally occupied by the acetyl
group in the natural substrate. Cells were treated with the ManLev, thereby incorporating a
ketone group onto the cell surface. See, also Saxon et ah. Science 287: 2007 (2000): Hang et
ah,./. Am. Chem. Sot: 123: 1242 (2001); Yarema et ah, J. Biol. Chem. 273: 31168 (1998);
and Charter et ah. Glycobiology 10: 1049 (2000).
The methods of modifying cell surfaces have not been applied in the absence of a cell
to modify a glycosylated or non-glycosylated peptide. Further, the methods of cell surface
modification are not utilized for the enzymatic incorporation preformed modified glycosyl
donor moiety into a peptide. Moreover, none of the cell surface modification methods are
practical for producing glycosyl-modified peptides on an industrial scale.
Despite the efforts directed toward the enzymatic elaboration of saccharide structures,
there remains still a need for an industrially practical method for the modification of
glycosylated and non-glycosylated peptides with modifying groups such as water-soluble
polymers, therapeutic moieties, biomolecules and the like. Of particular interest are methods
in which the modified peptide has improved properties, which enhance its use as a
therapeutic or diagnostic agent. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The invention includes a multitude of methods of remodeling a peptide to have
a specific glycan structure attached thereto. Although specific glycan structures are described
herein, the invention should not be construed to be limited to any one particular structure. In
addition, although specific peptides are described herein, the invention should not be limited
by the nature of the peptide described, but rather should encompass any and all suitable
peptides and variations thereof.
The description which follows discloses the preferred embodiments of the
invention and provides a written description of the claims appended hereto. The invention
encompasses any and all variations of these embodiments that are or become apparent
following a reading of the present specification.
The invention includes a cell-free, in vitro method of remodeling an
erythropoietin (EPO) peptide, the peptide having the formula:

wherein
AA is a terminal or internal amino acid residue of the peptide;
X1-X2 is a saccharide covalently linked to the AA, wherein
X1 is a first glycosyl residue; and
X2 is a second glycosyl residue covalently linked to X1, wherein X1 and X2 are
selected from monosaccharyl and oligosaccharyl residues;
the method comprising:
(a) removing X2 or a saccharyl subunit thereof from the peptide, thereby
forming a truncated glycan; and
(b) contacting the truncated glycan with at least one glycosyltransferase and at
least one glycosyl donor under conditions suitable to transfer the at least
one glycosyl donor to the truncated glycan, thereby remodeling the EPO
peptide.
The method further comprises:
(c) removing X1, thereby exposing the AA; and
(d) contacting the AA with at least one glycosyltransferase and at least one
glycosyl donor under conditions suitable to transfer the at least one
glycosyl donor to the AA, thereby remodeling the EPO peptide.
The method additionally comprises:
(e) prior to step (b), removing a group added to the saccharide during post-
translational modification.
In one aspect, the group is a member selected from phosphate, sulfate,
carboxylate and esters thereof.
In one aspect of the method, the peptide has the formula:

wherein
Z is a member selected from O, S, NH and a crosslinker.
In one embodiment, at least one of the glycosyl donors comprises a modifyin
group.
In this and in other embodiments, the modifying group is a member selected
from the group consisting of a polymer, a therapeutic moiety, a detectable label, a reactive
linker group, a targeting moiety, and a peptide. Preferably, the modifying group is a water
soluble polymer, preferably, poly(ethylene glycol), wherein preferably, the poly(ethylene
glycol) has a molecular weight distribution that is essentially homodisperse.
There is also provided a cell-free in vitro method of remodeling an EPO
peptide, the peptide having the formula:
wherein
X'1. X'1 X5, X6, X7, and X17are independently selected monosaccharyl or
oligosaccharyl residues; and
a, b. c. d. e and x are independently selected from the integers 0, 1 and 2, with
the proviso that at least one member selected from a, b, c, d, and e and
x are 1 or 2; the method comprising:
(a) removing at least one of X3, X4 X5, X6, X7, or X17, a saccharyl subunit
thereof from the peptide, thereby forming a truncated glycan; and
(b) contacting the truncated glycan with at least one glycosyltransferase and at
least one glycosyl donor under conditions suitable to transfer the at
least one glycosyl donor to the truncated glycan, thereby remodeling
the EPO peptide.
In one embodiment, the removing of step (a) produces a truncated glycan in
which a, b, c, e and x are each 0.
In another embodiment, X3, X5, and X7, are selected from the group consisting
of (mannose)z and (mannose)z-(X8)y
wherein
X8 is a glycosyl moiety selected from mono- and oligo-saccharides;
y is an integer selected from 0 and 1; and
z is an integer between 1 and 20, wherein
when z is 3 or greater, (mannose), is selected from linear and branched
structures.
In a further embodiment, X4 is selected from the group consisting of GlcNAc
and xylose.
In yet another embodiment. X3, X5. and X7 are (mannose)u. wherein
u is selected from the integers between I and 20, and when u is 3 or greater.
(mannose)u is selected from linear and branched structures.
In one aspect, at least one of the glycosyl donors comprises a modifying
group.
There is further provided a cell-free in vitro method of remodeling an EPO
peptide comprising a glycan having the formula:

wherein
r, s. and t are integers independently selected from 0 and 1,
the method comprising:
(a) contacting the peptide with at least one glycosyltransferase and at least
one glycosyl donor under conditions suitable to transfer the at least one
glycosyl donor to the glycan, thereby remodeling the EPO peptide.
In one aspect, at least one of the glycosyl donors comprises a modifying
group.
In another aspect, the peptide has the formula:

wherein
X9 and X10 are independently selected monosaccharyl or oligosaccharyl
residues; and
m, n and f are integers selected from 0 and 1.
Further, the peptide has the formula:

wherein
X and X are independently selected glycosyl moieties; and
r and x are integers independently selected from 0 and 1.
In another aspect, X" and X12 are (mannose)q, wherein
q is selected from the integers between 1 and 20, and when q is three or
greater. (mannosc)q is selected from linear and branched structures.
In addition, the peptide has the formula:

wherein
X13,X14, and X15 are independently selected glycosyl residues; and
g. h, i. j, k. and p are independently selected from the integers 0 and I. with
the proviso that at least one of g, h. i, j, k and p is I.
The method also includes wherein
X11 and X15 are members independently selected from GlcNAc and Sia: and i
and k are independently selected from the integers 0 and I. with the
proviso that at least one of i and k is 1 and if k is 1, g, h and j arc 0.
In an additional aspect, the peptide has the formula:

wherein
X16 is a member selected from:

wherein
s and i are integers independently selected from 0 and 1.
In one embodiment, the removing utilizes a glycosidase.
Also included is a cell-free, in vitro method of remodeling an EPO peptide
having the formula:
wherein
AA is a terminal or internal amino acid residue of the peptide;
X is a glycosyl residue covalently linked to the AA, selected from
monosaccharyl and oligosaccharyl residues; and
u is an integer selected from 0 and 1.
the method comprising:
contacting the peptide with at least one glycosyltransferase and at least one
glycosyl donor under conditions suitable to transfer the at least one glycosyl donor to the
truncated glycan, wherein the glycosyl donor comprises a modifying group, thereby
remodeling the EPO peptide.
The invention further provides a covalent conjugate between an EPO peptide
and a modifying group that alters a property of the peptide, wherein the modifying group is
covalently attached to the peptide at a preselected glycosyl or amino acid residue of the
peptide via an intact glycosyl linking group.
In one aspect, the modifying group is a member selected from the group
consisting of a polymer, a therapeutic moiety, a detectable label, a reactive linker group, a
targeting moiety, and a peptide. In another aspect, the modifying group and an intact
glycosyl linking group precursor are linked as a covalently attached unit to the peptide via the
action of an enzyme, the enzyme converting the precursor to the intact glycosyl linking
group, thereby forming the conjugate. In a further aspect, the covalent conjugate comprises a
first modifying group covalently linked to a first residue of the peptide via a first intact
glycosyl linking group, and a second glycosyl linking group linked to a second residue of the
peptide via a second intact glycosyl linking group. In one embodiment, the first residue and
the second residue are structurally identical. In another embodiment, the first residue and the
second residue have different structures. In an additional embodiment, the first residue and
the second residue are glycosyl residues. Further, the first residue and the second residue are
amino acid residues. Also, the peptide may be remodeled prior to forming the conjugate. In
another aspect, the remodeled peptide is remodeled to introduce an acceptor moiety for the
.intact glycosyl linking group. In yet a further aspect, themodifying group is a water-soluble
polymer. In one aspect, the intact glycosyl linking unit is a member selected from the group
consisting of a sialic acid residue, a Gal residue, a GlcNAc residue, and a GalNAc residue.
There is a also provided a method of forming a covalent conjugate between a
polymer and a glycosylated or non-glycosylated peptide, wherein the polymer is conjugated
to the peptide via an intact glycosyl linking group interposed between and covalently linked
to both the peptide and the polymer, the method comprising:
contacting the peptide with a mixture comprising a nucleotide sugar covalently
linked to the polymer and a glycosyltransferase for which the nucleotide sugar is a substrate
under conditions sufficient to form the conjugate, wherein the peptide is E^PO.
In one aspect, the polymer is a water-soluble polymer. In another aspect, the
glycosyl linking group is covalently attached to a glycosyl residue covalently attached to the
peptide. In yet a further aspect, the glycosyl linking group is covalently attached to an amino
acid residue of the peptide. In an additional aspect, the polymer comprises a member
selected from the group consisting of a polyalkylene oxide and a polypeptide. An in another
aspect, the polyalkylene oxide is poly(ethylene glycol).
In yet a further aspect, the glycosyltransferase is selected from the group
consisting of sialyltransferase, galactosyltransferase, glucosyltransferase. GalNAc

transferase, and a GlcNAc transferase. Additionally, the glycosyltransferase is recombinantly
produced and may be either a recombinant prokaryotic or eukaryotic enzyme.
In one embodiment, the nucleotide sugar is selected from the group consisting
of UDP-glycoside. CMP-glycoside, and GDP-glycoside. Further, the nucleotide sugar is
selected from the group consisting of UDP-galactose, UDP-galactosamine, UDP-glucose,
UDP-giucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylglucosamine, GDP-mannose,
GDP-fucose, CMP-sialic acid, CMP-NeuAc. In addition, the glycosylated peptide is partially
deglycosylated prior to the contacting. Further, the intact glycosyl linking group is a sialic
acid residue. In addition, the method is performed in a cell-free environment. Also, the
covalent conjugate is isolated, preferably by membrane filtration.
The invention further provides a composition for forming a conjugate between
a peptide and a modified sugar, the composition comprising: an admixture of a modified
sugar, a glycosyltransferase, and a peptide acceptor substrate, wherein the modified sugar has
covalently attached thereto a member selected from a polymer, a therapeutic moiety and a
biomolecule. wherein the peptide is EPO.
In addition, there is provided an EPO peptide remodeled by the methods of the
invention and pharmaceutical compositions comprising such EPO peptides.
Also provided is a cell-free, in vitro method of remodeling a peptide having
the formula:
wherein
A A is a terminal or internal amino acid residue of the peptide,
the method comprising:
contacting the peptide with at least one glycosyltransferase and at least one
glycosyl donor under conditions suitable to transfer the at least one glycosyl donor to the
amino acid residue, wherein the glycosyl donor comprises a modifying group, thereby
remodeling the peptide, wherein the peptide is EPO.
In addition, there is provided a method for forming a conjugate between an EPO
peptide and a modifying group, wherein the modifying group is covalently attached to the
EPO peptide through an intact glycosyl linking group, the EPO peptide comprising a glycosyl
residue having a formula which is a member selected from:

wherein
a, b, c, d, i, n, o, p, q, r, s, t. and u are members independently selected
from 0 and 1;
c, f, g. and h are members independently selected from the integers
between 0 and 4;
j. k. 1. and m are members independently selected from the integers
between 0 and 20:
v, w, x, y, and z are 0; and
R is a modifying group, a mannose or an oligomannose;
the method comprising:
(a) contacting the EPO peptide with a glycosyltransferase and a modified
glycosyl donor, comprising a glycosyl moiety which is a substrate for
the glycosyltransferase covalently linked to the modifying group,
under conditions appropriate for the formation of the intact glycosyl
linking group.
The method further comprises:
(b) prior to step (a), contacting the EPO peptide with a sialidase under conditions
appropriate to remove sialic acid from the EPO peptide.
The method additionally comprises:
(c) contacting the product of step (a) with a sialyltransferase and a sialic acid donor
under conditions appropriate to transfer sialic acid to the product.
The method further comprises:
(d) prior to step (a), contacting the EPO peptide with a galactosidase operating
synthetically under conditions appropriate to add a galactose to the EPO
peptide.
In addition, the method comprises:
(e) prior to step (a), contacting the EPO peptide with a galactosyl transferase and a
galactose donor under conditions appropriate to transfer the galactose to the
EPO peptide.
The method also comprises:
(f) contacting the product from step (e) with ST3Gal3 and a sialic acid donor under
conditions appropriate to transfer sialic acid to the product.
The method also comprises:
(g) contacting the product from step (a) with a moiety that reacts with the modifying
group, thereby forming a conjugate between the intact glycosyl linking group
and the moiety.
In addition, the method comprises:
(h) prior to step (a), contacting the EPO peptide with N-acetylglucosamine transferase
and a GlcNAc donor under conditions appropriate to transfer GlcNAc to the
EPO peptide.
In one asepct. the modifying group is a member selected from a polymer, a
toxin, a radioisotope, a therapeutic moiety and a glycoconjugate.
In another aspect,
a, b, c. d, e, f, g, n, and q are 1;
h is a member selected from the integers between 1 and 3;
i, j. k, 1, m. o. p. r. s, t, and u are members independently selected from 0 and I: and
v, w, x, y and /. are 0.
In yet another aspect,
a. b, c, d, f, h. j, k, 1, m, q, s, u, v, w, x, y, and z are 0; and
e, g, i, r, and t are members independently selected from 0 and I.
In an additional aspect.
a, b. c. d. e. f. g, h, i, j, k, I. m. n. o. p, q. r, s, t and u are members independently
selected from 0 and 1; and
v, w, x, y, and z are 0.
In a further aspect,
a, b, c, d. e. f. g, n. and q arc 1;
h is a member selected from the integers between 1 and 3;
i, j, k, 1, m, o, p, r, s, t, and u are members independently selected from 0 and 1; and
v, w, x, y and z are 0.
In another aspect,
a, b. c, d. f, h. j. k, I, m, o, p, s, u, v, w, x, y, and z are 0; and
e, g, i. n, q, r, and t are independently selected from 0 and 1.
Additionally,
a, b, c, d. f, h, j, k. I, m, n, o, p. s, u, v, w. x, y, and z are 0; and
c, g. i. q, r, and t are members independently selected from 0 and 1.
Further
q is 1;
a, b, c, d, e. f, g, h, i, n, r, s, t, and u are members independently selected from 0 and 1;
and
j. k, 1, m, o, p. v, w. x, y, and z are 0.
There is also provided an EPO peptide conjugate formed by the methods of
the invention.
Further provided is an EPO peptide comprising one or more glycans, having a
glycoconjugatc molecule covalcntly attached to the peptide. In one aspect, the one or more
glycans is a monoantennary glycan. In another aspect, the one or more glycans is a
biantennary glycan. In a further aspect, the one or more glycans is a triantennary glycan. In
yet another aspect, the one or more glycans is at least a triantennary glycan. In a further
aspect, the one or more glycans comprises at least two glycans comprising a mixture of mono
or multiantennary glycans. In yet another aspect, the one or more glycans is selected from an
N-linkcd glycan and an O-linked glycan. In a further aspect, the one or more glycans is at
least two glycans selected from an N-linked and an O-linked glycan. Additionally, the
peptide is expressed in a cell selected from the group consisting of a prokaryotic cell and a
eukaryotic cell, and the eukaryotic cell is selected from the group consisting of a mammalian
cell, an insect cell and a yeast cell.
Also provided in the invention is a glycoPEGylated EPO peptide comprising
an EPO peptide and at least one glycan and at least one poly(ethylene glycol) molecule
covalently attached to the glycan, wherein the poly(ethylene glycol) molecule is added to the
F.PO using a glycosyltransferase. In one aspect, the glycoPEGylated EPO peptide comprises
at least one mono-antennary glycan. In another aspect, all of the glycans are N-linked and are
mono-antennary. In a further aspect, all of the glycans are N-linked and at least one of the
glycans comprise the poly(ethylene glycol). In an additional aspect, more than one of the
glycans comprises the poly(ethylene glycol). In a further aspect, all of the glycans are re-
linked and all of the glycans comprise the poly(ethylene glycol). Additionally, the
glycoPEGylated peptide comprises at least three mono-antennary glycans having the
poly(ethylene glycol) covalently attached thereto.
There is further provided a glycoPEGylated EPO peptide, wherein the EPO
peptide comprises three or more glycans. In one aspect, at least one of the glycans
comprises the poly(ethylene glycol) covalently attached thereto. In another aspect, more than
one of the glycans comprises the poly(ethylene glycol) covalently attached thereto. In a
further aspect, all of the glycans comprise the poly(ethylene glycol) covalently attached
thereto. Additionally, the poly(ethylene glycol) is linked to at least one sugar moiety selected
from the group consisting of fucose (Fuc), N-acetylglucosamine (GlcNAc). galactose (Gal)
and a sialic acid (SA). Additionally, the sialic acid is N-acetylneuraminic acid. Further, the
EPO peptide does not comprise an O-linked glycan. In addition, the EPO peptide comprises
at least one O-linked glycan. Further, the O-linked peptide comprises the poly(ethylene
glycol) covanently attached thereto. In addition, the EPO peptide is recombinantly expressed
in a cell. Further, the cell is selected from the group consisting of an insect cell, a yeast cell
and a mammalian cell. Preferably, the the mammalian cell is a CHO cell. In one aspect, the
polyCethylene glycol) has a molecular weight selected from the group consisting of about 1
kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa and 40 kDa. In another aspect, the EPO peptide
is selected from the group consisting of a naturally occurring EPO peptide and a mutated
EPO peptide. In another aspect, mutated EPO peptide comprises the amino acid sequence of
SEQ ID NO:73 having at least one mutation selected from the group consisting of Arg'39 to
Ala139. Arg143 to Ala143 and Lys154 to Ala154.
There is also provided a method of making a glycoPEGylated EPO peptide,
the method comprising the steps of:
(a) contacting an EPO peptide with a mixture comprising a nucleotide sugar
covalently linked to poly(ethylene glycol) and a glycosyltransferase under conditions
sufficient to transfer the poly(ethylene glycol) to the EPO peptide.
In one embodiment, the sugar of the nucleotide sugar is selected from the
group consisting of fucose (Fuc), N-acetylglucosamine (GlcNAc), galactose (Gal) and a sialic
acid (SA). In another embodiment, the sialic acid is N-acetyineuraminic acid (NAN). In a
further embodiment, the poly(ethylene glycol) has a molecular weight selected from the
group consisting of about 1 kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa and 40 kDa. In an
additional embodiment, the EPO peptide is recombinantly expressed in a cell, where the cell
may be selected from the group consisting of an insect cell, a yeast cell and a mammalian
cell. In one aspect, the EPO peptide is selected from the group consisting of a naturalK
occurring EPO peptide and a mutated EPO peptide. In another aspect, the mature EPO has
the sequence of SEQ ID NO:73. In a further aspect, the mutated EPO peptide comprises the
amino acid sequence of SEQ ID NO: 73 having at least one mutation selected from the group
consisting of Arg13" to Ala139, Arg143 to Ala143 and Lys154 to Ala154.
In another aspect of the method of the invention, before step (a): the method
comprises:
(b) contacting the EPO peptide with a mixture comprising a nucleotide-N-
acetylglucosamine (GlcNAc) molecule and an N-acetylglucosamine transferase (GnT) for
which the nucleotide-GlcNAc is a substrate under conditions sufficient to form a bond
between the GlcNAc and the EPO. wherein the GnT is selected from the group consisting of
GnT I, GnT II, GnT III. GnT IV, GnT V and GnT VI.
In one aspect, the mixture comprises one GnT selected from the group
consisting of GnT I, GnT II, GnT IV, GnT V and GnT VI. In another aspect, the
glycoPEGylated EPO peptide comprises at least one mono-antennary glycan. In a further
aspect, the sugar of the nucleotide sugar is galactose and the glycosyltransferase is galactosyl
transferase I (GalT I).
In an additional aspect of the method, before step (a) but after step (b):
(c) contacting the EPO peptide with a mixture comprising a nucleotide galactose (Gal)
and galactosyl transferase 1 (GalT I) under conditions sufficient to transfer galactose to the
EPO peptide.
In one embodiment, in step (a), the sugar of the nucleotide sugar is sialic acid
and the glycosyltransferase is a sialyltransferase. In another embodiment, the sialic acid is N-
acetylneuraminic acid (NAN). In a further embodiment, the sialyltransferase is selected from
the group consisting of a(2,3)sialyltransferase, a(2,6)sialyltransferase and
(2,8)sialyltransferase.
There is also provided a glycoPEGylated EPO, the EPO comprising the
sequence of SEQ ID NO:73. In one aspect, the EPO comprises the sequence of SEQ ID
NO:73 and further comprising a mutation in the sequence.
There is further provided in the invention a method of making a
glycoPEGylated EPO peptide, the method comprising the steps of:
(a) contacting an EPO peptide with a mixture comprising a nucleotide sugar
covalently linked to poly(ethylene glycol) and a glycosyltransferase under conditions
sufficient to transfer the poly(ethylene glycol) to the EPO peptide, wherein the
glycosyltransferase is a fucosyltransferase.
In one aspect, the fucosyltransferase is selected from the group consisting of
fucosyltransferase I, fucosyltransferase 111, fucosyltransferase IV, fucosyltransferase V,
fucosyltransferase VI and fucosyltransferase VII, and in another, EPO is expressed in a CHO
cell.
There is further provided a method of treating a mammal having anemia, the
method comprising administering to the mammal an EPO peptide having one or more
glycans having a glycoconjugate molecule attached to the peptide, wherein the EPO is
administered in an amount effective to increase the hematocrit level in the mammal. In this
and in other embodiments, the mammal is a human.
Also provided is a method of providing erythropoietin therapy to a mammal,
the method comprising administering an effective amount of a glycoPEGylated EPO peptide
comprising an EPO peptide and at least one glycan and at least one poly(ethylene glycol)
molecule covalently attached to the glycan, wherein the poly(ethylene glycol) molecule is
added to the EPO using a glycosyltransferase, wherein the EPO is administered in an amount
effective to increase the hematocrit level in the mammal.
In addition, there is provided a method of treating a mammal having anemia,
the method comprising administering to the mammal a glycoPEGylated EPO peptide
comprising an EPO peptide and at least one glycan and at least one poly(ethylene glycol)
molecule covalently attached to the glycan. wherein the poly(ethylene glycol) molecule is
added to the EPO using a glycosyltransferase, wherein the EPO is administered in an amount
effective to increase the hematocrit level in the mammal. In one aspect, the anemia is
associated with chemotherapy.
Further provided is a method of treating a kidney dialysis patient, the method
comprising administering to the patient a glycoPEGylated EPO peptide comprising an EPO
peptide and at least one glycan and at least one poly(ethylene glycol) molecule covalently
attached to the glycan, wherein the poly(ethylene glycol) molecule is added to the EPO using
a glvcosyltransferase. wherein the EPO is administered in an amount effective to increase the
hematocrit level in the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
For the purpose of illustrating the invention, there are depicted in the drawings certain
embodiments of the invention. However, the invention is not limited to the precise
arrangements and instrumentalities of the embodiments depicted in the drawings.
Figure I is a scheme depicting a trimannosyl core glycan (left side) and the enzymatic
process for the generation of a glycan having a bisecting GlcNAc (right side).
Figure 2 is a scheme depicting an elemental trimannosyl core structure and complex
chains in various degrees of completion. The in vitro enzymatic generation of an elemental
trimannosyl core structure from a complex carbohydrate glycan structure which does not
contain a bisecting GlcNAc residue is shown, as is the generation of a glycan structure
therefrom which contains a bisecting GlcNAc. Symbols: squares: GlcNAc; light circles:
Man; dark circles: Gal; triangles: NeuAc.
Figure 3 is a scheme for the enzymatic generation of a sialylated glycan structure
(right side) beginning with a glycan having a trimannosyl core and a bisecting GlcNAc (left
side).
Figure 4 is a scheme of a typical high mannose containing glycan structure (left side)
and the enzymatic process for reduction of this structure to an elemental trimannosyl core
structure. In this scheme, X is mannose as a monosaccharide, an oligosaccharide or a
polysaccharide.
Figure 5 is a diagram of a fucose and xylose containing N-linked glycan structure
typically produced in plant cells.
Figure 6 is a diagram of a fucose containing N-linked glycan structure typically
produced in insect cells. Note that the glycan may have no core fucose, it amy have a single
core fucose with either linkage, or it may have a single core fucose having a preponderance
of one linkage.
Figure 7 is a scheme depicting a variety of pathways for the trimming of a high
mannose structure and the synthesis of complex sugar chains therefrom. Symbols: squares:
GlcNAc; circles: Man; diamonds: fucose; pentagon: xylose.
Figure 8 is a scheme depicting in vitro strategigg for the synthesis of complex
structures from an elemental trimannosyl core structure. Symbols: Squares: GlcNAc; light
circles: Man; dark circles: Gal; dark triangles: NeuAc; GnT: N-acetyl
glucosaminyltransferase; GalT: galactosyltransferase; ST: sialyltransferase.
Figure 9 is a scheme depicting two in vitro strategies for the synthesis of
monoantennary glycans, and the optional glycoPEGylation of the same. Dark squares:
GlcNAc; dark circles: Man; light circles: Gal; dark triangles: sialic acid.
Figure 10 is a scheme depicting two in vitro strategies for the synthesis of
monoantennary glycans, and the optional glycoPEGylation of the same. Dark squares:
GlcNAc; dark circles: Man; light circles: Gal; dark triangles: sialic acid.
Figure II is a scheme depicting various complex structures, which may be
synthesized from an elemental trimannosyl core structure. Symbols: Squares: GlcNAc; light
circles: Man; dark circles: Gal; triangles: NeuAc; diamonds: fucose; FT and FucT:
fucosyltransferasc; GalT: galactosyltransferase; ST: sialyltransferase; Le: Lewis antigen;
SLe: sialylated Lewis antigen.
Figure 12 is an exemplary scheme for preparing O-linked glycopeptides originating
with serine or threonine. Optionally, a water soluble polymer (WSP) such as poly(ethylene
glycol) is added to the final glycan structure.
Figure 13 is a series of diagrams depicting the four types of O-glycan structures,
termed cores 1 through 4. The core structure is outlined in dotted lines.
Figure 14, comprising Figure I4A and Figure 14B, is a series of schemes showing an
exemplary embodiment of the invention in which carbohydrate residues comprising complex
carbohydrate structures and/or high man nose high mannose structures are trimmed back to
the first generation biantennary structure. Optionally, fucose is added only after reaction with
GnT I. A modified sugar bearing a water-soluble polymer (WSP) is then conjugated to one
or more of the sugar residues exposed by the trimming back process.
Figure 15 is a scheme similar to that shown in Figure 4, in which a high mannose or
complex structure is "trimmed back" to the mannose beta-linked core and a modified sugar
bearing a water soluble polymer is then conjugated to one or more of the sugar residues
exposed by the trimming back process. Sugars are added sequentially using
glycosyltransferases.
Figure 16 is a scheme similar to that shown in Figure 4, in which a high mannose or
complex structure is trimmed back to the GlcNAc to which the first mannose is attached, and
a modified sugar bearing a water soluble polymer is then conjugated to one or more of the
sugar residues exposed by the trimming back process. Sugars are added sequentially using
glycosyltransferases.
Figure 17 is a scheme similar to that shown in Figure 4. in which a high mannose or
cpomplex structure is trimmed back to the first GlcNAc attached to the Asn of the peptide,
following which a water soluble polymer is conjugated to one or more sugar residues which
have subsequently been added on. Sugars are added sequentially using glycosyltransferases.
Figure 18, comprising Figure 18A and 18B, is a scheme in which an N-linked
carbohydrate is optionally trimmed back from a high mannose or cpmplex structure, and
subsequently derivatized with a modified sugar moiety (Gal or GlcNAc) bearing a water-
soluble polymer.
Figure 19, comprising Figure 19A and 19B, is a scheme in which an N-linked
carbohydrate is trimmed back from a high mannose or complex structure and subsequently
derivatized with a sialic acid moiety bearing a water-soluble polymer. Sugars are added
sequentially using glycosyltransferases.
Figure 20 is a scheme in which an N-linked carbohydrate is optionally trimmed back
from a high mannose oor complex structure and subsequently derivatized with one or more
sialic acid moieties, and terminated with a sialic acid derivatized with a water-soluble
polymer. Sugars are added sequentially using glycosyltransferases.
Figure 21 is a scheme in which an O-linked saccharide is "trimmed back" and
subsequently conjugated to a modified sugar bearing a water-soluble polymer. In the
exemplary scheme, the carbohydrate moiety is "trimmed back" to the first generation of the
biantennary structure.
Figure 22 is an exemplary scheme for trimming back the carbohydrate moiety of an
O-linked glycopeptide to produce a mannose available for conjugation with a modified sugar
having a water-soluble polymer attached thereto.
Figure 23. comprising Figure 23A to Figure 23C, is a series of exemplary schemes.
Figure 23 A is a scheme that illustrates addition of a PEGylated sugar, followed by the
addition of a non-modified sugar. Figure 23B is a scheme that illustrates the addition of more
that one kind of modified sugar onto one glycan. Figure 23C is a scheme that illustrates the
addition of different modified sugars onto O-linked glycans and N-linked glycans.
Figure 24 is a diagram of various methods of improving the therapeutic function of a
peptide by glycan remodeling, including conjugation.
Figure 25 is a set of schemes for glycan remodeling of a therapeutic peptide to treat
Gaucher Disease.
Figure 26 is a scheme for glycan remodeling to generate glycans having a terminal
mannose-6-phosphate moiety.
Figure 27 is a diagram illustrating the array of glycan structures found on CHO-
produced glucocerebrosidase (Cerezyme™) after sialylation.
Figure 28, comprising Figure 28A to Figure 28Z and Figure 28AA to Figure 28CC. is
a list of peptides useful in the methods of the invention.
Figure 29, comprising Figures 29A to 29G, provides exemplary schemes for
remodeling glycan structures on granulocyte colony stimulating factor (G-CSF). Figure 29A
is a diagram depicting the G-CSF peptide indicating the amino acid residue to which a glycan
is bonded, and an exemplary glycan formula linked thereto. Figure 29B to 29G are diagrams
of contemplated remodeling steps of the glycan of the peptide in Figure 29A based on the
type of cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 30, comprising Figures 30A to 30EE sets forth exemplary schemes for
remodeling glycan structures on interferon-alpha. Figure 30A is a diagram depicting the
interferon-alpha isoform 14c peptide indicating the amino acid residue to which a glycan is
bonded, and an exemplary glycan formula linked thereto. Figure 30B to 30D are diagrams of
contemplated remodeling steps of the glycan of the peptide in Figure 30A based on the type
of cell the peptide is expressed in and the desired remodeled glycan structure. Figure 301: is
a diagram depicting the interferon-alpha isoform 14c peptide indicating the amino acid
residue to which a glycan is linked, and an exemplary glycan formula linked thereto. Figure
30F to 30N are diagrams of contemplated remodeling steps of the glycan of the peptide in
Figure 30E based on the type of cell the peptide is expressed in and the desired remodeled
glycan structure. Figure 30O is a diagram depicting the interferon-alpha isoform 2a or 2b
peptides indicating the amino acid residue to which a glycan is linked, and an exemplary
glycan formula linked thereto. Figure 30P to 30W are diagrams of contemplated remodeling
steps of the glycan of the peptide in Figure 30O based on the type of cell the peptide is
expressed in and the desired remodeled glycan structure. Figure 30X is a diagram depicting
the interferon-alpha-mucin fusion peptides indicating the residue(s) which is linked to
glycans contemplated for remodeling, and exemplary glycan formulas linked thereto. Figure
30Y to 30AA are diagrams of contemplated remodeling steps of the glycan of the peptides in
Figure 30X based on the type of cell the peptide is expressed in and the desired remodeled
glycan structure. Figure 30BB is a diagram depicting the interferon-alpha-mucin fusion
peptides and interferon-alpha peptides indicating the residue(s) which bind to glycans
contemplated for remodeling, and formulas for the glycans. Figure 30CC to 30EE are
diagrams of contemplated remodeling steps of the glycan of the peptides in Figure 30BB
based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure.
Figure 31. comprising Figures 31A to 31S, sets forth exemplary schemes for
remodeling glycan structures on interferon-beta. Figure 31A is a diagram depicting the
interferon-beta peptide indicating the amino acid residue to which a glycan is linked, and an
exemplary glycan formula linked thereto. Figure 31B to 31O are diagrams of contemplated
remodeling steps of the glycan of the peptide in Figure 31A based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure. Figure 31P is a diagram
depicting the interferon-beta peptide indicating the amino acid residue to which a glycan is
linked, and an exemplary glycan formula linked thereto. Figure 31Q to 31S are diagrams of
contemplated remodeling steps of the glycan of the peptide in Figure 31P based on the type
of cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 32, comprising Figures 32A to 32D, sets forth exemplary schemes for
remodeling glycan structures on Factor VII and Factor Vila. Figure 32A is a diagram
depicting the Factor-VII and Factor-Vila peptides A (solid line) and B (dotted line) indicating
the residues which bind to glycans contemplated for remodeling, and the formulas for the
glycans. Figure 32B to 32D are diagrams of contemplated remodeling steps of the glycan of
the peptide in Figure 32A based on the type of cell the peptide is expressed in and the desired
remodeled glycan structure.
Figure 33, comprising Figures 33A to 33G, sets forth exemplary schemes for
remodeling glycan structures on Factor IX. Figure 33A is a diagram depicting the Factor-IX
peptide indicating residues which bind to glycans contemplated for remodeling, and formulas
of the glycans. Figure 33B to 33G are diagrams of contemplated remodeling steps of the
glycan of the peptide in Figure 33A based on the type of cell the peptide is expressed in and
the desired remodeled glycan structure.
Figure 34. comprising Figures 34A to 34J. sets forth exemplary schemes for
remodeling glycan structures on follicle stimulating hormone (FSII). comprising a and [}
subunits. Figure 34A is a diagram depicting the Follicle Stimulating Hormone peptides
FSHa and FSHP indicating the residues which bind to glycans contemplated for remodeling,
and exemplary glycan formulas linked thereto. Figure 34B to 34J are diagrams of
contemplated remodeling steps of the glycan of the peptides in Figure 34A based on the type
of cell the peptides are expressed in and the desired remodeled glycan structures.
Figure 35. comprising Figures 35A to 35AA, sets forth exemplary schemes for
remodeling glycan structures on Erythropoietin (EPO). Figure 35A is a diagram depicting
the EPO peptide indicating the residues which bind to glycans contemplated for remodeling,
and formulas for the glycans. Figure 35B to 35S are diagrams of contemplated remodeling
steps of the glycan of the peptide in Figure 35A based on the type of cell the peptide is
expressed in and the desired remodeled glycan structure. Figure 35T is a diagram depicting
the EPO peptide indicating the residues which bind to glycans contemplated for remodeling,
and formulas for the glycans. Figure 35U to 35 W are diagrams of contemplated remodeling
steps of the glycan of the peptide in Figure 35T based on the type of cell the peptide is
expressed in and the desired remodeled glycan structure. Figure 35X is a diagram depicting
the EPO peptide indicating the residues which bind to glycans contemplated for remodeling,
and formulas for the glycans. Figure 35Y to 35AA are diagrams of contemplated remodeling
steps of the glycan of the peptide in Figure 35X based on the type of cell the peptide is
expressed in and the desired remodeled glycan structure.
Figure 36, comprising Figures 36A to 36K sets forth exemplary schemes for
remodeling glycan structures on Granulocyte-Macrophage Colony Stimulating Factor (GM-
CSF). Figure 36A is a diagram depicting the GM-CSF peptide indicating the residues which
bind to glycans contemplated for remodeling, and formulas for the glycans. Figure 36B to
36G are diagrams of contemplated remodeling steps of the glycan of the peptide in Figure
36A based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure. Figure 36H is a diagram depicting the GM-CSF peptide indicating the residues
which bind to glycans contemplated for remodeling, and formulas for the glycans. Figure 361
to 36K are diagrams of contemplated remodeling steps of the glycan of the peptide in Figure
36H based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure.
Figure 37. comprising Figures 37A to 37N, sets forth exemplary schemes for
remodeling glycan structures on interferon-gamma. Figure 37A is a diagram depicting an
interferon-gamma peptide indicating the residues which bind to glycans contemplated for
remodeling, and exemplary glycan formulas linked thereto. Figure 37B to 37G are diagrams
of contemplated remodeling steps of the peptide in Figure 37A based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure. Figure 37H is a diagram
depicting an interferon-gamma peptide indicating the residues which bind to glycans
contemplated for remodeling, and exemplary glycan formulas linked thereto. Figure 371 to
37N are diagrams of contemplated remodeling steps of the peptide in Figure 37H based on
the type of cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 38, comprising Figures 38A to 38N, sets forth exemplary schemes for
remodeling glycan structures on a1-antitrypsin (ATT, or a-1 protease inhibitor). Figure 38A
is a diagram depicting an AAT peptide indicating the residues which bind to glycans
contemplated for remodeling, and exemplary glycan formulas linked thereto. Figure 3813 to
38F are diagrams of contemplated remodeling steps of the glycan of the peptide in Figure
38A based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure. Figure 38G is a diagram depicting an AAT peptide indicating the residues which
bind to glycans contemplated for remodeling, and exemplary glycan formulas linked thereto.
Figure 38H to 38J are diagrams of contemplated remodeling steps of the peptide in Figure
38G based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure. Figure 38K is a diagram depicting an AAT peptide indicating the residues which
bind to glycans contemplated for remodeling, and exemplary glycan formulas linked thereto.
Figure 38L to 38N are diagrams of contemplated remodeling steps of the peptide in Figure
38K based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure.
Figure 39. comprising Figures 39A to 39J sets forth exemplary schemes for
remodeling glycan structures on glucocerebrosidase. Figure 39A is a diagram depicting the
glucocerebrosidase peptide indicating the residues which bind to glycans contemplated for
remodeling, and exemplary glycan formulas linked thereto. Figure 39B to 39F are diagrams
of contemplated remodeling steps of the glycan of the peptide in Figure 39A based on the
type of cell the peptide is expressed in and the desired remodeled glycan structure. Figure
39G is a diagram depicting the glucocerebrosidase peptide indicating the residues which bind
to glycans contemplated for remodeling, and exemplary glycan formulas linked thereto.
Figure 39H to 39K. are diagrams of contemplated remodeling steps of the glycan of the
peptide in Figure 39G based on the type of cell the peptide is expressed in and the desired
remodeled glycan structure.
Figure 40, comprising Figures 40A to 40W, sets forth exemplary schemes for
remodeling glycan structures on Tissue-Type Plasminogen Activator (TPA). Figure 40A is a
diagram depicting the TPA peptide indicating the residues which bind to glycans
contemplated for remodeling, and formulas for the glycans. Figure 40B to 40G are diagrams
of contemplated remodeling steps of the peptide in Figure 40A based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure. Figure 40H is a diagram
depicting the TPA peptide indicating the residues which bind to glycans contemplated for
remodeling, and formulas for the glycans. Figure 401 to 40K are diagrams of contemplated
remodeling steps of the peptide in Figure 40H based on the type of cell the peptide is
expressed in and the desired remodeled glycan structure. Figure 40L is a diagram depicting a
mutant TPA peptide indicating the residues which bind to glycans contemplated for
remodeling, and the formula for the glycans. Figure 40M to 40O are diagrams of
contemplated remodeling steps of the peptide in Figure 40L based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure. Figure 40P is a diagram
depicting a mutant TPA peptide indicating the residues which bind to glycans contemplated
for remodeling, and formulas for the glycans. Figure 40Q to 40S are diagrams of
contemplated remodeling steps of the peptide in Figure 40P based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure. Figure 40T is a diagram
depicting a mutant TPA peptide indicating the residues which links to glycans contemplated
for remodeling, and formulas for the glycans. Figure 40U to 40W are diagrams of
contemplated remodeling steps of the peptide in Figure 40T based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure.
Figure 41, comprising Figures 41A to 41G, sets forth exemplary schemes for
remodeling glycan structures on Interleukin-2 (IL-2). Figure 41A is a diagram depicting the
lnterleukin-2 peptide indicating the amino acid residue to which a glycan is linked, and an
exemplary glycan formula linked thereto. Figure 41B to 41G are diagrams of contemplated
remodeling steps of the glycan of the peptide in Figure 41A based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure.
Figure 42, comprising Figures 42A to 42M, sets forth exemplary schemes for
remodeling glycan structures on Factor VIII. Figure 42A are the formulas for the glycans
that bind to the N-linked glycosylation sites (A and A') and to the O-linked sites (B) of the
Factor VIII peptides. Figure 42B to 42F are diagrams of contemplated remodeling steps of
the peptides in Figure 42A based on the type of cell the peptide is expressed in and the
desired remodeled glycan structure. Figure 42G are the formulas for the glycans that bind to
the N-linked glycosylation sites (A and A') and to the O-linked sites (B) of the Factor VIII
peptides. Figure 42H to 42M are diagrams of contemplated remodeling steps of the peptides
in Figure 42G based on the type of cell the peptide is expressed in and the desired remodeled
glycan structures.
Figure 43, comprising Figures 43A to 43L, sets forth exemplary schemes for
remodeling glycan structures on urokinase. Figure 43A is a diagram depicting the urokinase
peptide indicating a residue which is linked to a glycan contemplated for remodeling, and an
exemplary glycan formula linked thereto. Figure 43B to 43F are diagrams of contemplated
remodeling steps of the peptide in Figure 43A based on the type of cell the peptide is
expressed in and the desired remodeled glycan structure. Figure 43G is a diagram depicting
the urokinase peptide indicating a residue which is linked to a glycan contemplated for
remodeling, and an exemplary glycan formula linked thereto. Figure 43H to 43L are
diagrams of contemplated remodeling steps of the peptide in Figure 43G based on the type of
cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 44, comprising Figures 44A to 44J, sets forth exemplary schemes for
remodeling glycan structures on human DNase (hDNase). Figure 44A is a diagram depicting
the human DNase peptide indicating the residues which bind to glycans contemplated for
remodeling, and exemplary glycan formulas linked thereto. Figure 44B to 44F are diagrams
of contemplated remodeling steps of the peptide in Figure 44A based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure. Figure 44G is a diagram
depicting the human DNase peptide indicating residues which bind to glycans contemplated
for remodeling, and exemplary glycan formulas linked thereto. Figure 44H to 44J are
diagrams of contemplated remodeling steps of the peptide in Figure 44F based on the type of
cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 45, comprising Figures 45A to 45L, sets forth exemplary schemes for
remodeling glycan structures on insulin. Figure 45A is a diagram depicting the insulin
peptide mutated to contain an N glycosylation site and an exemplary glycan formula linked
thereto. Figure 45B to 45 D are diagrams of contemplated remodeling steps of the peptide in
Figure 45A based on the type of cell the peptide is expressed in and the desired remodeled
glycan structure. Figure 45E is a diagram depicting insulin-mucin fusion peptides indicating
a residue(s) which is linked to a glycan contemplated for remodeling, and an exemplary
glycan formula linked thereto. Figure 45F to 45H are diagrams of contemplated remodeling
steps of the peptide in Figure 45E based on the type of cell the peptide is expressed in and the
desired remodeled glycan structure. Figure 451 is a diagram depicting the insulin-mucin
fusion peptides and insulin peptides indicating a residue(s) which is linked to a glycan
contemplated for remodeling, and formulas for the glycan. Figure 45J to 45L are diagrams of
contemplated remodeling steps of the peptide in Figure 451 based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure.
Figure 46, comprising Figures 46A to 46K, sets forth exemplary schemes for
remodeling glycan structures on the M-antigen (preS and S) of the Hepatitis B surface protein
(HbsAg). Figure 46A is a diagram depicting the M-antigen peptide indicating the residues
which bind to glycans contemplated for remodeling, and formulas for the glycans. Figure
46B to 46G are diagrams of contemplated remodeling steps of the peptide in Figure 46A
based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure. Figure 46H is a diagram depicting the M-antigen peptide indicating the residues
which bind to glycans contemplated for remodeling, and formulas for the glycans. Figure 461
to 46K are diagrams of contemplated remodeling steps of the peptide in Figure 46H based on
the type of cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 47. comprising Figures 47A to 47K, sets forth exemplary schemes for
remodeling glycan structures on human growth hormone, including N, V and variants
thereof. Figure 47A is a diagram depicting the human growth hormone peptide indicating a
residue which is linked to a glycan contemplated for remodeling, and an exemplary glycan
formula linked thereto. Figure 47B to 47D are diagrams of contemplated remodeling steps oi~
the glycan of the peptide in Figure 47A based on the type of cell the peptide is expressed in
and the desired remodeled glycan structure. Figure 47E is a diagram depicting the three
fusion peptides comprising the human growth hormone peptide and part or all of a mucin
peptide, and indicating a residue(s) which is linked to a glycan contemplated for remodeling,
and exemplary glycan formula(s) linked thereto. Figure 47F to 47K are diagrams of
contemplated remodeling steps of the glycan of the peptides in Figure 47E based on the type
of cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 48, comprising Figures 48A to 48G, sets forth exemplary schemes for
remodeling glycan structures on a TNF Receptor-IgG Fc region fusion protein (Enbrel™).
Figure 48A is a diagram depicting a TNF Receptor—IgG Fc region fusion peptide which may
be mutated to contain additional N-glycosylation sites indicating the residues which bind to
glycans contemplated for remodeling, and formulas for the glycans. The TNF receptor
peptide is depicted in bold line, and the IgG Fc regions is depicted in regular line. Figure
48B to 48G are diagrams of contemplated remodeling steps of the peptide in Figure 48 A
based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure.
Figure 49, comprising Figures 49A to 49D, sets forth exemplary schemes for
remodeling glycan structures on an anti-HER2 monoclonal antibody (Herceptin™). Figure
49A is a diagram depicting an anti-HER2 monoclonal antibody which has been mutated to
contain an N-glycosylation site(s) indicating a residue(s) on the antibody heavy chain which
is linked to a glycan contemplated for remodeling, and an exemplary glycan formula linked
thereto. Figure 49B to 49D are diagrams of contemplated remodeling steps of the glycan of
the peptides in Figure 49A based on the type of cell the peptide is expressed in and the
desired remodeled glycan structure.
Figure 50. comprising Figures 50A to 50D, sets forth exemplary schemes for
remodeling glycan structures on a monoclonal antibody to Protein F of Respiratory Syncytial
Virus (Synagis™). Figure 50A is a diagram depicting a monoclonal antibody to Protein F
peptide which is mutated to contain an N-glycosylation site(s) indicating a residue(s) which is
linked to a glycan contemplated for remodeling,, and an exemplary glycan formula linked
thereto. Figure 50B to 50D are diagrams of contemplated remodeling steps of the peptide in
Figure 50A based on the type of cell the peptide is expressed in and the desired remodeled
glycan structure.
Figure 51, comprising Figures 51A to 5ID, sets forth exemplary schemes for
remodeling glycan structures on a monoclonal antibody to TNF-a (Remicade™). Figure 51A
is a diagram depicting a monoclonal antibody to TNF-a which has an N-glycosylation site(s)
indicating a residue which is linked to a glycan contemplated for remodeling, and an
exemplary glycan formula linked thereto. Figure 5 IB to 51D are diagrams of contemplated
remodeling steps of the peptide in Figure 51A based on the type of cell the peptide is
expressed in and the desired remodeled glycan structure.
Figure 52. comprising Figures 52A to 52L, sets forth exemplary schemes for
remodeling glycan structures on a monoclonal antibody to glycoprotein IIb/IIIa (Reopro™).
Figure 52A is a diagram depicting a mutant monoclonal antibody to glycoprotein IIb-IIIa
peptides which have been mutated to contain an N-glycosylation site(s) indicating the
residue(s) which bind to glycans contemplated for remodeling, and exemplary glycan
formulas linked thereto. Figure 52B to 52D are diagrams of contemplated remodeling steps
based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure. Figure 52E is a diagram depicting monoclonal antibody to glycoprotein IIb/IIIa-
mucin fusion peptides indicating the residues which bind to glycans contemplated for
remodeling, and exemplary glycan formulas linked thereto. Figure 52F to 52H are diagrams
of contemplated remodeling steps based on the type of cell the peptide is expressed in and the
desired remodeled glycan structure. Figure 521 is a diagram depicting monoclonal antibody
to glycoprotein IIb/IIIa- mucin fusion peptides and monoclonal antibody to glycoprotein
IIb/IIIa peptides indicating the residues which bind to glycans contemplated for remodeling,
and exemplary glycan formulas linked thereto. Figure 52J to 52L are diagrams of
contemplated remodeling steps based on the type of cell the peptide is expressed in and the
desired remodeled glycan structure.
Figure 53, comprising Figures 53A to 53G, sets forth exemplary schemes for
remodeling glycan structures on a monoclonal antibody to CD20 (Rituxan™). Figure 53A is
a diagram depicting monoclonal antibody to CD20 which have been mutated to contain an N-
glycosylation site(s) indicating the residue which is linked to glycans contemplated for
remodeling, and exemplary glycan formulas linked thereto. Figure 53B to 53D are diagrams
of contemplated remodeling steps of the glycan of the peptides in Figure 53A based on the
type of cell the peptide is expressed in and the desired remodeled glycan structure. Figure
53H is a diagram depicting monoclonal antibody to CD20 which has been mutated to contain
an N-glycosylation site(s) indicating the residue(s) which is linked to glycans contemplated
for remodeling, and exemplary glycan formulas linked thereto. Figure 53F to 53G are
diagrams of contemplated remodeling steps of the glycan of the peptides in Figure 53E based
on the type of cell the peptide is expressed in and the desired remodeled glycan structure.
Figure 54, comprising Figures 54A to 540, sets forth exemplary schemes for
remodeling glycan structures on anti-thrombin III (AT III). Figure 54A is a diagram
depicting the anti-thrombin III peptide indicating the amino acid residues to which an N-
linked glycan is linked, and an exemplary glycan formula linked thereto. Figure 54B to 54G
are diagrams of contemplated remodeling steps of the glycan of the peptide in Figure 54A
based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure. Figure 5411 is a diagram depicting the anti-thrombin III peptide indicating the
amino acid residues to which an N-Iinkcd glycan is linked, and an exemplary glycan formula
linked thereto. Figure 541 to 54K are diagrams of contemplated remodeling steps of the
glycan of the peptide in Figure 54H based on the type of cell the peptide is expressed in and
the desired remodeled glycan structure. Figure 54L is a diagram depicting the anti-thrombin
HI peptide indicating the amino acid residues to which an N-linked glycan is linked, and an
exemplary glycan formula linked thereto. Figure 54M to 540 are diagrams of contemplated
remodeling steps of the glycan of the peptide in Figure 54L based on the type of cell the
peptide is expressed in and the desired remodeled glycan structure.
Figure 55, comprising Figures 55A to 55J, sets forth exemplary schemes for
remodeling glycan structures on subunits a and B of human Chorionic Gonadotropin (hCG).
Figure 55A is a diagram depicting the hCGct and hCG(3 peptides indicating the residues
which bind to N-linked glycans (A) and O-linked glycans (B) contemplated for remodeling,
and formulas for the glycans. Figure 55B to 55J arc diagrams of contemplated remodeling
steps based on the type of cell the peptide is expressed in and the desired remodeled glycan
structure.
Figure 56. comprising Figures 56A to 56J, sets forth exemplary schemes for
remodeling glycan structures on alpha-galactosidase (Fabrazyme™). Figure 56A is a
diagram depicting the alpha-galactosidase A peptide indicating the amino acid residues which
bind to N-linked glycans (A) contemplated for remodeling, and formulas for the glycans.
Figure 56B to 56J are diagrams of contemplated remodeling steps based on the type of cell
the peptide is expressed in and the desired remodeled glycan structure.
Figure 57, comprising Figures 57A to 57J, sets forth exemplary schemes for
remodeling glycan structures on alpha-iduronidase (Aldurazyme™). Figure 57A is a diagram
depicting the alpha-iduronidase peptide indicating the amino acid residues which bind to N-
linked glycans (A) contemplated for remodeling, and formulas for the glycans. Figure 57B to
57J are diagrams of contemplated remodeling steps based on the type of cell the peptide is
expressed in and the desired remodeled glycan structure.
Figure 58, comprising Figures 58A and 58B, is an exemplary nucleotide and
corresponding amino acid sequence of granulocyte colony stimulating factor (G-CSF) (SFQ
ID NOS: 1 and 2, respectively).
Figure 59, comprising Figures 59A through 59D, is an exemplary nucleotide and
corresponding amino acid sequence of interferon alpha (IFN-alpha) (Figures 59A and 59 B,
SEQ ID NOS: 3 and 4, respectively), and an exemplary nucleotide and corresponding amino

acid sequence of interferon-omega (IFN-omega) (Figures 59C and 59 D, SEQ ID NOS: 74
and 75. respectively).
Figure 60. comprising Figures 60A and 60B, is an exemplary nucleotide and
corresponding amino acid sequence of interferon beta (IFN-beta) (SEQ ID NOS: 5 and 6,
respectively).
Figure 61. comprising Figures 61A and 6IB, is an exemplary nucleotide and
corresponding amino acid sequence of Factor Vila (SEQ ID NOS: 7 and 8, respectively).
Figure 62, comprising Figures 62A and 62B, is an exemplary nucleotide and
corresponding amino acid sequence of Factor IX (SEQ ID NOS: 9 and 10, respectively).
Figure 63. comprising Figures 63A through 63D, is an exemplary nucleotide and
corresponding amino acid sequence of the alpha and beta chains of follicle stimulating
hormone (FSH), respectively (SEQ ID NOS: 11 through 14, respectively).
Figure 64, comprising Figures 64A and 64B, is an exemplary nucleotide and
corresponding amino acid sequence of erythropoietin (EPO) (SEQ ID NOS: 15 and 16,
respectively).
Figure 65 is an amino acid sequence of mature EPO, i.e. 165 amino acids (SEQ ID
NO:73).
Figure 66. comprising Figures 66A and 66B, is an exemplary nucleotide and
corresponding amino acid sequence of granulocyte-macrophage colony stimulating factor
(GM-CSF) (SEQ ID NOS: 17 and 18, respectively).
Figure 67, comprising Figures 67A and 67B, is an exemplary nucleotide and
corresponding amino acid sequence of interferon gamma (IFN-gamma) (SEQ ID NOS: 19
and 20. respectively).
Figure 68, comprising Figures 68A and 68B, is an exemplary nucleotide and
corresponding amino acid sequence of a-1-protease inhibitor (A-l-PI, or a-antitrypsin) (SEQ
ID NOS: 21 and 22. respectively).
Figure 69, comprising Figures 69A-1 to 69A-2, and 69B, is an exemplary nucleotide
and corresponding amino acid sequence of glucocerebrosidase (SEQ ID NOS: 23 and 24,
respectively).
Figure 70, comprising Figures 70A and 70B. is an exemplary nucleotide and
corresponding amino acid sequence of tissue-type plasminogen activator (TPA) (SEQ ID
NOS: 25 and 26, respectively).
Figure 71, comprising Figures 71A and 71B, is an exemplary nucleotide and
corresponding amino acid sequence of lnterleukin-2 (IL-2) (SEQ ID NOS: 27 and 28.
respectively).
Figure 72. comprising Figures 72A-1 through 72A-4 and Figure 72B-1 through 72B-
4. is an exemplary nucleotide and corresponding amino acid sequence of Factor VIII (SEQ
ID NOS: 29 and 30, respectively).
Figure 73. comprising Figures 73A and 73B. is an exemplary nucleotide and
corresponding amino acid sequence of urokinase (SEQ ID NOS: 33 and 34, respectively).
Figure 74, comprising Figures 74A and 74B, is an exemplary nucleotide and
corresponding amino acid sequence of human recombinant DNase (hrDNase) (SEQ ID NOS:
39 and 40, respectively).
Figure 75, comprising Figures 75A and 75B, is an exemplary nucleotide and
corresponding amino acid sequence of an insulin molecule (SEQ ID NOS: 43 and 44,
respectively).
Figure 76, comprising Figures 76A and 76B, is an exemplary nucleotide and
corresponding amino acid sequence of S-protein from a Hepatitis B virus (FIbsAg) (SEQ ID
NOS: 45 and 46, respectively).
Figure 77, comprising Figures 77A and 77B. is an exemplary nucleotide and
corresponding amino acid sequence of human growth hormone (hGH) (SEQ ID NOS: 47 and
48, respectively).
Figure 78, comprising Figures 78A and 78B, are exemplary nucleotide and
corresponding amino acid sequences of anti-thrombin III. Figures 78A and 78B, are an
exemplary nucleotide and corresponding amino acid sequences of "WT" anti-thrombin III
(SEQ ID NOS: 63 and 64, respectively).
Figure 79, comprising Figures 79A to 79D, are exemplary nucleotide and
corresponding amino acid sequences of human chorionic gonadotropin (hCG) a and [3
subunits. Figures 79A and 79B are an exemplary nucleotide and corresponding amino acid
sequence of the a-subunit of human chorionic gonadotropin (SEQ ID NOS: 69 and 70,
respectively). Figures 79C and 79D are an exemplary nucleotide and corresponding amino
acid sequence of the beta subunit of human chorionic gonadotrophin (SEQ ID NOS: 71 and
72, respectively).
Figure 80. comprising Figures 80A and 80B, is an exemplary nucleotide and
corresponding amino acid sequence of a-iduronidase (SBQ ID NOS: 65 and 66,
respectively).
Figure 81. comprising Figures 81A and 8IB, is an exemplary nucleotide and
corresponding amino acid sequence of a-galactosidase A (SEQ ID NOS: 67 and 68,
respectively).
Figure 82, comprising Figures 82A and 82B, is an exemplary nucleotide and
corresponding amino acid sequence of the 75 kDa tumor necrosis factor receptor (TNF-R).
which comprises a portion of Enbrel™ (tumor necrosis factor receptor (TNF-R)/IgG fusion)
(SEQ ID NOS: 31 and 32, respectively).
Figure 83, comprising Figures 83A and 83B, is an exemplary amino acid sequence of
the light and heavy chains, respectively, of Herceptin™ (monoclonal antibody (MAb) to Her-
2. human epidermal growth factor receptor) (SEQ ID NOS: 35 and 36, respectively).
Figure 84, comprising Figures 84A and 84B, is an exemplary amino acid sequence the
heavy and light chains, respectively, of Synagis™ (MAb to F peptide of Respiratory
Syncytial Virus) (SEQ ID NOS: 37 and 38, respectively).
Figure 85. comprising Figures 85A and 85B, is an exemplary nucleotide and
corresponding amino acid sequence of the non-human variable regions of Remicadc™ (MAb
to TNFa) (SEQ ID NOS: 41 and 42, respectively).
Figure 86, comprising Figures 86A and 86B, is an exemplary nucleotide and
corresponding amino acid sequence of the Fc portion of human IgG (SEQ ID NOS: 49 and
50, respectively).
Figure 87 is an exemplary amino acid sequence of the mature variable region light
chain of an anti-glycoprotein Ilb/IIIa murine antibgcly (SEQ ID NO: 52).
Figure 88 is an exemplary amino acid sequence of the mature variable region heavy
chain of an anti-glycoprotein Ilb/IIIa murine antibody (SEQ ID NO: 54).
Figure 89 is an exemplary amino acid sequence of variable region light chain of a
human IgG (SEQ ID NO: 51).
Figure 90 is an exemplary amino acid sequence of variable region heavy chain of a
human IgG (SEQ ID NO:53).
Figure 91 is an exemplary amino acid sequence of a light chain of a human IgG (SFQ
IDNO:55).
Figure 92 is an exemplary amino acid sequence of a heavy chain of a human IgG
(SEQIDNO:56).
Figure 93. comprising Figures 93A and 93B, is an exemplary nucleotide and
corresponding amino acid sequence of the mature variable region of the light chain of an anti-
CD20 murine antibody (SFQ ID NOS: 59 and 60, respectively).
Figure 94, comprising Figures 94A and 94B, is an exemplary nucleotide and
corresponding amino acid sequence of the mature variable region of the heavy chain of an
anti-CD20 murine antibody (SEQ ID NOS: 61 and 62, respectively).
Figure 95, comprising Figures 95A through 95E, is the nucleotide sequence of the
tandem chimeric antibody expression vector TCAE 8 (SEQ ID NO:57).
Figure 96, comprising Figures 96A through 96E, is the nucleotide sequence of the
tandem chimeric antibody expression vector TCAE 8 containing the light and heavy variable
domains of the anti-CD20 murine antibody (SEQ ID NO:58).
Figure 97, comprising Figures 97A to 97C, are graphs depicting 2-AA HPLC analysis
of glycans released by PNGaseF from myeloma-expressed Cri-IgGl antibody. The structure
of the glycans is determined by retention time: the GO glycoform elutes at 30 min., the G1
glycoform elutes at ~ 33 min., the G2 glycoform elutes at about approximately 37 min. and
the Sl-Gl glycoform elutes at ~ 70 min. Figure 97A depicts the analysis of the DEAE
antibody sample. Figure 97B depicts the analysis of the SPA antibody sample. Figure 97C
depicts the analysis of the Fc antibody sample. The percent area under the peaks for these
graphs is summarized in Table 14.
Figure 98. comprising Figures 98A to 98C, are graphs depicting the MALDI analysis
of glycans released by PNGaseF from myeloma-expressed Cri-IgGl antibody. The glycans
were derivatized with 2-AA and then analyzed by MALDI. Figure 98A depicts the analysis
of the DEAE antibody sample. Figure 98B depicts the analysis of the SPA antibody sample.
Figure 98C depicts the analysis of the Fc antibody sample.
Figure 99. comprising Figures 99A to 99D, are graphs depicting the capillary
electrophoresis analysis of glycans released from Cri-IgGl antibodies that have been
glycoremodeled to contain M3N2 glycoforms. A graph depicting the capillary
electrophoresis analysis of glycan standards derivatized with APTS is shown in Figure 99A.
Figure 99B depicts the analysis of the DEAE antibody sample. Figure 99C depicts the
analysis of the SPA antibody sample. Figure 99D depicts the analysis of the Fc antibody
sample. The percent area under the peaks for these graphs is summarized in Table 15.
Figure 100, comprising Figures 100A to 100D, are graphs depicting the capillary
electrophoresis analysis of glycans released from Cri-IgGl antibodies that have been
glycoremodeled to contain GO glycoforms. A graph depicting the capillary electrophoresis
analysis of glycan standards derivatized with APTS is shown in Figure 100A. Figure 100B
depicts the analysis of the DEAE antibody sample. Figure 100C depicts the analysis of the
SPA antibody sample. Figure 100D depicts the analysis of the Fc antibody sample. The
percent area under the peaks for these graphs is summarized in Table 16.
Figure 101, comprising Figures lOlAto 101C, are graphs depicting 2-AA HPLC
analysis of glycans released from Cri-IgGl antibodies that have been glycoremodeled to
contain GO glycoforms. The released glycans were labeled with 2AA and separated by
HPLC on a NH2P-50 4D amino column. Figure 101A depicts the analysis of the DEAE
antibody sample. Figure 101B depicts the analysis of the SPA antibody sample. Figure
101C depicts the analysis of the Fc antibody sample. The percent area under the peaks for
these graphs is summarized in Table 16
Figure 102, comprising Figures I02A to 102C, are graphs depicting the MALDI
analysis of glycans released from Cri-IgGl antibodies that have been glycoremodeled to
contain GO glycoforms. The released glycans were derivatized with 2-AA and then analyzed
by MALDI. Figure 102A depicts the analysis of the DEAE antibody sample. Figure 102B
depicts the analysis of the SPA antibody sample. Figure 102C depicts the analysis of the Fc
antibody sample.
Figure 103, comprising Figures I03A to 103D, are graphs depicting the capillary
electrophoresis analysis of glycans released from Cri-IgGl antibodies that have been
glycoremodeled to contain G2 glycoforms. A graph depicting the capillary electrophoresis
analysis of glycan standards derivatized with APTS is shown in Figure 103 A. Figure 103B
depicts the analysis of the DEAE antibody sample. Figure 103C depicts the analysis of the
SPA antibody sample. Figure 103D depicts the analysis of the Fc antibody sample. The
percent area under the peaks for these graphs is summarized in Table 17.
Figure 104, comprising Figures I04A to 104C, are graphs depicting the 2-AA HPLC
analysis of glycans released from remodeled Cri-lgGI antibodies that have been
glycoremodeled to contain G2 glycoforms. The released glycans were labeled with 2AA and
then separated by HPLC on a NH2P-50 4D amino column. Figure 104A depicts the analysis
of the DEAE antibody sample. Figure 104B depicts the analysis of the SPA antibody sample.
Figure 104C depicts the analysis of the Fc antibody sample. The percent area under the peaks
for these graphs is summarized in Table 17.
Figure 105, comprising Figures 105A to 105C, are graphs depicting MALDI analysis
of glycans released from Cri-lgGI antibodies that have been glycoremodeled to contain G2
glycoforms. The released glycans were derivatized with 2-AA and then analyzed by
MALDI. Figure 105A depicts the analysis of the DEAE antibody sample. Figure 105B
depicts the analysis of the SPA antibody sample. Figure 105C depicts the analysis of the Fc
antibody sample.
Figure 106, comprising Figures 106A to 106D, are graphs depicting capillary
electrophoresis analysis of glycans released from Cri-lgGI antibodies that have been
glycoremodeled by GnT-I treatment of M3N2 glycoforms. A graph depicting the capillary
electrophoresis analysis of glycan standards derivatized with APTS is shown in Figure 106A.
Figure 106B depicts the analysis of the DEAE antibody sample. Figure 106C depicts the
analysis of the SPA antibody sample. Figure 106D depicts the analysis of the Fc antibody
sample.
Figure 107. comprising Figures 107A to 107C, are graphs depicting 2-AA HPLC
analysis of glycans released from Cri-lgGI antibodies that have been remodeled by GnT-I
treatment of M3N2 glycoforms. The released glycans were labeled with 2-AA and separated
by HPLC on a NI12P-50 4D amino column. Figure 107A depicts the analysis of the DEAE
antibody sample. Figure 107B depicts the analysis of the SPA antibody sample. Figure
107C depicts the analysis of the Fc antibody sample.
Figure 108, comprising Figures 108A to 108C, are graphs depicting MALDI analysis
of glycans released from Cri-lgGI antibodies that have been glycoremodeled by GnT-I
treatment of M3N2 glycofbrms. The released glycans were derivatized with 2-AA and then
analyzed by MALDI. Figure 108A depicts the analysis of the DEAE antibody sample.
Figure 108B depicts the analysis of the SPA antibody sample. Figure 108C depicts the
analysis of the Fc antibody sample.
Figure 109. comprising Figures 109A to 109D, are graphs depicting capillary
electrophoresis of glycans released from Cri-IgGI antibodies that have been glycoremodelcd
by GnT-I, II and III treatment of M3N2 glycoforms. A graph depicting the capillary
electrophoresis analysis of glycan standards derivatized with APTS is shown in Figure 109A.
Figure I09B depicts the analysis of the DEAE antibody sample. Figure 109C depicts the
analysis of the SPA antibody sample. Figure 109D depicts the analysis of the Fc antibody
sample. The percent area under the peaks for these graphs is summarized in Table 18.
Figure 110, comprising Figures 110A to 1 IOC, are graphs depicting 2-AA HPLC
analysis of glycans released from Cri-IgGI antibodies that have been glycoremodeled by
GnT-I, II and III treatment of M3N2 glycoforms. The released glycans were labeled with
2AA and then separated by HPLC on a NH2P-50 4D amino column. Figure 110A depicts the
analysis of the DEAE antibody sample. Figure 110B depicts the analysis of the SPA
antibody sample. Figure 1 IOC depicts the analysis of the Fc antibody sample. The percent
area under the peaks for these graphs is summarized in Table 18.
Figure 111, comprising Figures 111A to 1 11C, are graphs depicting MALDI analysis
of glycans released from Cri-IgGI antibodies that have been glycoremodeled by
galactosyltransferase treatment of NGA2F glycoforms. The released glycans were
derivatized with 2-AA and then analyzed by MALDI. Figure 111A depicts the analysis of
the DEAE antibody sample. Figure 11 IB depicts the analysis of the SPA antibody sample.
Figure 111C depicts the analysis of the Fc antibody sample.
Figure 112, comprising 112A to 112D, are graphs depicting 2-AA HPLC analysis of
glycans released from Cri-IgGI antibodies containing NGA2F isoforms before GalTl
treatment (Figures 112A and 112C) and after GalTl treatment (Figures 112B and I 12D).
Figures 112 A and 112B depict the analysis of the DEAE sample of antibodies. Figures 112C
and 112D depict the analysis of the Fc sample of antibodies. The released glycans were
labeled with 2AA and separated by HPLC on a NH2P-50 4D amino column.
Figure I 13, comprising 113A to 113C, are graphs depicting 2-AA HPLC analysis of
glycans released from Cri-IgGl antibodies that have been glycoremodeled by ST3Gal3
treatment of G2 glycoforms. The released glycans are labeled with 2-AA and then separated
by HPLC on a NH2P-50 4D amino column. Figure 113A depicts the analysis of the DEAL
antibody sample. Figure 113B depicts the analysis of the SPA antibody sample. Figure
113C depicts the analysis of the Fc antibody sample. The percent area under the peaks for
these graphs is summarized in Table 19.
Figure 114, comprising Figures 114A to 114C, are graphs depicting MALDI analysis
of glycans released from Cri-IgGl antibodies that had been glycoremodeled by ST3Gal3
treatment of G2 glycoforms. The released glycans were derivatized with 2-AA and then
analyzed by MALDI. Figure 114A depicts the analysis of the DEAE antibody sample.
Figure 114B depicts the analysis of the SPA antibody sample. Figure 114C depicts the
analysis of the Fc antibody sample.
Figure 115, comprising Figures 115A to 115D, are graphs depicting capillary
electrophoresis analysis of glycans released from Cri-IgGl antibodies that had been
glycoremodeled by ST6Gal 1 treatment of G2 glycoforms. A graph depicting the capillary
electrophoresis analysis of glycan standards derivatized with APTS is shown in Figure 115A.
Figure 115B depicts the analysis of the DEAL antibody sample. Figure 115C depicts the
analysis of the SPA antibody sample. Figure 115D depicts the analysis of the Fc antibody
sample.
Figure 116. comprising Figures 116A to 116C, are graphs depicting 2-AA HPLC
analysis of glycans released from Cri-IgGl antibodies that had been glycoremodeled bST6Gall treatment of G2 glycoforms. The released glycans were labeled with 2-AA and
separated by HPLC on a NH2P-50 4D amino column. Figure 116A depicts the analysis of
the DEAE antibody sample. Figure 116B depicts the analysis of the SPA antibody sample.
Figure 116C depicts the analysis of the Fc antibody sample.
Figure 117, comprising Figures 117A to 117C, are graphs depicting MALDI analysis
of glycans released from Cri-IgGl antibodies that had been glycoremodeled by ST6Gall
treatment of G2 glycoforms. The released glycans were derivatized with 2-AA and then
analyzed by MALDI. Figure 117A depicts the analysis of the DEAE antibody sample.
Figure 117B depicts the analysis of the SPA antibody sample. Figure 117C depicts the
analysis of the Fc antibody sample.
Figure 118, comprising Figures 118A to 118E, depicts images of SDS-PAGE
analysis of the glycoremodeled of Cri-IgGl antibodies with different glycoforms under non-
reducing conditions. Bovine serum albumin (BSA) was run under reducing conditions as a
quantitative standard. Protein molecular weight standards are displayed and their size is
indicated in kDa. Figure 118A depicts SDS-PAGE analysis of the DEAE, SPA and Fc Cri-
IgGl antibodies glycoremodeled to contain GO and G2 glycoforms. Figure 118B depicts
SDS-PAGE analysis of the DEAF), SPA and Fc Cri-IgGl antibodies glycoremodeled to
contain NGA2F (bisecting) and GnT-I-M3N2 (GnTl) glycoforms. Figure 1 18C depicts
SDS-PAGE analysis of the DEAE, SPA and Fc Cri-IgGl antibodies glycoremodeled to
contain S2G2 (ST6Gall) glycoforms. Figure 118D depicts SDS-PAGE analysis of the
DEAE, SPA and Fc Cri-IgGl antibodies glycoremodeled to contain M3N2 glycoforms, and
BSA. Figure 118E depicts SDS-PAGE analysis of the DEAE, SPA and Fc Cri-IgGl
antibodies glycoremodeled to contain Gal-NGA2F (Gal-bisecting) glycoforms, and BSA.
Figure 119 is an image of an acrylamide gel depicting the results of FACE analysis of
the pre- and post-sialylation of TP10. The BiNAo species has no sialic acid residues. The
BiNAi species has one sialic acid residue. The BiNA2 species has two sialic acid residues. Bi
= biantennary; NA = neuraminic acid.
Figure 120 is a graph depicting the plasma concentration in u,g/ml over time of pre -
and post-sialylation TP10 injected into rats.
Figure 121 is a graph depicting the area under the plasma concentration-time curve
(AUC) in u.g/hr/ml for pre- and post sialylated TP10.
Figure 122 is an image of an acrylamide gel depicting the results of FACE glycan
analysis of the pre- and post-fucosylation of TP10 and FACE glycan analysis of CHO cell
produced TP-20. The BiNA2F2 species has two neuraminic acid (NA) residues and two
fucose residues (F).
Figure 123 is a graph depicting the in vitro binding of TP20 (sCRlsLex) glycosylated
in vitro (diamonds) and in vivo in Lecl 1 CHO cells (squares).
Figure 124 is a graph depicting the analysis by 2-AA HPLC of glycoforms from the
+GlcNAc-ylationofEPO.
Figure 125, comprising Figures 125A and 125B, are graphs depicting the 2-AA J-IPLC
analysis of two lots of EPO to which N-acetylglucosamine was been added. Figure 125A
depicts the analysis of lot A, and Figure 125B depicts the analysis of lot B.
Figure 126 is a graph depicting the 2-AA HPLC analysis of the products the reaction
introducing a third glycan branch to EPO with GnT-V.
Figure 127 is a graph depicting a MALD1-TOF spectrum of the glycans of the EPO
preparation after treatment with GnT-l, GnT-11, GnT-IIl, GnT-V and GalTl, with appropriate
donor groups.
Figure 128 is a graph depicting a MALDI spectrum the glycans of native EPO.
Figure 129 is an image of an SDS-PAGE gel of the products of the PEGylation
reactions using CMP-SA-PEG (1 kDa), and CMP-SA-PEG (10 kDa).
Figure 130 is a graph depicting the results of the in vitro bioassay of PEGylated EPO.
Diamonds represent the data from sialylated EPO having no PEG molecules. Squares
represent the data obtained using EPO with PEG (1 kDa). Triangles represent the data
obtained using EPO with PEG (10 kDa).
Figure 131 is a diagram of CHO-expressed EPO. The EPO polypeptide is 165 amino
acids in length, with a molecular weight of 18 kDa without glycosylation. The glycosylated
forms of EPO produced in CHO cells have a molecular weight of about 33 kDa to 39 kDa.
The shapes which represent the sugars in the glycan chains are identified in the box at the
lower edge of the drawing.
Figure 132 is a diagram of insect cell expressed EPO. The shapes that represent the
sugars in the glycan chains are identified in the box at the lower edge of FIG. 131.
Figure 133 is a bar graph depicting the molecular weights of the EPO peptides
expressed in insect cells which were remodeled to form complete mono-, bi- and tri-
antennary glycans, with optional glycoPEGylation with 1 kDa, 10 kDa or 20 kDa PEG.
Epoetin™ is EPO expressed in mammalian cells without further glycan modification or
PEGylation. NESP (Aranesp™, Amgen, Thousand Oaks, CA) is a form of EPO having 5 N-
linked glycan sites that is also expressed in mammalian cells without further glycan
modification or PEGylation.
Figure 134. comprising Figures 134A and 134B, depicts one scheme for the
remodeling and glycoPEGylation of insect cell expressed EPO. Figure 134A depicts the
remodeling and glycoPEGylation steps that remodel the insect expressed glycan to a mono-
antennary glycoPEGylated glycan. Figure 134B depicts the remodeled EPO polypeptide
having a completed glycoPEGylated mono-antennary glycan at each N-linked glycan site of
the polypeptide. The shapes that represent the sugars in the glycan chains are identified in
the box at the lower edge of FIG. 131, except that the triangle represents sialic acid.
Figure 135 is a graph depicting the in vitro bioactivities of EPO-SA and EPO-SA-
PEiG constructs. The in vitro assay measured the proliferation of TF-1 erythroleukemia cells
which were maintained for 48 hr in RBMI + FBS 10% + GM-CSF (12 ng/ml) after the EPO
construct was added at 10.0, 5.0, 2.0, 1.0, 0.5, and 0 u.g/ml. Tri-SA refers to EPO constructs
where the glycans are tri-antennary and have SA. Tri-SA IK PEG refers to EPO constructs
where the glycans are tri-antennary and have Gal and are then glycoPEGylated with SA-PEG
1 kDa. Di-SA 10K PEG refers to EPO constructs where the glycans are bi-antennary and
have Gal and are then glycoPEGylated with SA-PEG 10 kDa. Di-SA IK PEG refers to EPO
constructs where the glycans are bi-antennary and have Gal and are then glycoPEGylated
with SA-PEG 1 kDa. Di-SA refers to EPO constructs where the glycans are bi-antennary and
are built out to SA. Epogen™ is EPO expressed in CHO cells with no further glycan
modification.
Figure 136 is a graph depicting the pharmacokinetics of the EPO constructs in rat.
Rats were bolus injected with [l125]-labeled glycoPEGylated and non-glycoPEGylated EPO.
The graph shows the concentration of the radio-labeled EPO in the bloodstream of the rat at 0
to about 72 minutes after injection. "Biant-1 OK" refers to EPO with biantennary glycan
structures with terminal 10 kDa PEG moieties. "Mono-20K" refers to EPO with
monoantennary glycan structures with terminal 20 kDa PEG moieties. NESP refers to the
commercially available Aranesp. "Biant-1 K" refers to EPO with biantennary glycan
structures with terminal 1 kDa PEG moieties. "Biant-SA" refers to EPO with biantennary
glycan structures with terminal 1 kDa moieties. The concentration of the EPO constructs in
the bloodstream at 72 hr. is as follows: Biant-1 OK, 5.1 cpm/ml; Mono-20K, 3.2 cpm/ml;
NESP, 1 cpm/ml: and Biant-1 K, 0.2 cpm/ml; Biant-SA, 0.1 cpm/ml. The relative area under
the curve of the EPO constructs is as follows: Biant-IOK, 2.9; Mono-20K, 2.1: NESP, 1;
Biant-1 K, 0.5: and Biant-SA, 0.2.
Figure 137 is a bar graph depicting the ability of the EPO constructs to stimulate
reticulocytosis in vivo. Each treatment group is composed of eight mice. Mice were given a
single subcutaneous injection of 10 µg protein / kg body weight. The percent reticulocytosis
was measured at 96 hr. Tri-antennary-SA2,3(6) construct has the SA molecule bonded in a
2.3 or 2.6 linkage (see. Example 18 herein for preparation) wherein the glycan on EPO is tri-
antennary N-glycans with SA-PEG 10 K is attached thereon. Similarly, bi-antennary-10K
PEG is EPO having a bi-antennary N-glycan with SA-PEG at 10 K PEG attached thereon.
Figure 138 is a bar graph depicting the ability of EPO constructs to increase the
hematocrit of the blood of mice in vivo. CD-I female mice were injected i.p. with 2.5 u,g
protein/kg body weight. The hematocrit of the mice was measured on day 15 after the EPO
injection. Bi-1 k refers to EPO constructs where the glycans are bi-antennary and are built out
to the Gal and then glycoPEGylated with SA-PEG 1 kDa. Mono-20k refers to EPO
constructs where the glycans are mono-antennary and are built out to the Gal and then
glycoPEGylated with SA-PEG 20 kDa.
Figure 139, comprising Figures 139A and 139B, depicts the analysis of glycans
enzymatically released from EPO expressed in insect cells (Protein Sciences, Lot # 060302).
Figure 139A depicts the 1IPLC analysis of the released glycans. Figure 139B depicts the
MALDI analysis of the released glycans. Diamonds represent fucose, and squares represent
GlcNAc, circles represent mannose.
Figure 140 depicts the MALDI analysis of glycans released from EPO after the GnT-
1/GalT-l reaction. The structures of the glycans have been determined by comparison of the
peak spectrum with that of standard glycans. The glycan structures are depicted beside the
peaks. Diamonds represent fucose, and squares represent GlcNAc, circles represent
mannose, stars represent galactose.
Figure 141 depicts the SDS-PAGE analysis of EPO after the GnT-l/GalT-1 reaction.
Superdex 75 purification, ST3Gal3 reaction with SA-PEG (10 kDa) and SA-PEG (20 kDa).
Figure 142 depicts the results of the TF-1 cell in vitro bioassay of PEGylated mono-
antennary EPO.
Figure 143, comprising Figures 143A and 143B, depicts the analysis of glycan
released from EPO after the GnT-l/GnT-11 reaction. Figure 143 A depicts the HPLC analysis
of the released glycans, where peak 3 represents the bi-antennary GlcNAc glycan. Figure
I43B depicts the MALDI analysis of the released glycans. The structures of the glycans have
been determined by comparison of the peak spectrum with that of standard glycans. The
glycan structures are depicted beside the peaks. Diamonds represent fucose, and squares
represent GlcNAc, circles represent mannose.
Figure 144, comprising Figures 144A and 144B, depict the HPLC analysis of glycans
released from EPO after the GalT-1 reaction. Figure 144A depicts the glycans released after
the small scale GalT-1 reaction. Figure 144B depicts the glycans released after the large
scale GalT-1 reaction. In both figures, Peak 1 is the bi-antennary glycan with terminal
galactose moieties and Peak 2 is the bi-antennary glycan without terminal galactose moieties.
Figure 145 depicts the Superdex 75 chromatography separation of EPO species after
the GalT-1 reaction. Peak 2 contains EPO with bi-antennary glycans with terminal galactose
moieties.
Figure 146 depicts the SDS-PAGE analysis of each of the products of the
glycoremodeling process to make bi-antennary glycans with terminal galactose moieties.
Figure 147 depicts the SDS-PAGE analysis of EPO after ST3Gal3 sialylation or
PEGylation with SA-PEG (1 kDa) or SA-PEG (10 kDa).
Figure 148 depicts the HPLC analysis of glycans released from EPO after the GnT-
I/GnT-II reaction. The structures of the glycans have been determined by comparison of the
peak retention with that of standard glycans. The glycan structures are depicted beside the
peaks. Diamonds represent fucose, and squares represent GlcNAc, circles represent
mannose.
Figure 149 depicts the HPLC analysis of glycans released from EPO after the GnT-V
reaction. The structures of the glycans have been determined by comparison of the peak
retention with that of standard glycans. The glycan structures are depicted beside the peaks.
Diamonds represent fucose, and squares represent GlcNAc, circles represent mannose.
Figure 150 depicts the HPLC analysis of glycans released from EPO after the GalT-1
reaction. The structures of the glycans have been determined by comparison of the peak
retention with that of standard glycans. The glycan structures are depicted beside the peaks.
Diamonds represent fucose, and squares represent GlcNAc, circles represent mannose, open
circles represent galactose and triangles represent sialic acid.
Figure 151 depicts the HPLC analysis of glycans released from EPO after the
ST3Gal3 reaction. The structures of the glycans have been determined by comparison of the
peak retention with that of standard glycans. The glycan structures are depicted beside the
peaks. Diamonds represent fucose, and squares represent GlcNAc, circles represent mannose,
open circles represent galactose and triangles represent sialic acid.
Figure 152 depicts the HPLC analysis of glycans released from EPO after the
ST6Gall reaction. The structures of the glycans have been determined by comparison of the
peak retention with that of standard glycans. The glycan structures are depicted beside the
peaks.
Figure 153 depicts the results of the TF-1 cells in vitro bioassay of EPO with bi-
antennary and triantennary glycans. "Di-SA" refers to EPO with bi-antennary glycans that
terminate in sialic acid. "Di-SA 10K PEG" refers to EPO with bi-antennary glycans that
terminate in sialic acid derivatized with PEG (10 kDa). "Di-SA 1K PEG" refers to EPO with
bi-antennary glycans that terminate in sialic acid derivatized with PEG (1 kDa). "Tri-SA ST6
+ ST3" refers to EPO with tri-antennary glycans terminating in 2,6-SA capped with 2.3-SA.
"Tri-SA ST3" refers to EPO with tri-antennary glycans terminating in 2,3-SA.
Figure 154 is an image of an 1EF gel depicting the pi of the products of the
desialylation procedure. Lanes 1 and 5 are IEF standards. Lane 2 is Factor IX protein. Lane
3 is rFactor IX protein. Lane 4 is the desialylation reaction of rFactor IX protein at 20 hr.
Figure 155 is an image of an SDS-PAGE gel depicting the molecular weight of Factor
IX conjugated with either SA-PEG (1 kDa) or SA-PEG (10 kDa) after reaction with CMP-
SA-PEG. Lanes 1 and 6 are SeeBlue +2 molecular weight standards. Lane 2 is rF-IX. Lane
3 is desialylated rF-IX. Lane 4 is rFactor IX conjugated to SA-PEG (1 kDa). Lane 5 is
rFactor IX conjugated to SA-PEG (10 kDa).
Figure 156 is an image of an SDS-PAGE gel depicting the reaction products of direct-
sialylation of Factor-IX and sialic acid capping of Factor-lX-SA-PEG. Lane 1 is protein
standards, lane 2 is blank; lane 3 is rFactor-lX; lane 4 is SA capped rFactor-IX-SA-PEG (10
kDa); lane 5 is rFactor-IX-SA-PEG (10 kDa); lane 6 is ST3Gall; lane 7 is ST3Gal3; lanes 8.
9, 10 are rFactor-IX-SA-PEG(10 kDa) with no prior sialidase treatment.
Figure 157 is an image of an isoelectric focusing gel (pH 3-7) of asialo-Factor VIa.
Lane 1 is rFactor VIIa; lanes 2-5 are asialo-Factor VIIa.
Figure 158 is a graph of a MALDI spectra of Factor V[Ia.
Figure 159 is a graph of a MALDI spectra of Factor VIIa-PEG (1 kDa).
Figure 160 is a graph depicting a MALDI spectra of Factor VIIa-PEG (10 kDa).
Figure 161 is an image of an SDS-PAGE gel of PEGylated Factor VIIa. Lane 1 is
asialo-Factor VIIa. Lane 2 is the product of the reaction of asialo-Factor VIIa and CMP-SA-
PFXj(1 kDa) with ST3Gal3 after 48 hr. Lane 3 is the product of the reaction of asialo-Factor
VIIa and CMP-SA-PEG (1 kDa) with ST3Gal3 after 48 hr. Lane 4 is the product of the
reaction of asialo-Factor VIIa and CMP-SA-PEG (10 kDa) with ST3Gal3 at % hr.
Figure 162 is an image of an isoelectric focusing (IEF) gel depicting the products of
the desialylation reaction of human pituitary FSH. Lanes 1 and 4 are isoelectric focusing
(IEF) standards. Lane 2 is native FSH. Lane 3 is desialylated FSH.
Figure 163 is an image of an SDS-PAGE gel of the products of the reactions to make
PEG-sialylation of rFSH. Lanes 1 and 8 are SeeBlue+2 molecular weight standards. Lane 2
is 15 ugof native FSH. Lane 3 is 15 u.gof asialo-FSH (AS-FSH). Lane 4 is 15 ugof the
products of the reaction of AS-FSH with CMP-SA. Lane 5 is 15 jj.g of the products of the
reaction of AS-FSH with CMP-SA-PEG (1 kDa). Lane 6 is 15 ug of the products of the
reaction of AS-FSH with CMP-SA-PEG (5 kDa). Lane 7 is 15 ug of the products of the
reaction of AS-FSH with CMP-SA-PEG (10 kDa).
Figure 164 is an image of an isoelectric focusing gel of the products of the reactions
to make PEG-sialylation of FSH. Lanes 1 and 8 are IEF standards. Lane 2 is 15 ug of native
FSH. Lane 3 is 15 ug of asialo-FSH (AS-FSH). Lane 4 is 15 ug of the products of the
reaction of AS-FSH with CMP-SA. Lane 5 is 15 ug of the products of the reaction of AS-
FSH with CMP-SA-PEG (1 kDa). Lane 6 is 15 ug of the products of the reaction of AS-FSH
with CMP-SA-PEG (5 kDa). Lane 7 is 15 ug of the products of the reaction of AS-FSH with
CMP-SA-PEG (10 kDa).
Figure 165 is an image of an SDS-PAGE gel of native non-recombinant FSH
produced in human pituitary cells. Lanes 1, 2 and 5 are SeeBlue™+2 molecular weight
standards. Lanes 3 and 4 are native FSH at 5 u\g and 25 ug, respectively.
Figure 166 is an image of an isoelectric focusing gel (pFl 3-7) depicting the products
of the asialylation reaction of rFSH. Lanes 1 and 4 are IEF standards. Lane 2 is native rFSH.
Lane 3 is asialo-rFSH.
Figure 167 is an image of an SDS-PAGE gel depicting the results of the PEG-
sialylation of asialo-rFSH. Lane 1 is native rFSH. Lane 2 is asialo-FSH. Lane 3 is the
products of the reaction of asialo-FSH and CMP-SA. Lanes 4-7 are the products of the
reaction between asialoFSH and 0.5 mM CMP-SA-PEG (10 kDa) at 2 hr, 5 hr, 24 hr. and 48
hr, respectively. Lane 8 is the products of the reaction between asialo-FSH and 1.0 mM
CMP-SA-PEG (10 kDa) at 48 hr. Lane 9 is the products of the reaction between asialo-FSH
and 1.0 mM CMP-SA-PEG (1 kDa) at 48 hr.
Figure 168 is an image of an isoelectric focusing gel showing the products of PEG-
sialylation of asialo-rFSH with a CMP-SA-PEG (1 kDa). Lane 1 is native rFSH. Lane 2 is
asialo-rFSH. Lane 3 is the products of the reaction of asialo-rFSH and CMP-SA at 24 hr.
Lanes 4-7 are the products of the reaction of asialo-rFSH and 0.5 mM CMP-SA-PEG (1 kDa)
at 2 hr, 5 hr, 24 hr, and 48 hr, respectively. Lane 8 is blank. Lanes 9 and 10 are the products
of the reaction at 48 hr of asialo-rFSH and CMP-SA-PEG (10 kDa) at 0.5 mM and 1.0 mM.
respectively.
Figure 169 is graph of the pharmacokinetics of rFSH and rFSH-SA-PEG (1 kDa and
10 kDa). This graph illustrates the relationship between the time a rFSH compound is in the
blood stream of the rat, and the mean concentration of the rFSH compound in the blood for
glycoPEGylated rFSH as compared to non-PEGylated rFSH.
Figure 170 is a graph of the results of the FSH bioassay using Sertoli cells. This
graph illustrates the relationship between the FSH concentration in the Sertoli cell incubation
medium and the amount of 17-p estradiol released from the Sertoli cells.
Figure 171 is a graph depicting the results of the Steelman-Pohley bioassay of
glycoPEGylated and non-glycoPEGylated FSH. Rats were subcutaneously injected with
human chorionic gonadotropin and varying amounts of FSH for three days, and the average
ovarian weight of the treatment group determined on day 4. rFSH-SA-PFG refers to
recombinant FSH that has been glycoPEGylated with PEG (1 kDa). rFSH refers to non-
glycoPEiGylated FSH. Each treatment group contains 10 rats.
Figure 172, comprising Figures 172A and 172B, depicts the chromatogram of INF-P
clution from a Superdex-75 column. Figure 172A depicts the entire chromatogram. Figure
172B depicts the boxed area of Figure 172A containing peaks 4 and 5 in greater detail.
Figure 173, comprising Figures 173A and 173B, depict MALD1 analysis of glycans
cnzymatically released from INF-p. Figure 173A depicts the MALDI analysis glycans
released from native INF-P. Figure 173B depicts the MALDI analysis of glycans released
from desialylated INF-p. The structures of the glycans have been determined by comparison
of the peak spectrum with that of standard glycans. The glycan structures are depicted beside
the peaks. Squares represent GlcNAc, triangles represent fucose, circles represent mannose.
diamonds represent galactose and stars represent sialic acid.
Figure 174 depicts the lectin blot analysis of the sialylation of the desialylated lNF-(3.
The blot on the right side is detected with Maackia amurensis agglutinin (MAA) labeled with
digoxogenin (DIG) (Roche Applied Science, Indianapolis, 1L) to detect a2,3-sialylation. The
blot on the left is detected with Erthrina cristagalli lectin (ECL) labeled with biotin (Vector
Laboratories, Burlingame, CA) to detect exposed galactose residues.
Figure 175 depicts the SDS-PAGE analysis of the products of the PEG (10 kDa)
PEGylation reaction of INF-p. "-PEG" refers to INF-P before the PEGylation reaction.
"+PEG'" refers to INF-P after the PEGylation reaction.
Figure 176 depicts the SDS-PAGE analysis of the products of the PEG (20 kDa)
PEGylation reaction of INF-p. "Unmodified" refers to INF-P before the PEGylation
reaction. "Pegylated" refers to INF-p after the PEGylation reaction.
Figure 177 depicts the chromatogram of PEG (10 kDa) PEGylated INF-P elution from
a Superdex-200 column.
Figure 178 depicts the results of a bioassay of peak fractions of PEG (10 kDa)
PEGylated INF-P shown in the chromatogram depicted Figure INF-PEG 6.
Figure 179 depicts the chromatogram of PEG (20 kDa) PEGylated INF-p elution from
a Superdex-200 column.
Figure 180, comprising Figures 180A and 180B, is two graphs depicting the MALD1-
TOF spectrum of RNaseB (Figure 180A) and the HPLC profile of the oligosaccharides
cleaved from RNaseB by N-Glycanase (Figure 180B). The majority of N-glycosylation sites
of the peptide are modified with high mannose oligosaccharides consisting of 5 to 9 mannose
residues.
Figure 181 is a scheme depicting the conversion of high mannose N-Glycans to
hybrid N-Glycans. Enzyme 1 is al,2-mannosidase, from Trichodoma reesei or Aspergillus
sciitoi. Enzyme 2 is GnT-I ((3-1,2-iV-acetyl glucosaminyl transferase I). Enzyme 3 is GalT-I
(P1.4-galactosyltransfease I). Enzyme 4 is ot2.3-sialyltransferase or ct2,6-sialyltransferase.
Figure 182. comprising Figures 182A and 182B, is two graphs depicting the MALDI-
TOF spectrum of RNaseB treated with a recombinant T. reesei a 1,2-mannosidase (Figure
182A) and the HPLC profile of the oligosaccharides cleaved by N-Glycanase from the
modified RNaseB (Figure 182B).
Figure 183 is a graph depicting the MALDI-TOF spectrum of RNaseB treated with a
commercially available a 1,2-mannosidase purified from A. saitoi (Glyko & CalBioChem).
Figure 184 is a graph depicting the MALDI-TOF spectrum of modified RNaseB by
treating the product shown in Figure 182 with a recombinant Gn'I'-l (GlcNAc transferase-1).
Figure 185 is a graph depicting the MALDI-TOF spectrum of modified RNaseB by
treating the product shown in Figure 184 with a recombinant GalT 1 (galactosyltransferase
1).
Figure 186 is a graph depicting the MALDI-TOF spectrum of modified RNaseB by
treating the product shown in Figure 185 with a recombinant ST3Gal III (a2,3-
sialyltransferase III) using CMP-SA as the donor for the transferase.
Figure 187, comprising Figures 187A and 187B, are graphs depicting the MALDI-
TOF spectrum of modified RNaseB by treating the product shown in Figure 185 with a
recombinant ST3Gal III (a2,3-sialyltransferase III) using CMP-SA-PEG (10 kDa) as the
donor for the transferase.
Figure 188 is a series of schemes depicting the conversion of high mannose N-glycans
to complex N-glycans. Enzyme 1 is a 1,2-mannosidase from Trichoderma reesei or
Aspergillus saitoi. Enzyme 2 is GnT-I. Enzyme 3 is GalT 1. Enzyme 4 is a2,3-
sialyltransferase or o,2,6-sialyltransferase. Enzyme 5 is a-mannosidase II. Enzyme 6 is a-
mannosidase. Enzyme 7 is GnT-I I. Enzyme 8 is ctl,6-mannosidase. Enzyme 9 is al.3-
mannosidase.
Figure 189 is a diagram of the linkage catalyzed by M-acetylglucosaminyltransferase I
to VI (GnT 1-V1). R - GlcNAcpl,4GlcNAc-Asn-X.
Figure 190 is an image of an SDS-PAGE gel: standard (Lane 1); native transferrin
(Lane 2); asialotransferrin (Lane 3); asialotransferrin and CMP-SA (Lane 4); Lanes 5 and 6,
asialotransferrin and CMP-SA-PEG (1 kDa) at 0.5 mM and 5 mM, respectively; Lanes 7 and
8, asialotransferrin and CMP-SA-PEG (5 kDa) at 0.5 mM and 5 mM, respectively; Lanes 9
and 10. asialotransferrin and CMP-SA-PEG (10 kDa) at 0.5 mM and 5 mM, respectively.
Figure 191 is an image of an IEF gel: native transferrin (Lane 1); asialotransferrin
(Lane 2); asialotransferrin and CMP-SA, 24 hr (Lane 3); asialotransferrin and CMP-SA, 96
hr (Lane 4) Lanes 5 and 6, asialotransferrin and CMP-SA-PEG (1 kDa) at 24 hr and 96 hr,
respectively; Lanes 7 and 8, asialotransferrin and CMP-SA-PEG (5 kDa) at 24 hr and 96 hr,
respectively; Lanes 9 and 10, asialotransferrin and CMP-SA-PEG (10 kDa) at 24 hr and 96
hr. respectively.
Figure 192 is a graph depicting the effects of EPO-SA-PEG (10 kDa), EPO-SA-PEG
(20 kDa), and NESP on hemoglobin response in Sprague Dawley rats. Rats were dosed
(indicated by arrows) by subcutaneous injection of 50 u,g protein per Kg body weight.
Values plotted are mean (n=5/group) increment in total blood hemoglobin baseline corrected
against values obtained for control animals injected with vehicle alone.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes methods and compositions for the cell free in vitro
addition and/or deletion of sugars to or from a peptide molecule in such a manner as to
provide a glycopeptide molecule having a specific customized or desired glycosylation
pattern, wherein the glycopeptide is produced at an industrial scale. In a preferred
embodiment of the invention, the glycopeptide so produced has attached thereto a modified
sugar that has been added to the peptide via an enzymatic reaction. A key feature of the
invention is to take a peptide produced by any cell type and generate a core glycan structure
on the peptide, following which the glycan structure is then remodeled in vitro to generate a
glycopeptide having a glycosylation pattern suitable for therapeutic use in a mammal. More
specifically, it is possible according to the present invention, to prepare a glycopeptide
molecule having a modified sugar molecule or other compound conjugated thereto, such that
the conjugated molecule confers a beneficial property on the peptide. According to the
present invention, the conjugate molecule is added to the peptide enzymatically because
enzyme-based addition of conjugate molecules to peptides has the advantage of
regioselectivity and stereoselectivity. The glycoconjugate may be added to the glycan on a
peptide before or after glycosylation has been completed. In other words, the order of
glycosylation with respect to glycoconjugation may be varied as described elsewhere herein.
It is therefore possible, using the methods and compositions provided herein, to remodel a
peptide to confer upon the peptide a desired glycan structure preferably having a modified
sugar attached thereto. It is also possible, using the methods and compositions of the
invention to generate peptide molecules having desired and or modified glycan structures at
an industrial scale, thereby, for the first time, providing the art with a practical solution for
the efficient production of improved therapeutic peptides.
Definitions
Unless defined otherwise, all technical and scientific terms used herein generally have
the same meaning as commonly understood by one of ordinary skill in the art to which this
invention belongs. Generally, the nomenclature used herein and the laboratory procedures in
cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and
hybridization are those well known and commonly employed in the art. Standard techniques
are used for nucleic acid and peptide synthesis. The techniques and procedures are generally
performed according to conventional methods in the art and various general references (e.g..
Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2d ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY), which are provided throughout this document.
The nomenclature used herein and the laboratory procedures used in analytical chemistry and
organic syntheses described below are those well known and commonly employed in the art.
Standard techniques or modifications thereof, are used for chemical syntheses and chemical
analyses.
The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at
least one) of the grammatical object of the article. By way of example, "an element" means
one element or more than one element.
The term "antibody," as used herein, refers to an immunoglobulin molecule which is
able to specifically bind to a specific epitope on an antigen. Antibodies can be intact
immunoglobulins derived from natural sources or from recombinant sources and can be
immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of
immunoglobulin molecules. The antibodies in the present invention may exist in a variety of
forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and
F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999,
Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow
et al.. 1989, Antibodies: A Laboratory Manual. Cold Spring Harbor. New York; Houston et
al.. 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
By the term "synthetic antibody" as used herein, is meant an antibody which is
generated using_recombinant DNA technology, such as, for example, an antibody expressed
by a bacteriophage as described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA molecule encoding the
antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence
specifying the antibody, wherein the DNA or amino acid sequence has been obtained using
synthetic DNA or amino acid sequence technology which is available and well known in the
art.
As used herein, a "functional" biological molecule is a biological molecule in a form
in which it exhibits a property by which it is characterized. A functional enzyme, for
example, is one which exhibits the characteristic catalytic activity by which the enzyme is
characterized.3
As used herein, the structure , is the point of connection between an
amino acid or an amino acid sidechain in the peptide chain and the glycan structure.
"N-linked" oligosaccharides are those oligosaccharides that are linked to a peptide
backbone through asparagine, by way of an asparagine-N-acetylglucosamine linkage. N-
linked oligosaccharides are also called "N-glycans." All N-linked oligosaccharides have a
common pentasaccharide core of Man3GlcNAc2. They differ in the presence of, and in the
number of branches (also called antennae) of peripheral sugars such as N-acetylglucosamine,
galactose. N-acetylgalactosamine, fucose and sialic acid. Optionally, this structure may also
contain a core fucose molecule and/or a xylose molecule.
An "elemental trimannosyl core structure" refers to a glycan moiety comprising soleK
a trimannosyl core structure, with no additional sugars attached thereto. When the term
"elemental" is not included in the description of the "trimannosyl core structure," then the
glycan comprises the trimannosyl core structure with additional sugars attached thereto.
Optionally, this structure may also contain a core fucose molecule and/or a xylose molecule.
The term 'elemental trimannosyl core glycopeptide" is used herein to refer to a
glycopeptide having glycan structures comprised primarily of an elemental trimannosyl core
structure. Optionally, this structure may also contain a core fucose molecule and/or a xylose
molecule.
"O-linked" oligosaccharides are those oligosaccharides that are linked to a peptide
backbone through threonine, serine, hydroxyproline, tyrosine, or other hydroxy-containing
amino acids.
All oligosaccharides described herein are described with the name or abbreviation for
the non-reducing saccharide (i.e., Gal), followed by the configuration of the glycosidic bond
(a or P), the ring bond (1 or 2), the ring position of the reducing saccharide involved in the
bond (2, 3, 4, 6 or 8), and then the name or abbreviation of the reducing saccharide (i.e..
GlcNAc). Each saccharide is preferably a pyranose. For a review of standard glycobiology
nomenclature see. Essentials of Glycobiology Varki et al. eds., 1999, CSHL Press.
The term 'sialic acid" refers to any member of a family of nine-carbon carboxylated
sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-
keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-l-onic acid (often
abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is N-glycolyl-
neuraminic acid (Neu5Gc or NeuGc). in which the N-acetyl group of NeuAc is hydroxylase!.
A third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al.
(1986). J. Biol. Chem.261: 11550-11557; Kanamori et al, J. Biol. Chem.265: 21811-21819
(1990)). Also included are 9-substituted sialic acids such as a 9-0-C|-C6 acyl-Neu5Ac like
9-0-lactyl-Neu5Ac or 9-0-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-
Neu5Ac. For review of the sialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);
Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, New
York (1992)). The synthesis and use of sialic acid compounds in a sialylation procedure is
disclosed in international application WO 92/16640, published October 1, 1992.
A peptide having "desired glycosylation'", as used herein, is a peptide that comprises
one or more oligosaccharide molecules which are required for efficient biological activity of
the peptide.
A "disease" is a state of health of an animal wherein the animal cannot maintain
homeostasis, and wherein if the disease is not ameliorated then the animal's health continues
to deteriorate.
The "area under the curve" or "AUC", as used herein in the context of administering a
peptide drug to a patient, is defined as total area under the curve that describes the
concentration of drug in systemic circulation in the patient as a function of time from zero to
infinity.
The term "half-life" or "t½", as used herein in the context of administering a peptide
drug to a patient, is defined as the time required for plasma concentration of a drug in a
patient to be reduced by one half. There may be more than one half-life associated with the
peptide drug depending on multiple clearance mechanisms, redistribution, and other
mechanisms well known in the art. Usually, alpha and beta half-lives are defined such that
the alpha phase is associated with redistribution, and the beta phase is associated with
clearance. However, with protein drugs that are, for the most part, confined to the
bloodstream, there can be at least two clearance half-lives. For some glycosylated peptides,
rapid beta phase clearance may be mediated via receptors on macrophages, or endothelial
cells that recognize terminal galactose, N-acetylgalactosamine, N-acetylglucosamine,
mannose, or fucose. Slower beta phase clearance may occur via renal glomerular filtration
for molecules with an effective radius specific uptake and metabolism in tissues. GlycoPEGylation may cap terminal sugars (e.g.
galactose or N-acetylgalactosamine) and thereby block rapid alpha phase clearance via
receptors that recognize these sugars. It may also confer a larger effective radius and thereby
decrease the volume of distribution and tissue uptake, thereby prolonging the late beta phase.
Thus, the precise impact of glycoPEGylation on alpha phase and beta phase half-lives will
vary depending upon the size, state of glycosylation, and other parameters, as is well known
in the art. Further explanation of "half-life" is found in Pharmaceutical Biotechnology
(1997. DFA Crommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp 101 —
120).
The term "residence time", as used herein in the context of administering a peptide
drug to a patient, is defined as the average time that drug stays in the body of the patient after
dosing.
An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has
been separated from sequences which flank it in a naturally occurring slate, e.g.. a DNA
fragment which has been removed from the sequences which are normally adjacent to the
fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally
occurs. The term also applies to nucleic acids which have been substantially purified from
other components which naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, which naturally accompany it in the cell. The term therefore includes, for example,
a recombinant DNA which is incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a
separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or
restriction enzyme digestion) independent of other sequences. It also includes a recombinant
DNA which is part of a hybrid nucleic acid encoding additional peptide sequence.
A "polynucleotide" means a single strand or parallel and anti-parallel strands of a
nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded
nucleic acid.
The term "nucleic acid" typically refers to large polynucleotides. The term
"oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50
nucleotides.
Conventional notation is used herein to describe polynucleotide sequences: the left-
hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of
a double-stranded polynucleotide sequence is referred to as the 5'-direction. The direction of
5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription
direction. The DNA strand having the same sequence as an mRNA is referred to as the
"coding strand"; sequences on the DNA strand which are located 5' to a reference point on the
DNA are referred to as "upstream sequences"; sequences on the DNA strand which are 3' to a
reference point on the DNA are referred to as "downstream sequences.'"
"Encoding" refers to the inherent property of specific sequences of nucleotides in a
polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of
other polymers and macromolecules in biological processes having either a defined sequence
of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the
biological properties resulting therefrom. Thus, a nucleic acid sequence encodes a protein if
transcription and translation of mRNA corresponding to that nucleic acid produces the
protein in a cell or other biological system. Both the coding strand, the nucleotide sequence
of which is identical to the mRNA sequence and is usually provided in sequence listings, and
the non-coding strand, used as the template for transcription of a gene or cDNA. can be
referred to as encoding the protein or other product of that nucleic acid or cDNA.
Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence"
includes all nucleotide sequences that are degenerate versions of each other and that encode
the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may
include introns.
"Homologous" as used herein, refers to the subunit sequence similarity between two
polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or
two RNA molecules, or between two peptide molecules. When a subunit position in both of
the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of
two DNA molecules is occupied by adenine, then they are homologous at that position. The
homology between two sequences is a direct function of the number of matching or
homologous positions, e.g.. if half (e.g.. five positions in a polymer ten subunits in length) of
the positions in two compound sequences are homologous then the two sequences are 50%
homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two
sequences share 90% homology. By way of example, the DNA sequences 3'ATTGCC5' and
3TATGGC share 50% homology.
As used herein, "homology" is used synonymously with "identity."
The determination of percent identity between two nucleotide or amino acid
sequences can be accomplished using a mathematical algorithm. For example, a
mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and
Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and
Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated
into the NBLAST and XBLAST programs of Altschul. et al. (1990, J. Mol. Biol. 215:403-
410), and can be accessed, for example at the National Center for Biotechnology Information
(NCBI) world wide web site having the universal resource locator
http://www.ncbi.nlm.nih.gov/BLAST/". BLAST nucleotide searches can be performed with
the NBLAST program (designated "blastn" at the NCB1 web site), using the following
parameters: gap penalty = 5; gap extension penalty = 2; mismatch penalty =- 3; match reward
- 1: expectation value 10.0; and word size = 11 to obtain nucleotide sequences homologous
to a nucleic acid described herein. BLAST protein searches can be performed with the
XBLAST program (designated "blastn" at the NCBI web site) or the NCBI "blastp" program,
using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to
obtain amino acid sequences homologous to a protein molecule described herein. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al. (1997. Nucleic Acids Res. 25:3389-3402). Alternatively, PSl-Blast or PH1-
Blast can be used to perform an iterated search which detects distant relationships between
molecules (Id.) and relationships between molecules which share a common pattern. When
utilizing BLAST, Gapped BLAST, PSl-Blast, and PHI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
The percent identity between two sequences can be determined using techniques
similar to those described above, with or without allowing gaps. In calculating percent
identity, typically exact matches are counted.
A "heterologous nucleic acid expression unit" encoding a peptide is defined as a
nucleic acid having a coding sequence for a peptide of interest operably linked to one or
more expression control sequences such as promoters and/or repressor sequences wherein at
least one of the sequences is heterologous, i. e., not normally found in the host cell.
By describing two polynucleotides as "operably linked" is meant that a single-
stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged
within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is
able to exert a physiological effect by which it is characterized upon the other. By way of
example, a promoter operably linked to the coding region of a nucleic acid is able to promote
transcription of the coding region.
As used herein, the term "promoter/regulatory sequence" means a nucleic acid
sequence which is required for expression of a gene product operably linked to the
promoter/regulator sequence. In some instances, this sequence may be the core promoter
sequence and in other instances, this sequence may also include an enhancer sequence and
other regulatory elements which are required for expression of the gene product. The
promoter/regulatory sequence may, for example, be one which expresses the gene product in
a tissue specific manner.
A "constitutive promoter is a promoter which drives expression of a gene to which it
is operably linked, in a constant manner in a cell. By way of example, promoters which drive
expression of cellular housekeeping genes are considered to be constitutive promoters.
An "inducible" promoter is a nucleotide sequence which, when operably linked with a
polynucleotide which encodes or specifies a gene product, causes the gene product to be
produced in a living cell substantially only when an inducer which corresponds to the
promoter is present in the cell.
A "tissue-specific" promoter is a nucleotide sequence which, when operably linked
with a polynucleotide which encodes or specifies a gene product, causes the gene product to
be produced in a living cell substantially only if the cell is a cell of the tissue type
corresponding to the promoter.
A "vector" is a composition of matter which comprises an isolated nucleic acid and
which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous
vectors are known in the art including, but not limited to, linear polynucleotides,
polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term
should also be construed to include non-plasmid and non-viral compounds which facilitate
transfer of nucleic acid into cells, such as. for example, polylysine compounds, liposomes,
and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors,
adeno-associated virus vectors, retroviral vectors, and the like.
"Expression vector" refers to a vector comprising a recombinant polynucleotide
comprising expression control sequences operatively linked to a nucleotide sequence to be
expressed. An expression vector comprises sufficient cis-acting elements for expression;
other elements for expression can be supplied by the host cell or in an in vitro expression
system. Expression vectors include all those known in the art, such as cosmids, plasmids
(e.g.. naked or contained in liposomes) and viruses that incorporate the recombinant
polynucleotide.
A "genetically engineered" or "recombinant" cell is a cell having one or more
modifications to the genetic material of the cell. Such modifications are seen to include, but
are not limited to, insertions of genetic material, deletions of genetic material and insertion of
genetic material that is extrachromasomal whether such material is stably maintained or not.
A "peptide" is an oligopeptide, polypeptide, peptide, protein or glycoprotein. The use
of the term "peptide" herein includes a peptide having a sugar molecule attached thereto
when a sugar molecule is attached thereto.
As used herein, "native form'" means the form of the peptide when produced by the
cells and/or organisms in which it is found in nature. When the peptide is produced by a
plurality of cells and/or organisms, the peptide may have a variety of native forms.
"Peptide" refers to a polymer in which the monomers are amino acids and are joined
together through amide bonds, alternatively referred to as a peptide. Additionally, unnatural
amino acids, for example, P-alanine, phenylglycine and homoarginine are also included.
Amino acids that are not nucleic acid-encoded may also be used in the present invention.
Furthermore, amino acids that have been modified to include reactive groups, glycosylation
sites, polymers, therapeutic moieties, biomolecules and the like may also be used in the
invention. All of the amino acids used in the present invention may be either the D - or L -
isomer thereof. The L -isomer is generally preferred. In addition, other peptidomimetics are
also useful in the present invention. As used herein, "peptide" refers to both glycosylated and
unglycosylated peptides. Also included are peptides that are incompletely glycosylated by a
system that expresses the peptide. For a general review, see, Spatola, A. F., in Chemistry
and Biochemistry OF Amino Acids, Peptides and Proteins, B. Weinstein, eds., Marcel
Dekker, New York, p. 267 (1983).
The term "peptide conjugate," refers to species of the invention in which a peptide is
conjugated with a modified sugar as set forth herein.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well
as amino acid analogs and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, (-
carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is
linked to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine.
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified
R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical
structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical
compounds that have a structure that is different from the general chemical structure of an
amino acid, but that function in a manner similar to a naturally occurring amino acid.
As used herein, amino acids are represented by the full name thereof, by the three
letter code corresponding thereto, or by the one-letter code corresponding thereto, as
indicated in the following Table 1:

The present invention also provides for analogs of proteins or peptides which
comprise a protein as identified above. Analogs may differ from naturally occurring proteins
or peptides by conservative amino acid sequence differences or by modifications which do

not affect sequence, or by both. For example, conservative amino acid changes may be
made, which although they alter the primary sequence of the protein or peptide, do not
normally alter its function. Conservative amino acid substitutions typically include
substitutions within the following groups:
glycine, alanine;
valine, isoleucine, leucine;
aspartic acid, glutamic acid;
asparagine, glutamine;
serine, threonine;
lysine, arginine;
phenylalanine, tyrosine.
Modifications (which do not normally alter primary sequence) include in vivo, or in
vitro, chemical derivatization of peptides, e.g., acetylation, or carboxylation. Also included
are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns
of a peptide during its synthesis and processing or in further processing steps; e.g., by-
exposing the peptide to enzymes which affect glycosylation, e.g., mammalian glycosylating
or deglycosylating enzymes. Also embraced are sequences which have phosphorylated
amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
It will be appreciated, of course, that the peptides may incorporate amino acid
residues which are modified without affecting activity. For example, the termini may be
dcrivatized to include blocking groups, i.e. chemical substituents suitable to protect and/or
stabilize the N- and C-termini from "undesirable degradation", a term meant to encompass
any type of enzymatic, chemical or biochemical breakdown of the compound at its termini
which is likely to affect the function of the compound, i.e. sequential degradation of the
compound at a terminal end thereof.
Blocking groups include protecting groups conventionally used in the art of peptide
chemistry which will not adversely affect the in vivo activities of the peptide. For example,
suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N-
terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or
unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted
forms thereof, such as the acetamidomethyl (Acm), Fmoc or Boc groups. Desamino analogs
of amino acids are also useful N-terminal blocking groups, and can either be coupled to the
N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal
blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not,
include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower
alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as
primary amines (-NH2). and mono- and di-alkylamino groups such as methylamino,
ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-
terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also
useful C-terminal blocking groups and can be either coupled to the peptide's C-terminal
residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl
groups at the termini can be removed altogether from the peptide to yield desamino and
descarboxylated forms thereof without affect on peptide activity.
Other modifications can also be incorporated without adversely affecting the activity
and these include, but are not limited to, substitution of one or more of the amino acids in the
natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide mainclude one or more D-amino acid resides, or may comprise amino acids which are all in the
D-form. Retro-inverso forms of peptides in accordance with the present invention are also
contemplated, for example, inverted peptides in which all amino acids are substituted with D-
amino acid forms.
Acid addition salts of the present invention are also contemplated as functional
equivalents. Thus, a peptide in accordance with the present invention treated with an
inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or
an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic,
succinic, maleic, fumaric, tataric, citric, benzoic, cinnamic, mandelic, methanesulfonic.
ethanesulfonic, p-toluenesulfonic, salicyclic and the like, to provide a water soluble salt of the
peptide is suitable for use in the invention.
Also included are peptides which have been modified using ordinary molecular
biological techniques so as to improve their resistance to proteolytic degradation or to
optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs
of such peptides include those containing residues other than naturally occurring L-amino
acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of
the invention are not limited to products of any of the specific exemplary processes listed
herein.
As used herein, the term "MALDI" is an abbreviation for Matrix Assisted Laser
Desorption Ionization. During ionization, SA-PEG (sialic acid-poly(ethylene glycol)) can be
partially eliminated from the N-glycan structure of the glycoprotein.
As used herein, the term "glycosyltransferase," refers to any enzyme/protein that has
the ability to transfer a donor sugar to an acceptor moiety.
As used herein, the term "modified sugar," refers to a naturally- or non-naturally-
occurring carbohydrate that is enzymatically added onto an amino acid or a glycosyl residue
of a peptide in a process of the invention. The modified sugar is selected from a number of
enzyme substrates including, but not limited to sugar nucleotides (mono-, di-, and tri-
phosphates), activated sugars (e.g., glycosyl halides, glycosyl mesylates) and sugars that are
neither activated nor nucleotides.
The "modified sugar" is covalently functionalized with a "modifying group.1' Useful
modifying groups include, but are not limited to, water-soluble polymers, therapeutic
moieties, diagnostic moieties, biomolecules and the like. The locus of functional ization with
the modifying group is selected such that it does not prevent the "modified sugar" from being
added enzymatically to a peptide.
The term ""water-soluble" refers to moieties that have some detectable degree of
solubility in water. Methods to detect and/or quantify water solubility are well known in the
art. Exemplary water-soluble polymers include peptides, saccharides, poly(ethers),
poly(amines), poly(carboxylic acids) and the like. Peptides can have mixed sequences or be
composed of a single amino acid, e.g. poly(lysine). Similarly, saccharides can be of mixed
sequence or composed of a single saccharide subunit, e.g., dextran, amylosc, chitosan, and
poly(sialic acid). An exemplary poly(ether) is polyethylene glycol). Polyethylene imine) is
an exemplary polyamine. and poly(aspartic) acid is a representative poly(carboxylic acid).
"Poly(alkylenc oxide)" refers to a genus of compounds having a polyether backbone.
Poly(alkylene oxide) species of use in the present invention include, for example, straight-
and branched-chain species. Moreover. exemplary poly(alkylcnc oxide) species can
terminate in one or more reactive, activatable, or inert groups. For example. poly(ethylene
glycol) is a poly(alkylene oxide) consisting of repeating ethylene oxide subunits, which may
or may not include additional reactive, activatable or inert moieties at either terminus. Useful
poly(alkylene oxide) species include those in which one terminus is "capped" by an inert
group, e.g., monomethoxy-poly(alkylenc oxide). When the molecule is a branched species, it
may include multiple reactive, activatable or inert groups at the termini of the alkylene oxide
chains and the reactive groups may be either the same or different. Derivatives of straight-
chain poly(alkylene oxide) species that are heterobifunctional are also known in the art.
The term, "glycosyl linking group," as used herein refers to a glycosyl residue to
which an agent (e.g.. water-soluble polymer, therapeutic moiety, biomolecule) is covalently
attached. In the methods of the invention, the "glycosyl linking group" becomes covalently
attached to a glycosylated or unglycosylated peptide, thereby linking the agent to an amino
acid and/or glycosyl residue on the peptide. A "glycosyl linking group" is generally derived
from a "modified sugar" by the enzymatic attachment of the "modified sugar" to an amino
acid and/or glycosyl residue of the peptide. More specifically, a "glycosyl linking group," as
used herein, refers to a moiety that covalently joins a "modifying group," as discussed herein,
and an amino acid residue of a peptide. The glycosyl linking group-modifying group adduct
has a structure that is a substrate for an enzyme. The enzymes for which the glycosyl linking
group-modifying group adduct are substrates are generally those capable of transferring a
saccharyl moiety onto an amino acid residue of a peptide, e.g, a glycosyltransferase, amidase,
glycosidase, trans-sialidase, etc. The "glycosyl linking group" is interposed between, and
covalently joins a "modifying group" and an amino acid residue of a peptide.
An "intact glycosyl linking group" refers to a linking group that is derived from a
glycosyl moiety in which the individual saccharide monomer that links the conjugate is not
degraded, e.g., oxidized, e.g., by sodium metaperiodate. "Intact glycosyl linking groups" of
the invention may be derived from a naturally occurring oligosaccharide by addition of
glycosyl unit(s) or removal of one or more glycosyl unit from a parent saccharide structure.
An exemplary "intact glycosyl linking group" includes at least one intact, e.g., non-degraded,
saccharyl moiety that is covalently attached to an amino acid residue on a peptide. The
remainder of the "linking group" can have substantially any structure. For example, the
modifying group is optionally linked directly to the intact saccharyl moiety. Alternatively.
the modifying group is linked to the intact saccharyl moiety via a linker arm. The linker arm
can have substantially any structure determined to be useful in the selected embodiment. In
an exemplary embodiment, the linker arm is one or more intact saccharyl moieties, i.e. "the
intact glycosyl linking group" resembles an oligosaccharide. Another exemplary intact
glycosyl linking group is one in which a saccharyl moiety attached, directly or indirectly, to
the intact saccharyl moiety is degraded and derivatized (e.g., periodate oxidation followed by
reductive amination). Still a further linker arm includes the modifying group attached to the
intact saccharyl moiety, directly or indirectly, via a cross-linker, such as those described
herein or analogues thereof.
"Degradation," as used herein refers to the removal of one or more carbon atoms from
a saccharyl moiety.
The terms "targeting moiety" and "targeting agent", as used herein, refer to species
that will selectively localize in a particular tissue or region of the body. The localization is
mediated by specific recognition of molecular determinants, molecular size of the targeting
agent or conjugate, ionic interactions, hydrophobic interactions and the like. Other
mechanisms of targeting an agent to a particular tissue or region are known to those of skill in
the art.
As used herein, "therapeutic moiety" means any agent useful for therapy including,
but not limited to. antibiotics, anti-inflammatory agents, anti-tumor drugs, cytotoxins, and
radioactive agents. "Therapeutic moiety" includes prodrugs of bioactive agents, constructs in
which more than one therapeutic moiety is linked to a carrier, e.g., multivalent agents.
Therapeutic moiety also includes peptides, and constructs that include peptides. Exemplary
peptides include those disclosed in Figure 28 and Tables 6 and 7, herein. "Therapeutic
moiety" thus means any agent useful for therapy including, but not limited to. antibiotics,
anti-inflammatory agents, anti-tumor drugs, cytotoxins, and radioactive agents. "Therapeutic
moiety" includes prodrugs of bioactive agents, constructs in which more than one therapeutic
moiety is linked to a carrier, e.g., multivalent agents.
As used herein, "anti-tumor drug" means any agent useful to combat cancer including,
but not limited to, cytotoxins and agents such as antimetabolites, alkylating agents,
anthracyclines. antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase,
corticosteroids, interferons and radioactive agents. Also encompassed within the scope of the
term "anti-tumor drug," are conjugates of peptides with anti-tumor activity, e.g. TNF-oc.
Conjugates include, but are not limited to those formed between a therapeutic protein and a
glycoprotein of the invention. A representative conjugate is that formed between PSGL-1
and TNF-oc.
As used herein, "a cytotoxin or cytotoxic agent" means any agent that is detrimental to
cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin, dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D. 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and
puromycin and analogs or homologs thereof. Other toxins include, for example, ricin, CC-
1065 and analogues, the duocarmycins. Still other toxins include diphtheria toxin, and snake
venom (e.g., cobra venom).
As used herein, "a radioactive agent" includes any radioisotope that is effective in
diagnosing or destroying a tumor. Examples include, but are not limited to, indium-111.
cobalt-60 and technetium. Additionally, naturally occurring radioactive elements such as
uranium, radium, and thorium, which typically represent mixtures of radioisotopes, are
suitable examples of a radioactive agent. The metal ions are typically chelated with an
organic chelating moiety.
Many useful chelating groups, crown ethers, cryptands and the like are known in the
art and can be incorporated into the compounds of the invention (e.g. EDTA, DTPA, DOTA.
NTA, HDTA, etc. and their phosphonate analogs such as DTPP, EDTP, HDTP, NIP. etc).
See, for example, Pitt et ai, "The Design of Chelating Agents for the Treatment of Iron
Overload," In, Inorganic Chemistry in Biology and Medicine; Martell, Ed.; American
Chemical Society. Washington, D.C., 1980, pp. 279-312; Lindoy, The Chemistry of
MACROCYCElC Ligand COMPLEXES; Cambridge University Press, Cambridge, 1989; Dugas,
Bioorganic Chemistry: Springer-Verlag, New York, 1989, and references contained
therein.
Additionally, a manifold of routes allowing the attachment of chelating agents, crown
ethers and cyclodextrins to other molecules is available to those of skill in the art. See, for
example. Meares el al., "'Properties of In Vivo Chelate-Tagged Proteins and Polypeptides."
In, MODiriCATION OK PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICAL ASPECTS;"
Feeney. et al., Eds.. American Chemical Society, Washington, D.C., 1982, pp. 370-387;
Kasina et al., Bioconjugate Chem., 9: 108-117 (1998); Song et ah, Bioconjugate Chem. 8:
249-255(1997).
As used herein, "pharmaceutically acceptable carrier" includes any material, which
when combined with the conjugate retains the activity of the conjugate activity and is non-
reactive with the subject's immune system. Examples include, but are not limited to. any of
the standard pharmaceutical carriers such as a phosphate buffered saline solution, water,
emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers
may also include sterile solutions, tablets including coated tablets and capsules. Typically
such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin,
stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums,
glycols, or other known excipients. Such carriers may also include flavor and color additives
or other ingredients. Compositions comprising such carriers are formulated by well known
conventional methods.
As used herein, "administering" means oral administration, administration as a
suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional,
intranasal or subcutaneous administration, intrathecal administration, or the implantation of a
slow-release device e.g., a mini-osmotic pump, to the subject.
The term "isolated" refers to a material that is substantially or essentially free from
components, which are used to produce the material. For peptide conjugates of the invention,
the term "isolated" refers to material that is substantially or essentially free from components,
which normally accompany the material in the mixture used to prepare the peptide conjugate.
"Isolated" and "pure" are used interchangeably. Typically, isolated peptide conjugates of the
invention have a level of purity preferably expressed as a range. The lower end of the range
of purity for the peptide conjugates is about 60%, about 70% or about 80% and the upper end
of the range of purity is about 70%, about 80%, about 90% or more than about 90%.
When the peptide conjugates are more than about 90% pure, their purities are also
preferably expressed as a range. The lower end of the range of purity is about 90%, about
92%. about 94%. about 96% or about 98%. The upper end of the range of purity is about
92%, about 94%, about 96%. about 98% or about ! 00% purity.
Purity is determined by any art-recognized method of analysis (e.g., band intensity on
a silver stained gel, polyacrylamide gel electrophoresis, HPLC, or a similar means).
"Commercial scale" as used herein means about one or more gram of final product
produced in the method.
"Essentially each member of the population," as used herein, describes a
characteristic of a population of peptide conjugates of the invention in which a selected
percentage of the modified sugars added to a peptide are added to multiple, identical acceptor
sites on the peptide. "Essentially each member of the population" speaks to the
"homogeneity" of the sites on the peptide conjugated to a modified sugar and refers to
conjugates of the invention, which are at least about 80%, preferably at least about 90% and
more preferably at least about 95% homogenous.
"Homogeneity." refers to the structural consistency across a population of acceptor
moieties to which the modified sugars are conjugated. Thus, in a peptide conjugate of the
invention in which each modified sugar moiety is conjugated to an acceptor site having the
same structure as the acceptor site to which every other modified sugar is conjugated, the
peptide conjugate is said to be about 100% homogeneous. Homogeneity is typically
expressed as a range. The lower end of the range of homogeneity for the peptide conjugates
is about 60%, about 70% or about 80% and the upper end of the range of purity is about 70%,
about 80%, about 90% or more than about 90%.
When the peptide conjugates are more than or equal to about 90% homogeneous, their
homogeneity is also preferably expressed as a range. The lower end of the range of
homogeneity is about 90%, about 92%), about 94%, about 96% or about 98%. The upper end
of the range of purity is about 92%, about 94%, about 96%, about 98% or about 100%
homogeneity. The purity of the peptide conjugates is typically determined by one or more
methods known to those of skill in the art, e.g., liquid chromatography-mass spectrometry
(LC-MS), matrix assisted laser desorption time of flight mass spectrometry (MALD1-TOF).
capillary electrophoresis, and the like.
"Substantially uniform glycoform" or a "substantially uniform glycosylation pattern."
when referring to a glycopeptide species, refers to the percentage of acceptor moieties that
are glycosylated by the glycosytransferase of interest {e.g., fucosytransferase). For
example, in the case of a a 1,2 fucosyltransferase, a substantially uniform fucosylation pattern
exists if substantially all (as defined below) of the Gal(31,4-GlcNAc-R and sialylated
analogues thereof are fucosylated in a peptide conjugate of the invention. It will be
understood by one of skill in the art, that the starting material may contain glycosylated
acceptor moieties (e.g., fucosylated Galß1,4-GlcNAc-R moieties). Thus, the calculated
percent glycosylation will include acceptor moieties that are glycosylated by the methods of
the invention, as well as those acceptor moieties already glycosylated in the starting material.
The term "substantially" in the above definitions of "substantially uniform' generally
means at least about 40%, at least about 70%, at least about 80%, or more preferably at least
about 90%, and still more preferably at least about 95% of the acceptor moieties for a
particular glycosyltransferase are glycosylated.
Description of the Invention
I. Method to Remodel Glycan Chains
The present invention includes methods and compositions for the in vitro addition
and/or deletion of sugars to or from a glycopeptide molecule in such a manner as to provide a
peptide molecule having a specific customized or desired glycosylation pattern, preferably
including the addition of a modified sugar thereto. A key feature of the invention therefore is
to take a peptide produced by any cell type and generate a core glycan structure on the
peptide, following which the glycan structure is then remodeled in vitro to generate a peptide
having a glycosylation pattern suitable for therapeutic use in a mammal.
The importance of the glycosylation pattern of a peptide is well known in the art as
are the limitations of present in vivo methods for the production of properly glycosylated
peptides, particularly when these peptides are produced using recombinant DNA
methodology. Moreover, until the present invention, it has not been possible to generate
glycopeptides having a desired glycan structure thereon, wherein the peptide can be produced
at industrial scale.
In the present invention, a peptide produced by a cell is enzymatically treated in vitro
by the systematic addition of the appropriate enzymes and substrates therefor, such that sugar
moieties that should not be present on the peptide are removed, and sugar moieties, optionally
including modified sugars, that should be added to the peptide are added in a manner to
provide a glycopeptide having "desired glycosylation" as defined elsewhere herein.
A. Method to remodel N-linked glycans
In one aspect, the present invention takes advantage of the fact that most peptides of
commercial or pharmaceutical interest comprise a common five sugar structure referred to
herein as the trimannosyl core, which is N-linked to asparagine at the sequence Asn-X-
Ser/Thr on a peptide chain. The elemental trimannosyl core consists essentially of two N-
acctylglucosamine (GlcNAc) residues and three mannose (Man) residues attached to a
peptide, i.e., it comprises these five sugar residues and no additional sugars, except that it
may optionally include a fucose residue. The first GlcNAc is attached to the amide group of
the asparagine and the second GlcNAc is attached to the first via a ß1,4 linkage. A mannose
residue is attached to the second GlcNAc via a ß1,4 linkage and two mannose residues are
attached to this mannose via an a1,3 and an a1,6 linkage respectively. A schematic depiction
of a trimannosyl core structure is shown in Figure 1, left side. While it is the case that glycan
structures on most peptides comprise other sugars in addition to the trimannosyl core, the
trimannosyl core structure represents an essential feature of N-linked glycans on mammalian
peptides.
The present invention includes the generation of a peptide having a trimannosyl core
structure as a fundamental element of the structure of the glycan molecules contained
thereon. Given the variety of cellular systems used to produce peptides, whether the systems
are themselves naturally occurring or whether they involve recombinant DNA methodology,
the present invention provides methods whereby a glycan molecule on a peptide produced in
any cell type can be reduced to an elemental trimannosyl core structure. Once the elemental
trimannosyl core structure has been generated then it is possible using the methods described
herein, to generate in vitro, a desired glycan structure on the peptide which confers on the
peptide one or more properties that enhances the therapeutic effectiveness of the peptide.
It should be clear from the discussion herein that the term "trimannosyl core" is used
to describe the glycan structure shown in Figure 1, left side. Glycopeptides having a
trimannosyl core structure may also have additional sugars added thereto, and for the most
part, do have additional structures added thereto irrespective of whether the sugars give rise
to a peptide having a desired glycan structure. The term "elemental trimannosyl core
structure" is defined elsewhere herein. When the term "elemental" is not included in the
description of the "trimannosyl core structure,'" then the glycan comprises the trimannosyl
core structure with additional sugars attached to the mannose sugars.
The term "elemental trimannosyl core glycopeptide" is used herein to refer to a
glycopeptide having glycan structures comprised primarily of an elemental trimannosyl core
structure. However, it may also optionally contain a fucose residue attached thereto. As
discussed herein, elemental trimannosyl core glycopeptides are one optimal, and therefore
preferred, starting material for the glycan remodeling processes of the invention.
Another optimal starting material for the glycan remodeling process of the invention
is a glycan structure having a trimannosyl core wherein one or two additional GlcNAc
residues are added to each of the al.3 and the a1,6 mannose residues (see for example, the
structure on the second line of Figure 2, second structure in from the left of the figure). This
structure is referred to herein as "Man3GlcNAc4." When the structure is monoantenary, the
structure is referred to herein as "Man3GlcNAc3." Optionally, this structure may also
contain a core fucose molecule. Once the Man3GlcNAc3 or Man3GlcNAc4 structure has
been generated then it is possible using the methods described herein, to generate in vitro, a
desired glycan structure on the glycopeptide which confers on the glycopeptide one or more
properties that enhances the therapeutic effectiveness of the peptide.
In their native form, the N-linked glycopeptides of the invention, and particularly the
mammalian and human glycopeptides useful in the present invention, are N-linked
glycosylated with a trimannosyl core structure and one or more sugars attached thereto.
The terms "glycopeptide" and "glycopolypeptide" are used synonymously herein to
refer to peptide chains having sugar moieties attached thereto. "No distinction is made herein
to differentiate small glycopdlypeptides or glycopeptides from large glycopolypeptides or
glycopeptides. Thus, hormone molecules having very few amino acids in their peptide chain
(e.g., often as few as three amino acids) and other much larger peptides are included in the
general terms "glycopolypeptide" and "glycopeptide," provided they have sugar moieties
attached thereto. However, the use of the term "peptide" does not preclude that peptide from
being a glycopeptide.
An example of an N-linked glycopeptide having desired glycosylation is a peptide
having an N-linked glycan having a trimannosyl core with at least one GlcNAc residue
attached thereto. This residue is added to the trimannosyl core using N-acetyl
glucosaminyltransferase 1 (GnT-1). If a second GlcNAc residue is added, N-acetyl
glucosaminyltransferase II (GnT-II) is used. Optionally, additional GlcNAc residues may be
added with GnT-IV and/or GnT-V, and a third bisecting GlcNAc residue may be attached to
the ß1,4 mannose of the trimannosyl core using N-acetyl glucosaminyltransferase III (GnT-
III). Optionally, this structure may be extended by treatment with ß1,4 galactosyltransferase
to add a galactose residue to each non-bisecting GlcNAc, and even further optionally, using
a2,3 or a2,6-sialyltransferase enzymes, sialic acid residues may be added to each galactose
residue. The addition of a bisecting GlcNAc to the glycan is not required for the subsequent
addition of galactose and sialic acid residues; however, with respect to the substrate affinity
of the rat and human GnT-II I enzymes, the presence of one or more of the galactose residues
on the glycan precludes the addition of the bisecting GlcNAc in that the galactose-coniaining
glycan is not a substrate for these forms of GnT-III. Thus, in instances where the presence of
the bisecting GlcNAc is desired and these forms of GnT-III are used, it is important should
the glycan contain added galactose and/or sialic residues, that they are removed prior to the
addition of the bisecting GlcNAc. Other forms of GnT-III may not require this specific order
of substrates for their activity. In the more preferred reaction, a mixture of GnT-I, GnT-II
and GnT-III is added to the reaction mixture so that the GlcNAc residues can be added in any
order.
Examples of glycan structures which represent the various aspects of peptides having
"desired glycosylation" are shown in the drawings provided herein. The precise procedures
for the in vitro generation of a peptide having "desired glycosylation" are described
elsewhere herein. However, the invention should in no way be construed to be limited solely
to any one glycan structure disclosed herein. Rather, the invention should be construed to
include any and all glycan structures which can be made using the methodology provided
herein.
In some cases, an elemental trimannosyl core alone may constitute the desired
glycosylation of a peptide. For example, a peptide having only a trimannosyl core has been
shown to be a useful component of an enzyme employed to treat Gaucher disease (Mistry et
al.. 1966, Lancet 348: 1555-1559; Bijsterbosch et al., 1996, Eur. J. Biochem. 237:344-349).
According to the present invention, the following procedures for the generation of
peptides having desired glycosylation become apparent.
a) Beginning with a glycopeptide having one or more glycan molecules which have
as a common feature a trimannosyl core structure and at least one or more of a heterogeneous
or homogeneous mixture of one or more sugars added thereto, it is possible to increase the
proportion of glycopeptides having an elemental trimannosyl core structure as the sole glycan
structure or which have Man3GIcNAc3 or Man3GlcNAc4 as the sole glycan structure. This
is accomplished in vitro by the systematic addition to the glycopeptide of an appropriate
number of enzymes in an appropriate sequence which cleave the heterogeneous or
homogeneous mixture of sugars on the glycan structure until it is reduced to an elemental
trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4 structure. Specific examples of how
this is accomplished will depend on a variety of factors including in large part the type of cell
in which the peptide is produced and therefore the degree of complexity of the glycan
structure(s) present on the peptide initially produced by the cell. Examples of how a complex
glycan structure can be reduced to an elemental trimannosyl core or a Man3GlcNAc3 or
Man3GlcNAc4 structure are presented in Figure 2 or are described in detail elsewhere herein.
b) It is possible to generate a peptide having an elemental trimannosyl core structure
as the sole glycan structure on the peptide by isolating a naturally occurring cell whose
glycosylation machinery produces such a peptide. DNA encoding a peptide of choice is then
transfected into the cell wherein the DNA is transcribed, translated and glycosylated such that
the peptide of choice has an elemental trimannosyl core structure as the sole glycan structure
thereon. For example, a cell lacking a functional GnT-I enzyme will produce several types of
glycopeptides. In some instances, these will be glycopeptides having no additional sugars
attached to the trimannosyl core. However, in other instances, the peptides produced may
have two additional mannose residues attached to the trimannosyl core, resulting in a Man5
glycan. This is also a desired starting material for the remodeling process of the present
invention. Specific examples of the generation of such glycan structures are described
herein.
c) Alternatively, it is possible to genetically engineer a cell to confer upon it a
specific glycosylation machinery such that a peptide having an elemental trimannosyl core or
Man3GlcNAc3 or Man3GlcNAc4 structure as the sole glycan structure on the peptide is
produced. DMA encoding a peptide of choice is then transfected into the cell wherein the
DNA is transcribed, translated and glycosylated such that the peptide of choice has an
increased number of glycans comprising solely an elemental trimannosyl core structure. For
example, certain types of cells that are genetically engineered to lack GnT-I. may produce a
glycan having an elemental trimannosyl core structure, or, depending on the cell, may
produce a glycan having a trimannosyl core plus two additional mannose residues attached
thereto (Man5). When the cell produces a Man5 glycan structure, the cell may be further
genetically engineered to express mannosidase 3 which cleaves off the two additional
mannose residues to generate the trimannosyl core. Alternatively, the Man5 glycan may be
incubated in vitro with mannosidase 3 to have the same effect.
d) When a peptide is expressed in an insect cell, the glycan on the peptide comprises
a partially complex chain. Insect cells also express hexosaminidase in the cells which trims
the partially complex chain back to a trimannosyl core structure which can then be remodeled
as described herein.
e) It is readily apparent from the discussion in b), c) and d) that it is not necessary
that the cells produce only peptides having elemental trimannosyl core or Man3GlcNAc3 or
Man3GleNAc4 structures attached thereto. Rather, unless the cells described in b) and c)
produce peptides having 100% elemental trimannosyl core structures (i.e., having no
additional sugars attached thereto) or 100% of Man3GlcNAc3 or Man3GlcNAc4 structures,
the cells in fact produce a heterogeneous mixture of peptides having, in combination,
elemental trimannosyl core structures, or Man3GlcNAc3 or Man3GlcNAc4 structures, as the
sole glycan structure in addition to these structures having additional sugars attached thereto.
The proportion of peptides having a trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4
structures having additional sugars attached thereto, as opposed to those having one structure,
will vary depending on the cell which produces them. The complexity of the glycans (i.e.
which and how many sugars are attached to the trimannosyl core) will also vary depending
on the cell which produces them.
1) Once a glycopeptide having an elemental trimannosyl core or a trimannosyl core
with one or two GlcNAc residues attached thereto is produced by following a), b) or c)
above, according to the present invention, additional sugar molecules are added in vitro to the
trimannosyl core structure to generate a peptide having desired glycosylation (i.e., a peptide
having an in vitro customized glycan structure).
g) However, when it is the case that a peptide having an elemental trimannosyl core
or Man3GlcNAc4 structure with some but not all of the desired sugars attached thereto is
produced, then it is only necessary to add any remaining desired sugars without reducing the
glycan structure to the elemental trimannosyl core or Man3GlcNAc4 structure. Therefore, in
some cases, a peptide having a glycan structure having a trimannosyl core structure with
additional sugars attached thereto, will be a suitable substrate for remodeling.
Isolation of an elemental trimannosyl core glycopeptide
The elemental trimannosyl core or Man3GlcNAc3 or Man3GlcNAc4 glycopeptides of
the invention may be isolated and purified, if necessary, using techniques well known in the
art of peptide purification. Suitable techniques include chromatographic techniques,
isoelectric focusing techniques, ultrafiltration techniques and the like. Using any such
techniques, a composition of the invention can be prepared in which the glycopeptides of the
invention are isolated from other peptides and from other components normally found within
cell culture media. The degree of purification can be, for example, 90% with respect to other
peptides or 95%, or even higher, e.g., 98%. See, e.g., Deutscher et al. (ed.. 1990, Guide to
Protein Purification, Harcourt Brace Jovanovich, San Diego).
The heterogeneity of N-linkcd glycans present in the glycopeptides produced b\ the
prior art methodology generally only permits the isolation of a small portion of the target
glycopeptides which can be modified to produce desired glycopeptides. In the present
methods, large quantities of elemental trimannosyl core glycopeptides and other desired
glycopeptides, including Man3GlcNAc3 or Man3GlcNAc4 glycans, can be produced which
can then be further modified to generate large quantities of peptides having desired
glycosylation.
Specific enrichment of any particular type of glycan linked to a peptide may be
accomplished using lectins which have an affinity for the desired glycan. Such techniques
are well known in the art of glycobiology.
A key feature of the invention which is described in more detail below, is that once a
core glycan structure is generated on any peptide, the glycan structure is then remodeled m
vitro to generate a peptide having desired glycosylation that has improved therapeutic use in a
mammal. The mammal may be any type of suitable mammal, and is preferably a human.
The various scenarios and the precise methods and compositions for generating
peptides with desired glycosylation will become evident from the disclosure which follows.
The ultimate objective of the production of peptides for therapeutic use in mammals is
that the peptides should comprise glycan structures that facilitate rather than negate the
therapeutic benefit of the peptide. As disclosed throughout the present specification, peptides
produced in cells may be treated in vitro with a variety of enzymes which catalyze the
cleavage of sugars that should not be present on the glycan and the addition of sugars which
should be present on the glycan such that a peptide having desired glycosylation and thus
suitable for therapeutic use in mammals is generated. The generation of different glycoforms
of peptides in cells is described above. A variety of mechanisms for the generation of
peptides having desired glycosylation is now described, where the starting material i.e., the
peptide produced by a cell may differ from one cell type to another. As will become apparent
from the present disclosure, it is not necessary that the starting material be uniform with
respect to its glycan composition. However, it is preferable that the starting material be
enriched for certain glycoforms in order that large quantities of end product, i.e., correctly
glycosylated peptides are produced.
In a preferred embodiment according to the present invention, the degradation and
synthesis events that result in a peptide having desired glycosylation involve at some point,
the generation of an elemental trimannosyf core structure or a Man3GlcNAc3 or
Man3GlcNAc4 structure on the peptide.
The present invention also provides means of adding one or more selected glycosyl
residues to a peptide, after which a modified sugar is conjugated to at least one of the selected
glycosyl residues of the peptide. The present embodiment is useful, for example, when it is
desired to conjugate the modified sugar to a selected glycosyl residue that is either not
present on a peptide or is not present in a desired amount. Thus, prior to coupling a modified
sugar to a peptide, the selected glycosyl residue is conjugated to the peptide by enzymatic or
chemical coupling. In another embodiment, the glycosylation pattern of a peptide is altered
prior to the conjugation of the modified sugar by the removal of a carbohydrate residue from
the peptide. See for example WO 98/3 1826.
Addition or removal of any carbohydrate moieties present on the peptide is
accomplished either chemically or enzymatically. Chemical deglycosylation is preferably
brought about by exposure of the peptide variant to the compound trifluoromethanesulfonic
acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars
except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the
peptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch.
Biochem. Biophys. 259: 52 and by Edge et al., 1981, Anal. Biochem. 118: 131. Enzymatic
cleavage of carbohydrate moieties on peptide variants can be achieved by the use of a variety
of endo- and exo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 138:
350.
Chemical addition of glycosyl moieties is carried out by any art-recognized method.
Enzymatic addition of sugar moieties is preferably achieved using a modification of the
methods set forth herein, substituting native glycosyl units for the modified sugars used in the
invention. Other methods of adding sugar moieties are disclosed in U.S. Patent No.
5.876,980. 6,030.815. 5,728,554, and 5.922,577.
Exemplary attachment points for selected glycosyl residue include, but are not limited
to: (a) sites for N- and O-glycosylation; (b) terminal glycosyl moieties that are acceptors for a
glycosyltransferase; (c) arginine, asparagine and histidine; (d) free carboxyl groups; (e) free
sulfhydryl groups such as those of cysteine; (f) free hydroxy! groups such as those of serine,
threonine, or hydroxyproline; (g) aromatic residues such as those of phenylalanine, tyrosine,
or tryptophan; or (h) the amide group of glutamine. Exemplary methods of use in the present
invention are described in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,
CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Dealing specifically with the examples shown in several of the figures provided
herein, a description of the sequence of in vitro enzymatic reactions for the production of
desired glycan structures on peptides is now presented. The precise reaction conditions for
each of the enzymatic conversions disclosed below are well known to those skilled in the art
of glycobiology and are therefore not repeated here. For a review of the reaction conditions
for these types of reactions, see Sadler etal., 1982. Methods in Enzymology 83:458-514 and
references cited therein.
In Figure 1 there is shown the structure of an elemental trimannosyl core glycan on
the left side. It is possible to convert this structure to a complete glycan structure having a
bisecting GlcNAc by incubating the elemental trimannosyl core structure in the presence of
GnT-I. followed by GnT-II, and further followed by GnT-III, and a sugar donor comprising
UDP-GlcNAc, wherein GlcNAc is sequentially added to the elemental trimannosyl core
structure to generate a trimannosyl core having a bisecting GlcNAc. In some instances, for
example when remodeling Fc glycans as described herein, the order of addition of GnT-1,
GnT-H and GnT-III may be contrary to that reported in the literature. The bisecting GlcNAc
structure may be produced by adding a mixture of GnT-I, GnT-II and GnT-III and UDP-
GlcNAc to the reaction mixture
In Figure 3 there is shown the conversion of a bisecting GlcNAc containing
trimannosyl core glycan to a complex glycan structure comprising galactose and N-acetyl
neuraminic acid. The bisecting GlcNAc containing trimannosyl core glycan is first incubated
with galactosyltransferase and UDP-Gal as a donor molecule, wherein two galactose residues
are added to the peripheral GlcNAc residues on the molecule. The enzyme NeuAc-
transferase is then used to add two NeuAc residues one to each of the galactose residues.
In Figure 4 there is shown the conversion of a high mannose glycan structure to an
elemental trimannosyl core glycan. The high mannose glycan (Man9) is incubated
sequentially in the presence of the mannosidase 1 to generate a Man5 structure and then in
the presence of mannosidase 3, wherein all but three mannose residues are removed from the
glycan. Alternatively, incubation of the Man9 structure may be trimmed back to the
trimannosyl core structure solely by incubation in the presence of mannosidase 3. According
to the schemes presented in Figures 1 and 3 above, conversion of this elemental trimannosyl
core glycan to a complex glycan molecule is then possible.
In Figure 5 there is shown a typical complex N-linked glycan structure produced in
plant cells. It is important to note that when plant cells are deficient in GnT-I enzymatic
activity, xylose and fucose cannot be added to the glycan. Thus, the use of GnT-1 knock-out
cells provides a particular advantage in the present invention in that these cells produce
peptides having an elemental trimannosyl core onto which additional sugars can be added
without performing any "trimming back" reactions. Similarly, in instances where the
structure produced in a plant cell may be of the Man5 variety of glycan, if GnT-I is absent in
these cells, xylose and fucose cannot be added to the structure. In this case, the Man5
structure may be trimmed back to an elemental trimannosyl core (Man3) using mannosidase
3. According to the methods provided herein, it is now possible to add desired sugar moieties
to the trimannosyl core to generate a desired glycan structure.
In Figure 6 there is shown a typical complex N-linked glycan structure produced in
insect cells. As is evident, additional sugars, such as, for example, fucose may also be
present. Further although not shown here, insect cells may produce high mannose glycans
having as many as nine mannose residues and may have additional sugars attached thereto. It
is also the case in insect cells that GnT-I knock out cells prevent the addition of fucose
residues to the glycan. Thus, production of a peptide in insect cells mav preferably be
accomplished in a GnT-I knock out cell. The glycan thus produced may then be trimmed
back in vitro if necessary using any of the methods and schemes described herein, and
additional sugars may be added in vitro thereto also using the methods and schemes provided
herein.
In Figure 2 there is shown glycan structures in various stages of completion.
Specifically, the in vitro enzymatic generation of an elemental trimannosyl core structure
from a complex carbohydrate glycan structure which does not contain a bisecting GlcNAc
residue is shown. Also shown is the generation of a glycan structure therefrom which
contains a bisecting GlcNAc. Several intermediate glycan structures which can be produced
are shown. These structures can be produced by cells, or can be produced in the in vitro
trimming back reactions described herein. Sugar moieties may be added in vitro to the
elemental trimannosyl core structure, or to any suitable intermediate structure in order that a
desired glycan is produced.
In Figure 7 there is shown a series of possible in vitro reactions which can be
performed to trim back and add onto glycans beginning with a high mannose structure. For
example, a Man9 glycan may be trimmed using mannosidase 1 to generate a Man5 glycan, or
it may be trimmed to a trimannosyl core using mannosidase 3 or one or more microbial
mannosidases. GnT-I and or GnT-II may then be used to transfer additional GlcNAc residues
onto the glycan. Further, there is shown the situation which would not occur when the glycan
molecule is produced in a cell that does not have GnT-I (see shaded box). For example,
fucose and xylose may be added to a glycan only when GnT-I is active and facilitates the
transfer of a GlcNAc to the molecule.
Figure 8 depicts well known strategies for the synthesis of biantennary. triantennary
and even tetraantennary glycan structures beginning with the trimannosyl core structure.
According to the methods of the invention, it is possible to synthesize each of these structures
in vitro using the appropriate enzymes and reaction conditions well known in the art of
glycobiology.
Figure 9 depicts two methods for synthesis of a monoantennary glycan structure
beginning from a high mannose (6 to 9 mannose moieties) glycan structures. A terminal
sialic acid-PEG moiety may be added in place of the sialic acid moiety in accordance with
glycoPBGylation methodology described herein. In the first method, endo-II is used to
cleave the glycan structure on the peptide back to the first GlcNAc residue. Galactose is then
added using galactosyltransferase and sialylated-PEG is added as described elsewhere herein.
In the second method, mannosidase I is used to cleave mannose residues from the glycan
structure in the peptide. A galactose residue is added to one arm of the remaining mannose
residues which were cleaved off the glycan using Jack Bean oc-mannosidase. Sialylated-PEG
is then added to this structure as directed.
Figure 10 depicts two additional methods for synthesis of a monoantennary glycan
structures beginning from high mannose (6 to 9 mannose moieties) glycan structure. As in
Figure 9, a terminal sialic acid-PEG moiety may be added in place of the sialic acid moiety in
accordance with the glycoPEGylation methodology described herein. In the situation
described here, some of the mannose residues from the arm to which sialylated-PEG is not
added, are removed.
In Figure II there is shown a scheme for the synthesis of yet more complex
carbohydrate structures beginning with a trimannosyl core structure. For example, a scheme
for the in vitro production of Lewis x and Lewis a antigen structures, which may or may not
be sialylated is shown. Such structures when present on a peptide may confer on the peptide
immunological advantages for upregulating or downregulating the immune response. In
addition, such structures are useful for targeting the peptide to specific cells, in that these
types of structures are involved in binding to cell adhesion peptides and the like.
Figure 12 is an exemplary scheme for preparing an array of O-linked peptides
originating with serine or threonine.
Figure 13 is a series of diagrams depicting the four types of O-linked glycan structure
termed cores 1 through 4. The core structure is outlined in dotted lines. Sugars which may
also be included in this structure include sialic acid residues added to the galactose residues,
and fucose residues added to the GlcNAc residues.
Thus, in preferred embodiments, the present invention provides a method of making
an N-linked glycosylated glycopeptidc by providing an isolated and purified glycopeplide to
which is attached an elemental trimannosyl core or a Man3GlcNAc4 structure, contacting the
glycopeptide with a glycosyltransferase enzyme and a donor molecule having a glycosyl
moiety under conditions suitable to transfer the glycosyl moiety to the glycopeptide.
Customization of a trimannosyl core glycopeptide or Man3GlcNAc4 glycopeptide to produce
a peptide having a desired glycosylation pattern is then accomplished by the sequential
addition of the desired sugar moieties, using techniques well known in the art.
Determination of Glycan Primary Structure
When an N-linked glycopeptide is produced by a cell, as noted elsewhere herein, it
may comprise a heterogeneous mixture of glycan structures which must be reduced to a
common, generally elemental trimannosyl core or Man3GlcNAc4 structure, prior to adding
other sugar moieties thereto. In order to determine exactly which sugars should be removed
from any particular glycan structure, it is sometimes necessary that the primary glycan
structure be identified. Techniques for the determination of glycan primary structure are well
know in the art and are described in detail, for example, in Montreuil, "Structure and
Biosynthesis of Glycopeptides" In Polysaccharides in Medicinal Applications, pp. 273-327,
19%. Eds. Severian Damitriu, Marcel Dekker, NY. It is therefore a simple matter for one
skilled in the art of glycobiology to isolate a population of peptides produced by a cell and
determine the structure(s) of the glycans attached thereto. For example, efficient methods are
available for (i) the splitting of glycosidic bonds either by chemical cleavage such as
hydrolysis, acetolysis, hydrazinolysis, or by nitrous deamination; (ii) complete methylation
followed by hydrolysis or methanolysis and by gas-liquid chromatography and mass
spectroscopy of the partially methylated monosaccharides; and (iii) the definition of anomeric
linkages between monosaccharides using exoglycosidases. which also provide insight into the
primary glycan structure by sequential degradation. In particular, the techniques of mass
spectroscopy and nuclear magnetic resonance (NMR) spectrometry, especially high field
NMR have been successfully used to determine glycan primary structure.
Kits and equipment for carbohydrate analysis are also commercially available.
Fluorophore Assisted Carbohydrate Electrophoresis (FACE*) is available from Glyko, Inc.
(Novato, CA). In FACE analysis, glycoconjugates are released from the peptide with cither
Endo H or N-glycanase (PNGase F) for N-linked glycans, or hydrazine for Ser/Thr linked
glycans. The glycan is then labeled at the reducing end with a fluorophore in a non-structure
discriminating manner. The fluorophore labeled glycans are then separated in
polyacrylamide gels based on the charge/mass ratio of the saccharide as well as the
hydrodynamic volume. Images are taken of the gel under UV light and the composition of
the glycans are determined by the migration distance as compared with the standards.
Oligosaccharides can be sequenced in this manner by analyzing migration shifts due to the
sequential removal of saccharides by exoglycosidase digestion.
Exemplary embodiment
The remodeling of N-linked glycosylation is best illustrated with reference to Formula
1:
where X3, X4, X5. X6, X7 and X17 are (independently selected) monosaccharide or
oligosaccharide residues; and
a. b, c, d, e and x are (independently selected) 0, 1 or 2, with the proviso that at least
one member selected from a, b, c, d, e and x are 1 or 2.

Formula 1 describes glycan structure comprising the tri-mannosyl core, which is
preferably covalently linked to an asparagine residue on a peptide backbone. Preferred
expression systems will express and secrete exogenous peptides with N-linked glycans
comprising the tri-mannosyl core. Using the remodeling method of the invention, the glycan
structures on these peptides can be conveniently remodeled to any glycan structure desired.
Exemplary reaction conditions are found throughout the examples and in the literature.
In preferred embodiments, the glycan structures are remodeled so that the structure
described in Formula 1 has specific determinates. The structure of the glycan can be chosen
to enhance the biological activity of the peptide, give the peptide a new biological activity,
remove the biological activity of peptide, or better approximate the glycosylation pattern of
the native peptide, among others.
In the first preferred embodiment, the peptide N-linked glycans are remodeled to
better approximate the glycosylation pattern of native human proteins. In this embodiment,
the glycan structure described in Formula 1 is remodeled to have the following moieties:
X3 and X5 = |-GlcNAc-Gal-SA;
a and c = I;
d = 0 or 1;
b, e and x = 0.
This embodiment is particularly advantageous for human peptides expressed in heterologous
cellular expression systems. By remodeling the N-linked glycan structures to this
configuration, the peptide can be made less immunogenic in a human patient, and/or more
stable, among others.
In the second preferred embodiment, the peptide N-linked glycans are remodeled to
have a bisecting GlcNAc residue on the tri-mannosyl core. In this embodiment, the glycan
structure described in Formula I is remodeled to have the following moieties:
X3 and X5 are |-GlcNAc-Gal-SA;
a and c = 1:
X4 is GlcNAc;
b=l;
d = 0 or 1;
e and x = 0.
This embodiment is particularly advantageous for recombinant antibody molecules expressed
in heterologous cellular systems. When the antibody molecule includes a Fc-mediated
cellular cytotoxicity, it is known that the presence of bisected oligosaccharides linked the Fc
domain dramatically increased antibody-dependent cellular cytotoxicity.
In a third preferred embodiment, the peptide N-linked glycans are remodeled to have
a sialylated Lewis X moiety. In this embodiment, the glycan structure described in Formula
1 is remodeled to have the following moieties:

This embodiment is particularly advantageous when the peptide which is being remodeling is
intended to be targeted to selectin molecules and cells exhibiting the same.
In a fourth preferred embodiment, the peptide N-linked glycans are remodeled to have
a conjugated moiety. The conjugated moiety may be a PEG molecule, another peptide, a
small molecule such as a drug, among others. In this embodiment, the glycan structure
described in Formula I is remodeled to have the following moieties:
X3 and Xs are |-GlcNAc-Gal-SA-R:
a and c - 1 or 2;
d = 0 or 1;
b, d, e and x = 0;
where R = conjugate group.
The conjugated moiety may be a PEG molecule, another peptide, a small molecule such as a
drug, among others. This embodiment therefore is useful for conjugating the peptide to PEG
molecules that will slow the clearance of the peptide from the patient's bloodstream, to
peptides that will target both peptides to a specific tissue or cell, or to another peptide of
complementary therapeutic use.
It will be clear to one of skill in the art that the invention is not limited to the preferred
glycan molecules described above. The preferred embodiments are only a few of the many
useful glycan molecules that can be made by the remodeling method of the invention. Those
skilled in the art will know how to design other useful glycans.
In the first exemplary embodiments, the peptide is expressed in a CHO (Chinese
hamster ovarian cell line) according to methods well known in the art. When a peptide with
N-linked glycan consensus sites is expressed and secreted from CHO cells, the N-linked
glycans will have the structures depicted in top row of Figure 2, but also comprising a core
fucose. While all of these structures may be present, by far the most common structures are
the two at the right side. In the terms of Formula 1,
X3 and X5 are |-GlcNAc-Gal-(SA);
a and c = 1;
b, e and x = 0. and
d = 0 or 1.
Therefore, in one exemplary embodiment, the N-linked glycans of peptides expressed in
CHO cells are remodeled to the preferred humanized glycan by contacting the peptides with a
glycosyltransferase that is specific for a galactose acceptor molecule and a sialic acid donor
molecule. This process is illustrated in Figure 2 and Example 17. In another exemplary
embodiment, the N-linked glycans of a peptide expressed and secreted from CHO cells are
remodeled to be the preferred PEGylated structures. The peptide is first contacted with a
glycosidase specific for sialic acid to remove the terminal SA moiety, and then contacted
with a glycosyltransferase specific for a galactose acceptor moiety and an sialic acid acceptor
moiety, in the presence of PEG- sialic acid-nucleotide donor molecules. Optionally, the
peptide may then be contacted with a glycosyltransferase specific for a galactose acceptor
moiety and an sialic acid acceptor moiety, in the presence of sialic acid-nucleotide donor
molecules to ensure complete the SA capping of all of the glycan molecules.
In other exemplary embodiments, the peptide is expressed in insect cells, such as the
sf9 cell line, according to methods well known in the art. When a peptide with N-linked
glycan consensus sites is expressed and secreted from sf9 cells, the N-linked glycans will
often have the structures depicted in top row of Figure 6. In the terms of Formula 1:
X3 and X5 are |- GlcNAc;
a and c = 0 or 1;
b = 0;
X6 is fucose.
d = 0. I or 2: and
e and x = 0.
The trimannose core is present in the vast majority of the N-linked glycans made by
insect cells, and sometimes an antennary GlcNAc and/or fucose residue(s) are also present.
Note that the glycan may have no core fucose, it may have a single core fucose having either
linkage, or it may have a single core fucose with a perponderance of a single linkage. In one
exemplary embodiment, the N-linked glycans of a peptide expressed and secreted from insect
cells is remodeled to the preferred humanized glycan by first contacting the glycans with a
glycosidase specific to fucose molecules, then contacting the glycans with a
glycosyltransferases specific to the mannose acceptor molecule on each antennary of the
trimannose core, a GlcNAc donor molecule in the presence of nucleotide-GlcNAc molecules:
then contacting the glycans with a glycosyltransferase specific to a GlcNAc acceptor
molecule, a Gal donor molecule in the presence of nucleotide-Gal molecules; and then
contacting the glycans with a glycosyltransferase specific to a galactose acceptor molecule, a
sialic acid donor molecule in the presence of nucleotide-SA molecules. One of skill in the art
will appreciate that the fucose molecules, if any, can be removed at any time during the
procedure, and if the core fucose is of the same alpha 1,6 linkage as found in human glycans,
it may be left intact. In another exemplary embodiment, the humanized glycan of the
previous example is remodeled further to the sialylated Lewis X glycan by contacting the
glycan further with a glycosyltransferase specific to a GlcNAc acceptor molecule, a fucose
donor molecule in the presence of nucleotide-fucose molecules. This process is illustrated in
Figure 11 and Example 39.
In yet other exemplary embodiments, the peptide is expressed in yeast, such as
Saccharomyces cerevisiae, according to methods well known in the art. When a peptide with
N-linked glycan consensus sites is expressed and secreted from S. cerevisiae cells, the N-
linked glycans will have the structures depicted at the left in Figure 4. The N-linked glycans
will always have the trimannosyl core, which will often be elaborated with mannose or
related polysaccharides of up to 1000 residues. In the terms of Formula 1:
X-1 and X' = |-Man - Man - (Man)0-1000;
a and c =1 or 2:
b, d, e and x = 0.
In one exemplary embodiment, the N-linked glycans of a peptide expressed and
secreted from yeast cells are remodeled to the elemental trimannose core by first contacting
the glycans with a glycosidase specific to a2 mannose molecules, then contacting the glycans
with a glycosidase specific to a6 mannose molecules. This process is illustrated in Figure 4
and Example 38.
In another exemplary embodiment, the N-linked glycans are further remodeled to
make a glycan suitable for an recombinant antibody with Fc-mediated cellular toxicity
function by contacting the elemental trimannose core glycans with a glycosyltransferase
specific to the mannose acceptor molecule on each antennary of the trimannose core and a
GlcNAc donor molecule in the presence of nucleotide-GlcNAc molecules. Then, the glycans
are contacted with a glycosyltransferase specific to the acceptor mannose molecule in the
middle of the trimannose core, a GlcNAc donor molecule in the presence of nucleotide-
GlcNAc molecules and further contacting the glycans with a glycosyltransferase specific to a
GlcNAc acceptor molecule, a Gal donor molecule in the presence of nucleotide-Gal
molecules; and then optionally contacting the glycans with a glycosyltransferase specific to a
galactose acceptor molecule and further optionally a sialic acid donor molecule in the
presence of nucleotide-SA molecules. This process is illustrated in Figures 1, 2 and 3.
In another exemplary embodiment, the peptide is expressed in bacterial cells, in
particular E. coli cells, according to methods well known in the art. When a peptide with N-
linked glycans consensus sites is expressed in E. coli cells, the N-linked consensus sites will
not be glycosylated. In an exemplary embodiment, a humanized glycan molecule is built out
from the peptide backbone by contacting the peptides with a glycosyltransferase specific for a
N-linked consensus site and a GlcNAc donor molecule in the presence of nucleotide-
GlcNAc; and further sequentially contacting the growing glycans with glycosyltransferases
specific for the acceptor and donor moieties in the present of the required donor moiety until
the desired glycan structure is completed. When a peptide with N-linked glycans is
expressed in a eukaryotic cells but without the proper leader sequences that direct the nascent
peptide to the golgi apparatus, the mature peptide is likely not to be glycosylated. In this case
as well the peptide may be given N-linked glycosylation by building out from the peptide re-
linked consensus site as aforementioned. When a protein is chemically modified with a sugar
moiety, it can be built out as aforementioned.
These examples are meant to illustrate the invention, and not to limit it. One of skill
in the art will appreciate that the steps taken in each example may in some circumstances be
able to be performed in a different order to get the same result. One of skill in the art will
also understand that a different set of steps may also produce the same resulting glycan. The
preferred remodeled glycan is by no means specific to the expression system that the peptide
is expressed in. The remodeled glycans are only illustrative and one of skill in the art will
know how to take the principles from these examples and apply them to peptides produced in
different expression systems to make glycans not specifically described herein.
B. Method to remodel O-linked glycans
O-glycosylation is characterized by the attachment of a variety of monosaccharides in
an O-glycosidic linkage to hydroxy amino acids. O-glycosylation is a widespread post-
translational modification in the animal and plant kingdoms. The structural complexity of
glycans O-linked to proteins vastly exceeds that of N-linked glycans. Serine or threonine
residues of a newly translated peptide become modified by virtue of a peptidyl GalNAc
transferase in the cis to trans compartments of the Golgi. The site of O-glycosylation is
determined not only by the sequence specificity of the glycosyltransferase, but also
epigenetic regulation mediated by competition between different substrate sites and
competition with other glycosyltransferases responsible for forming the glycan.
The O-linked glycan has been arbitrarily defined as having three regions: the core, the
backbone region and the peripheral region. The "core'" region of an O-linked glycan is the
inner most two or three sugars of the glycan chain proximal to the peptide. The backbone
region mainly contributes to the length of the glycan chain formed by uniform elongation.
The peripheral region exhibits a high degree of structural complexity. The structural
complexity of the O-linked glycans begins with the core structure. In most cases, the first
sugar residue added at the O-linked glycan consensus site is GalNAc; however the sugar may
also be GlcNAc, glucose, mannose, galactose or fucose, among others. Figure 12 is a
diagram of some of the known O-linked glycan core structures and the enzymes responsible
for their in vivo synthesis.
In mammalian cells, at leasteight different O-linked core structures are found, all
based on a core-a-GalNAc residue. The four core structures depicted in Figure 13 are the
most common. Core 1 and core 2 are the most abundant structures in mammalian cells, and
core 3 and core 4 are found in more restricted, organ-characteristic expression systems. O-
linked glycans are reviewed in Montreuil, Structure and Synthesis of Glycopeptides, In
Polysaccharides in Medicinal Applications, pp. 273-327, 1996, Eds. Severian Damitriu.
Marcel Dekker, NY. and in Schachter and Brockhausen, The Biosynthesis of Branched O-
Linked Glycans, 1989, Society for Experimental Biology, pp. 1-26 (Great Britain).
It will be apparent from the present disclosure that the glycan structure of O-
glycosylated peptides can be remodeled using similar techniques to those described for N-
linked glycans. O-glycans differ from N-glycans in that they are linked to a serine or
threonine residue rather than an asparagine residue. As described herein with respect to N-
glycan remodeling, hydrolytic enzymes can be used to cleave unwanted sugar moieties in an
O-linked glycan and additional desired sugars can then be added thereto, to build a
customized O-glycan structure on the peptide (See Figures 12 and 13).
The initial step in O-glycosylation in mammalian cells is the attachment of N-
acetylgalactosamine (GalNAc) using any of a family of at least eleven known a-N-
acetylgalactosaminyitransferases, each of which has a restricted acceptor peptide specificity.
Generally, the acceptor peptide recognized by each enzyme constitutes a sequence of at least
ten amino acids. Peptides that contain the amino acid sequence recognized by one particular
GalNAc-transferase become O-glycosylated at the acceptor site if they are expressed in a cell
expressing the enzyme and if they are appropriately localized to the Golgi apparatus where
UDP-GalNAc is also present.
However, in the case of recombinant proteins, the initial attachment of the GalNAc
may not take place. The a-N-acetylgalactosaminyltransferase enzyme native to the
expressing cell may have a consensus sequence specificity which differs from that of the
recombinant peptide being expressed.
The desired recombinant peptide may be expressed in a bacterial cell, such as E. coli,
that does not synthesize glycan chains. In these cases, it is advantageous to add the initial
GalNAc moiety in vitro. The GalNAc moiety can be introduced in vitro onto the peptide
once the recombinant peptide has been recovered in a soluble form, by contacting the peptide
with the appropriate GalNAc transferase in the presence of UDP-GalNAc.
In one embodiment, an additional sequence of amino acids that constitute an effective
acceptor for transfer of an O-linked sugar may be present. Such an amino acid sequence is
encoded by a DNA sequence fused in frame to the coding sequence of the peptide, or
alternatively, may be introduced by chemical means. The peptide may be otherwise lacking
glycan chains. Alternately, the peptide may have N- and/or O-linked glycan chains but
require an additional glycosylation site, for example, when an additional glycan substituent is
desired.
In an exemplary embodiment, the amino acid sequence PTTTK-COOH. which is the
natural GalNAc acceptor sequence in the human mucin MUC-1, is added as a fusion tag. The
fusion protein is then expressed in E. coli and purified. The peptide is then contacted with
recombinant human GalNAc-transferases T3 or T6 in the presence of UDP-GalNAc to
transfer a GalNAc residue onto the peptide in vitro.
This glycan chain on the peptide may then be further elongated using the methods
described in reference to the N-linked or O-linked glycans herein. Alternatively, the GalNAc
transferase reaction can be carried out in the presence of UDP-GalNAc to which PEG is
covalently substituted in the 0-3. 4. or 6 positions or the N-2 position. Glycoconjugation is
described in detail elswhere herein. Any antigenicity introduced into the peptide by the new
peptide sequence can be conveniently masked by PEGylation of the associated glycan. The
acceptor site fusion technique can be used to introduce not only a PEG moiety, but to
introduce other glycan and non-glycan moieties, including, but not limited to. toxins, anti-
infectives, cytotoxic agents, chelators for radionucleotides, and glycans with other
functionalities, such as tissue targeting.
Exemplary Embodiments
The remodeling of O-linked glycosylation is best illustrated with reference to Formula
2:
-
Formula 2 describes a glycan structure comprising a GalNAc which is covalently linked
preferably to a serine or threonine residue on a peptide backbone. While this structure is used
to illustrate the most common forms of O-linked glycans, it should not be construed to limit
the invention solely to these O-linked glycans. Other forms of O-linked glycans are
illustrated in Figure 12. Preferred expression systems useful in the present invention express
and secrete exogenous peptides having O-linked glycans comprising the GalNAc residue.
Using the remodeling methods of the invention, the glycan structures on these peptides can
be conveniently remodeled to generate any desired glycan structure. One of skill in the art
will appreciate that O-linked glycans can be remodeled using the same principles, enzymes
and reaction conditions as those available in the art once armed with the present disclosure.
Exemplary reaction conditions are found throughout the Examples.
In preferred embodiments, the glycan structures are remodeled so that the structure
described in Formula 2 has specific moieties. The structure of the glycan may be chosen to
enhance the biological activity of the peptide, confer upon the peptide a new biological
activity, remove or alter a biological activity of peptide, or better approximate the
glycosylation pattern of the native peptide, among others.
In the first preferred embodiment, the peptide O-linked glycans are remodeled to
better approximate the glycosylation pattern of native human proteins. In this embodiment,
the glycan structure described in Formula 2 is remodeled to have the following moieties:
X2 is |-SA; or |-SA-SA;
f and n = 0 or 1;
X10 is SA;
m = 0.
This embodiment is particularly advantageous for human peptides expressed in heterologous
cellular expression systems. By remodeling the O-linked glycan structures to have this
configuration, the peptide can be rendered less immunogenic in a human patient and/or more
stable.
In the another preferred embodiment, the peptide O-linked glycans are remodeled to
display a sialylated Lewis X antigen. In this embodiment, the glyean structure described in
Formula 2 is remodeled to have the following moieties:
X2is|-SA:
X10 is Fuc or |-GlcNAc(Fuc)-Gal-SA;
fand n I;
m = 0.
This embodiment is particularly advantageous when the peptide which is being remodeled is
most effective when targeted to a selectin molecule and cells exhibiting the same.
In a yet another preferred embodiment, the peptide O-linked glycans are remodeled to
contain a conjugated moiety. The conjugated moiety may be a PEG molecule, another
peptide, a small molecule such as a drug, among others. In this embodiment, the glyean
structure described in Formula 2 is remodeled to have the following moieties:
X2 is |-SA-R;
f - 1;
n and m - 0:
where R is the conjugate group.
This embodiment is useful for conjugating the peptide to PHG molecules that will slow the
clearance of the peptide from the patient's bloodstream, to peptides that will target both
peptides to a specific tissue or cell or to another peptide of complementary therapeutic use.
It will be clear to one of skill in the art that the invention is not limited to the preferred
glyean molecules described above. The preferred embodiments are only a few of the many
useful glyean molecules that can be made using the remodeling methods of the invention.
Those skilled in the art will know how to design other useful glycans once armed with the
present invention.
In the first exemplary embodiment, the peptide is expressed in a CHO (Chinese
hamster cell line) according to methods well known in the art. When a peptide with O-linked
glyean consensus sites is expressed and secreted from CHO cells, the majority of the O-
linked glycans will often have the structure, in the terms of Formula 2.
X2-|-SA:
f= 1;
m and n = 0.
Therefore, most of the glycans in CHO cells do not require remodeling in order to be
acceptable for use in a human patient. In an exemplary embodiment, the O-linked glycans of
a peptide expressed and secreted from a CHO cell are remodeled to contain a sialylated
Lewis X structure by contacting the glycans with a glycosyltransferasc specific for the
GalNAc acceptor moiety and the fucose donor moiety in the presence of nucleotide-fucose.
This process is illustrated on N-Iinked glycans in Figure 11 and Example 39.
In other exemplary embodiments, the peptide is expressed in insect cells such as st9
according to methods well known in the art. When a peptide having O-linked glycan
consensus sites is expressed and secreted from most sf9 cells, the majority of the O-linked
glycans have the structure, in the terms of Formula 2:
X2 = H;
f =-0 or I;
n and m = 0.
See, for example, Marchal et al., (2001, Biol. Chem. 382:151-159). In one exemplary
embodiment, the O-linked glycan on a peptide expressed in an insect cell is remodeled to a
humanized glycan by contacting the glycans with a glycosyltransferase specific for a GalNAc
acceptor molecule and a galactose donor molecule in the presence of nucleotide-Gal; and
then contacting the glycans with a glycosyltransferase specific for a Gal acceptor molecule
and a SA donor molecule in the presence of nucleotide-SA. In another exemplary
embodiment, the O-linked glycans are remodeled further from the humanized form to the
sialylated Lewis X form by further contacting the glycans with a glycosyltransferase specific
for a GalNAc acceptor molecule and a fucose donor molecule in the presence of nucleotide-
fucose.
In yet another exemplary embodiment, the peptide is expressed in fungal cells, in
particular S. cerevisiae cells, according to methods well known in the art. When a peptide
with O-linked glycans consensus sites is expressed and secreted from S. cerevisiae cells, the
majority of the O-linked glycans have the structure:
| - AA-Man- Man1-2.
See Gemmill and Trimble (1999, Biochim. Biophys. Acta 1426:227-237). In order to
remodel these O-linked glycans for use in human, it is preferable that the glycan be cleaved at
the amino acid level and rebuilt from there.
In an exemplary embodiment, the glycan is the O-linked glycan on a peptide
expressed in a fungal cell and is remodeled to a humanized glycan by contacting the glycan
with an endoglycosylase specific for an amino acid - GalNAc bond; and then contacting the
glycan with a glycosyltransferase specific for a O-linked consensus site and a GalNAc donor
molecule in the presence of nucleotide-GalNAc; contacting the glycan with a
glycosyltransferase specific for a GalNAc acceptor molecule and a galactose donor molecule
in the presence of nucleotide-Gal; and then contacting the glycans with a glycosyltransferase
specific for a Gal acceptor molecule and a SA donor molecule in the presence of nucleotide-
SA.
Alternately, in another exemplary embodiment, the glycan is the O-linked glycan on a
peptide expressed in a fungal cell and is remodeled to a humanized glycan by contacting the
glycan with an protein O-mannose p-l,2-N-acetylglucosaminyltransferase (POMGnTI) in the
presence of GlcNAc-nucleotide; then contacting the glycan with an galactosyltransferase in
the presence of nucleotide-Gal; and then contracting the glycan with an sialyltransferase in
the presence of nucleotide-SA.
In another exemplary embodiment, the peptide is expressed in bacterial cells, in
particular E. coli cells, according to methods well known in the art. When a peptide with an
O-linked glycan consensus site is expressed in E. coli cells, the O-linked consensus site will
not be glycosylated. In this case, the desired glycan molecule must be built out from the
peptide backbone in a manner similar to that describe for S. cerevisiae expression above.
Further, when a peptide having an O-linked glycan is expressed in a eukaryotic cell without
the proper leader sequences to direct the nascent peptide to the golgi apparatus, the mature
peptide is likely not to be glycosylated. In this case as well, an O-linked glycosyl structure
may be added to the peptide by building out the glycan directly from the peptide O-linked
consensus site. Further, when a protein is chemically modified with a sugar moiety, it can
also be remodeled as described herein.
These examples are meant to illustrate the invention, and not to limit it in any way.
One of skill in the art will appreciate that the steps taken in each example may in some
circumstances be performed in a different order to achieve the same result. One of skill in the
art will also understand that a different set of steps may also produce the same resulting
glycan. Futher, the preferred remodeled glycan is by no means specific to the expression
system that the peptide is expressed in. The remodeled glycans are only illustrative and one
of skill in the art will know how to take the principles from these examples and apply them :o
peptides produced in different expression systems to generate glycans not specifically
described herein.
C. Glycoconjugation, in general
The invention provides methods of preparing a conjugate of a glycosylated or an
unglycosylated peptide. The conjugates of the invention are formed between peptides and
diverse species such as water-soluble polymers, therapeutic moieties, diagnostic moieties,
targeting moieties and the like. Also provided are conjugates that include two or more
peptides linked together through a linker arm, i.e., multifunctional conjugates. The multi-
functional conjugates of the invention can include two or more copies of the same peptide or
a collection of diverse peptides with different structures, and/or properties.
The conjugates of the invention are formed by the enzymatic attachment of a
modified sugar to the glycosylated or unglycosylated peptide. The modified sugar, when
interposed between the peptide and the modifying group on the sugar becomes what is
referred to herein as "an intact glycosyl linking group." Using the exquisite selectivity of
enzymes, such as glycosyltransferases, the present method provides peptides that bear a
desired group at one or more specific locations. Thus, according to the present invention, a
modified sugar is attached directly to a selected locus on the peptide chain or, alternatively,
the modified sugar is appended onto a carbohydrate moiety of a peptide. Peptides in which
modified sugars are linked to both a peptide carbohydrate and directly to an amino acid
residue of the peptide backbone are also within the scope of the present invention.
In contrast to known chemical and enzymatic peptide elaboration strategies, the
methods of the invention make it possible to assemble peptides and glycopeptides that have a
substantially homogeneous derivatization pattern; the enzymes used in the invention are
generally selective for a particular amino acid residue or combination of amino acid residues
of the peptide or particular glycan structure. The methods are also practical for large-scale
production of modified peptides and glycopeptides. Thus, the methods of the invention
provide a practical means for large-scale preparation of peptides having preselected
substantially uniform derivatization patterns. The methods are particularly well suited for
modification of therapeutic peptides, including but not limited to, peptides that are
incompletely glycosylated during production in cell culture cells (e.g.. mammalian cells,
insect cells, plant cells, fungal cells, yeast cells, or prokaryotic cells) or transgenic plants or
animals.
The methods of the invention also provide conjugates of glycosylated and
unglycosylated peptides with increased therapeutic half-life due to, for example, reduced
clearance rate, or reduced rate of uptake by the immune or reticuloendothelial system (RES).
Moreover, the methods of the invention provide a means for masking antigenic determinants
on peptides, thus reducing or eliminating a host immune response against the peptide.
Selective attachment of targeting agents can also be used to target a peptide to a particular
tissue or cell surface receptor that is specific for the particular targeting agent. Moreover,
there is provided a class of peptides that are specifically modified with a therapeutic moiety.
1. The Conjugates
In a first aspect, the present invention provides a conjugate between a peptide and a
selected moiety. The link between the peptide and the selected moiety includes an intact
glycosyl linking group interposed between the peptide and the selected moiety. As discussed
herein, the selected moiety is essentially any species that can be attached to a saccharide unit,
resulting in a "modified sugar' that is recognized by an appropriate transferase enzyme,
which appends the modified sugar onto the peptide. The saccharide component of the
modified sugar, when interposed between the peptide and a selected moiety, becomes an
"intact glycosyl linking group." The glycosyl linking group is formed from any mono- or
oligo-saccharide that, after modification with a selected moiety, is a substrate for an
appropriate transferase.
The conjugates of the invention will typically correspond to the general structure:
in which the symbols a, b, e, d and s represent a positive, non-zero integer; and I is
either 0 or a positive integer. The "agent" is a therapeutic agent, a bioactive agent, a
delectable label, water-soluble moiety or the like. The "agent" can be a peptide, e.g..
enzyme, antibody, antigen, etc. The linker can be any of a wide array of linking groups.
infra. Alternatively, the linker may be a single bond or a "zero order linker." The identity of
the peptide is without limitation. Exemplary peptides are provided in Figure 28.
In an exemplary embodiment, the selected moiety is a water-soluble polymer. The
water-soluble polymer is covalently attached to the peptide via an intact glycosyl linking
group. The glycosyl linking group is covalently attached to either an amino acid residue or a
glycosyl residue of the peptide. Alternatively, the glycosyl linking group is attached to one
or more glycosyl units of a glycopeptide. The invention also provides conjugates in which
the glycosyl linking group is attached to both an amino acid residue and a glycosyl residue.
In addition to providing conjugates that are formed through an enzymatically added
intact glycosyl linking group, the present invention provides conjugates that are highly
homogenous in their substitution patterns. Using the methods of the invention, it is possible
to form peptide conjugates in which essentially all of the modified sugar moieties across a
population of conjugates of the invention are attached to multiple copies of a structurally
identical amino acid or glycosyl residue. Thus, in a second aspect, the invention provides a
peptide conjugate having a population of water-soluble polymer moieties, which are
covalently linked to the peptide through an intact glycosyl linking group. In a preferred
conjugate of the invention, essentially each member of the population is linked via the
glycosyl linking group to a glycosyl residue of the peptide, and each glycosyl residue of the
peptide to which the glycosyl linking group is attached has the same structure.
Also provided is a peptide conjugate having a population of water-soluble polymer
moieties covalently linked thereto through an intact glycosyl linking group. In a preferred
embodiment, essentially every member of the population of water soluble polymer moieties
is linked to an amino acid residue of the peptide via an intact glycosyl linking group, and
each amino acid residue having an intact glycosyl linking group attached thereto has the same
structure.
The present invention also provides conjugates analogous to those described above in
which the peptide is conjugated to a therapeutic moiety, diagnostic moiety, targeting moiety.
toxin moiety or the like via an intact glycosyl linking group. Each of the above-recited
moieties can be a small molecule, natural polymer (e.g., peptide) or synthetic polymer.
In an exemplary embodiment, interleukin-2 (IL-2) is conjugated to transferrin via a
Afunctional linker that includes an intact glycosyl linking group at each terminus of the PEG
moiety (Scheme 1). For example, one terminus of the PEG linker is functionalized with an
intact sialic acid linker that is attached to transferrin and the other is functionalized with an
intact GalNAc linker that is attached to IL-2.
In another exemplary embodiment, EPO is conjugated to transferrin. In another
exemplary embodiment, EPO is conjugated to glial derived neurotropic growth factor
(GDNF). In these embodiments, each conjugation is accomplished via a bifunctional linker
that includes an intact glycosyl linking group at each terminus of the PEG moiety, as
aforementioned. Transferrin transfers the protein across the blood brain barrier.
As set forth in the Figures appended hereto, the conjugates of the invention can
include intact glycosyl linking groups that are mono- or multi-valent (e.g.. antennary
structures), see, Figures 14-22. The conjugates of the invention also include glycosyl linking
groups that are O-linked glycans originating from serine or threonine (Figure 11). Thus,
conjugates of the invention include both species in which a selected moiety is attached to a
peptide via a monovalent glycosyl linking group. Also included within the invention are
conjugates in which more than one selected moiety is attached to a peptide via a multivalent
linking group. One or more proteins can be conjugated together to take advantage of their
biophysical and biological properties.
In a still further embodiment, the invention provides conjugates that localize
selectively in a particular tissue due to the presence of a targeting agent as a component of the
conjugate. In an exemplary embodiment, the targeting agent is a protein. Exemplary
proteins include transferrin (brain, blood pool), human serum (HS)-glycoprotein (bone, brain,
blood pool), antibodies (brain, tissue with antibody-specific antigen, blood pool), coagulation
Factors V-XII (damaged tissue, clots, cancer, blood pool), serum proteins, e.g., a-acid
glycoprotein, fetuin. a-fetal protein (brain, blood pool). (32-glycoprotein (liver,
atherosclerosis plaques, brain, blood pool), G-CSF, GM-CSF, M-CSF, and EPO (immune
stimulation, cancers, blood pool, red blood cell overproduction, neuroprotection), and
albumin (increase in half-life).
In addition to the conjugates discussed above, the present invention provides methods
for preparing these and other conjugates. Thus, in a further aspect, the invention provides a
method of forming a covalent conjugate between a selected moiety and a peptide.
Additionally, the invention provides methods for targeting conjugates of the invention to a
particular tissue or region of the body.
In exemplary embodiments, the conjugate is formed between a water-soluble
polymer, a therapeutic moiety, targeting moiety or a biomolecule, and a glycosylated or non-
glycosylated peptide. The polymer, therapeutic moiety or biomolecule is conjugated to the
peptide via an intact glycosyl linking group, which is interposed between, and covalently
linked to both the peptide and the modifying group (e.g., water-soluble polymer). The
method includes contacting the peptide with a mixture containing a modified sugar and a
glycosyltransferase for which the modified sugar is a substrate. The reaction is conducted
under conditions sufficient to form a covalent bond between the modified sugar and the
peptide. The sugar moiety of the modified sugar is preferably selected from nucleotide
sugars, activated sugars and sugars, which are neither nucleotides nor activated.
In one embodiment, the invention provides a method for linking two or more peptides
through a linking group. The linking group is of any useful structure and may be selected
from straight-chain and branched chain structures. Preferably, each terminus of the linker,
which is attached to a peptide, includes a modified sugar (i.e., a nascent intact glycosyl
linking group).
In an exemplary method of the invention, two peptides are linked together via a linker
moiety that includes a PEG linker. The construct conforms to the general structure set forth
in the cartoon above. As described herein, the construct of the invention includes two intact
glycosyl linking groups (i.e., s +1 = 1). The focus on a PEG linker that includes two glycosyl
groups is for purposes of clarity and should not be interpreted as limiting the identity of linker
arms of use in this embodiment of the invention.
Thus, a PEG moiety is functionalized at a first terminus with a first glycosyl unit and
at a second terminus with a second glycosyl unit. The first and second glycosyl units are
preferably substrates for different transferases, allowing orthogonal attachment of the first
and second peptides to the first and second glycosyl units, respectively. In practice, the
(glycosyl)1-PEG-(glycosyl)2 linker is contacted with the first peptide and a first transferase
for which the first glycosyl unit is a substrate, thereby forming
(peptide)'-(glycosyl)'-PEG-(glycosyl)2. The first transferase and/or unreacted peptide is then
optionally removed from the reaction mixture. The second peptide and a second transferase
for which the second glycosyl unit is a substrate are added to the
(peptide)1-(glycosyl)1-PBG-(glycosyl)2 conjugate, forming
(peptide)1-(gIycosyl)1-PEG-(glycosyl)2-(peptide)2 . Those of skill in the art will appreciate
that the method outlined above is also applicable to forming conjugates between more than
two peptides by, for example, the use of a branched PEG, dendrimer, poly(amino acid),
polysaccharide or the like.
As noted previously, in an exemplary embodiment, interleukin-2 (IL-2) is conjugated
to transferrin via a bifunctional linker that includes an intact glycosyl linking group at each
terminus of the PEG moiety (Scheme 1). The IL-2 conjugate has an in vivo half-life that is
increased over that of IL-2 alone by virtue of the greater molecular size of the conjugate.
Moreover, the conjugation of IL-2 to transferrin serves to selectively target the conjugate to
the brain. For example, one terminus of the PEG linker is functionalized with a CMP-sialic
acid and the other is functionalized with an UDP-GalNAc. The linker is combined with IL-2
in the presence of a GalNAc transferase, resulting in the attachment of the GalNAc of the
linker arm to a serine and/or threonine residue on the IL-2.
In another exemplary embodiment, transferrin is conjugated to a nucleic acid for use
The processes described above can be carried through as many cycles as desired, and
is not limited to forming a conjugate between two peptides with a single linker. Moreover,
those of skill in the art will appreciate that the reactions functionalizing the intact glycosyl
linking groups at the termini of the PEG (or other) linker with the peptide can occur
simultaneously in the same reaction vessel, or they can be carried out in a step-wise fashion.
When the reactions are carried out in a step-wise manner, the conjugate produced at each step
is optionally purified from one or more reaction components (e.g., enzymes, peptides).
A still further exemplary embodiment is set forth in Scheme 2. Scheme 2 shows a
method of preparing a conjugate that targets a selected protein, e.g., EPO. to bone and
increases the circulatory half-life of the selected protein.
Scheme 2

The use of reactive derivatives of PEG (or other linkers) to attach one or more peptide
moieties to the linker is within the scope of the present invention. The invention is not
limited by the identity of the reactive PEG analogue. Many activated derivatives of
poly(ethylene glycol) are available commercially and in the literature. It is well within the
abilities of one of skill to choose, and synthesize if necessary, an appropriate activated PEG
derivative with which to prepare a substrate useful in the present invention. See, Abuchowski
el al. Cancer Biochem. Biophys., 7: 175-186 (1984); Abuchowski et al,./. Biol. Chem., 252:
3582-3586 (1977); Jackson et al, Anal. Biochem., 165: 114-127 (1987); Koide et al,
Biochem Biophys. Res. Commun., 111: 659-667 (1983)), tresylate (Nilsson et al. Methods
Enzymol, 104: 56-69 (1984); Delgado et al, Biotechnol. Appl. Biochem., 12: 119-128
(1990)); N-hydroxysuccinimide derived active esters (Buckmann et al, Makromol. Chem.,
182: 1379-1384(1981); Joppich / al, Makromol. Chem., 180: 1381-1384(1979);
Abuchowski et al. Cancer Biochem. Biophys., 7: 175-186 (1984); Katreet al. Proc. Natl.
Acad. Sci. U.S.A.,84: 1487-1491 (1987); Kitamura et al, Cancer Res., 51: 4310-4315

(1991); Boccu et al. Z Naturforsch., 38C: 94-99 (1983), carbonates (Zalipsky et al.
POLY(ETHYLENE-GLYCOL) CHEMISTRY: BlOI LCEN1CAL AND BIOMLUlCAL APPLICATIONS.
Harris. Ed., Plenum Press, New York. 1992, pp. 347-370; Zalipsky et al, Biotechnol. Appl.
Biochem., 15: 100-1 14(1992); Veronese et al, Appl. Biochem. Biotech., 11: 141-152
(1985)). imidazolyl formates (Beauchamp et al.,Anal. Biochem., 131: 25-33 (1983): Berger
et al. Blood, 71: 1641 -1647 (1988)), 4-dithiopyridines (Woghiren et al, Bioconjugate
Chem., 4: 314-318 (1993)), isocyanates (Byun et al.,ASAIO Journal, M649-M-653 (1992))
and epoxides (U.S. Pat. No. 4,806,595, issued to Noishiki et al, (1989). Other linking groups
include the urethane linkage between amino groups and activated PEG. See, Veronese, et al,
Appl. Biochem. Biotechnol., 11: 141-152 (1985).
In another exemplary embodiment in which a reactive PEG derivative is utilized, the
invention provides a method for extending the blood-circulation half-life of a selected
peptide, in essence targeting the peptide to the blood pool, by conjugating the peptide to a
synthetic or natural polymer of a size sufficient to retard the filtration of the protein by the
glomerulus (e.g., albumin). This embodiment of the invention is illustrated in Scheme 3 in
which erythropoietin (EPO) is conjugated to albumin via a PEG linker using a combination of
chemical and enzymatic modification.
Scheme 3

Thus, as shown in Scheme 3, an amino acid residue of albumin is modified with a
reactive PEG derivative, such as X-PEG-(CMP-sialic acid), in which X is an activating group
(e.g., active ester, isothiocyanate, etc). The PEG derivative and EPO are combined and
contacted with a transferase for which CMP-sialic acid is a substrate. In a further illustrative
embodiment, an e-amine of lysine is reacted with the N-hydroxysuccinimide ester of the

PEG-linker to form the albumin conjugate. The CMP-sialic acid of the linker is
enzymatically conjugated to an appropriate residue on EPO, e.g., Gal. thereby forming the
conjugate. Those of skill will appreciate that the above-described method is not limited to the
reaction partners set forth. Moreover, the method can be practiced to form conjugates that
include more than two protein moieties by. for example, utilizing a branched linker having
more than two termini.
2. Modified Sugars
Modified glycosyl donor species ("modified sugars"') are preferably selected from
modified sugar nucleotides, activated modified sugars and modified sugars that are simple
saccharides that are neither nucleotides nor activated. Any desired carbohydrate structure can
be added to a peptide using the methods of the invention. Typically, the structure will be a
monosaccharide, but the present invention is not limited to the use of modified
monosaccharide sugars; oligosaccharides and polysaccharides are useful as well.
The modifying group is attached to a sugar moiety by enzymatic means, chemical
means or a combination thereof, thereby producing a modified sugar. The sugars are
substituted at any position that allows for the attachment of the modifying moiety, yet which
still allows the sugar to function as a substrate for the enzyme used to ligate the modified
sugar to the peptide. In a preferred embodiment, when sialic acid is the sugar, the sialic acid
is substituted with the modifying group at either the 9-position on the pyruvyl side chain or at
the 5-position on the amine moiety that is normally acetylated in sialic acid.
In certain embodiments of the present invention, a modified sugar nucleotide is
utilized to add the modified sugar to the peptide. Exemplary sugar nucleotides that are used
in the present invention in their modified form include nucleotide mono-, di- or triphosphates
or analogs thereof. In a preferred embodiment, the modified sugar nucleotide is selected
from a UDP-glycoside, CMP-glycoside, or a GDP-glycoside. Even more preferably, the
modified sugar nucleotide is selected from an UDP-galactose, UDP-galactosamine, UDP-
glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid, or CMP-NeuAc.
N-acetylamine derivatives of the sugar nucleotides are also of use in the method of the
invention.
The invention also provides methods for synthesizing a modified peptide using a
modified sugar, e.g., modified-galactose, -fucose, and -sialic acid. When a modified sialic
acid is used, either a sialyltransferase or a trans-sialidase (for a2,3-Iinked sialic acid only) can
be used in these methods.
In other embodiments, the modified sugar is an activated sugar. Activated modified
sugars, which are useful in the present invention are typically glycosides which have been
synthetically altered to include an activated leaving group. As used herein, the term
"activated leaving group" refers to those moieties, which are easily displaced in enzyme-
regulated nucleophilic substitution reactions. Many activated sugars are known in the art.
See, for example, Vocadlo et al., In Carbohydrate Chemistry and Biology, Vol. 2. Ernst
et al. Ed., Wiley-VCH Verlag: Weinheim, Germany, 2000; Kodama el al.. Tetrahedron Lett.
34: 6419 (1993); Lougheed, et al., J. Biol. Chem. 274: 37717 (1999)).
Examples of activating groups (leaving groups) include fluoro, chloro. bromo,
tosylate ester, mesylate ester, triflate ester and the like. Preferred activated leaving groups,
for use in the present invention, are those that do not significantly sterically encumber the
enzymatic transfer of the glycoside to the acceptor. Accordingly, preferred embodiments of
activated glycoside derivatives include glycosyl fluorides and glycosyl mesylates, with
glycosyl fluorides being particularly preferred. Among the glycosyl fluorides, a-galactosyl
fluoride, a-mannosyl fluoride, a-glucosyl fluoride, a-fucosyl fluoride, a-xylosyl fluoride, a-
sialyl fluoride, a-N-acetylglucosaminyl fluoride, a-N-acetylgalactosaminyl fluoride, ß-
galactosyl fluoride, ß-mannosyl fluoride, ß-glucosyl fluoride, ß-fucosyl fluoride. P-xylosyl
fluoride, ß-sialyl fluoride, ß-N-acetylglucosaminyl fluoride and ß-"N-acetylgalactosaminyl
fluoride are most preferred.
By way of illustration, glycosyl fluorides can be prepared from the free sugar by first
acetylating the sugar and then treating it with HF/pyridine. This generates the
thermodynamically most stable anomer of the protected (acetylated) glycosyl fluoride (i.e.,
the a-glycosyl fluoride). If the less stable anomer (i.e., the p-glycosyl fluoride) is desired, it
can be prepared by converting the peracetylated sugar with HBr/HOAc or with HC1 to
generate the anomeric bromide or chloride. This intermediate is reacted with a fluoride salt
such as silver fluoride to generate the glycosyl fluoride. Acetylated glycosyl fluorides may
be deprotected by reaction with mild (catalytic) base in methanol (e.g. NaOMe/MeOH). In
addition, many glycosyl fluorides are commercially available.
Other activated glycosyl derivatives can be prepared using conventional methods
known to those of skill in the art. For example, glycosyl mesylates can be prepared by
treatment of the fully benzylated hemiacetal form of the sugar with mesyl chloride, followed
by catalytic hydrogenation to remove the benzyl groups.
In a further exemplary embodiment, the modified sugar is an oligosaccharide having
an antennary structure. In a preferred embodiment, one or more of the termini of the
antennae bear the modifying moiety. When more than one modifying moiety is attached to
an oligosaccharide having an antennary structure, the oligosaccharide is useful to "amplify"
the modifying moiety; each oligosaccharide unit conjugated to the peptide attaches multiple
copies of the modifying group to the peptide. The general structure of a typical chelate of the
invention as set forth in the drawing above, encompasses multivalent species resulting from
preparing a conjugate of the invention utilizing an antennary structure. Many antennary
saccharide structures are known in the art, and the present method can be practiced with them
without limitation.
Exemplary modifying groups are discussed below. The modifying groups can be
selected for one or more desirable property. Exemplary properties include, but are not
limited to, enhanced pharmacokinetics, enhanced pharmacodynamics, improved
biodistribution, providing a polyvalent species, improved water solubility, enhanced or
diminished lipophilicity. and tissue targeting.
P. Peptide Conjugates
a) Water-Soluble Polymers
The hydrophilicity of a selected peptide is enhanced by conjugation with polar
molecules such as amine-, ester-, hydroxyl- and polyhydroxyl-containing molecules.
Representative examples include, but are not limited to, polylysine, polyethyleneimine.
poly(ethylene glycol) and poly(propyleneglycol). Preferred water-soluble polymers are
essentially non-fluorescent, or emit such a minimal amount of fluorescence that they are
inappropriate for use as a fluorescent marker in an assay. Polymers that are not naturally
occurring sugars may be used. In addition, the use of an otherwise naturally occurring sugar
that is modified by covalent attachment of another entity (e.g., poly(ethylene glycol),
poly(propylene glycol), poly(aspartate), biomolecule, therapeutic moiety, diagnostic moiety.
etc.) is also contemplated. In another exemplary embodiment, a therapeutic sugar moiety is
conjugated to a linker arm and the sugar-linker arm is subsequently conjugated to a peptide
via a method of the invention.
Methods and chemistry for activation of water-soluble polymers and saccharides as
well as methods for conjugating saccharides and polymers to various species arc described in
the literature. Commonly used methods for activation of polymers include activation of
functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides,
epichlorohydrin, divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc. {see. R.
F. Taylor, (1991), Protein Immobilisation. Fundamentals and Applications, Marcel
Dekker, N.Y.; S. S. Wong, (1992), Chemistry ok Protein Conjugation and
Crosslinking, CRC Press, Boca Raton; G. T. Hermanson et al., (1993), IMMOBILIZED
Affinity Ligand Techniques, Academic Press, N.Y.; Dunn, R.L., et al., Eds. Polymeric-
Drugs And Drug Delivery Systems, ACS Symposium Series Vol. 469, American
Chemical Society, Washington, D.C. 1991).
Routes for preparing reactive PEG molecules and forming conjugates using the
reactive molecules are known in the art. For example. U.S. Patent No. 5.672.662 discloses a
water soluble and isolatable conjugate of an active ester of a polymer acid selected from
linear or branched poly(alkylene oxides), poly(oxyethylated polyols), poly(olefinic alcohols),
and poly(acrylomorpholine), wherein the polymer has about 44 or more recurring units.
U.S. Patent No. 6,376.604 sets forth a method for preparing a water-soluble 1-
benzotriazolylcarbonate ester of a water-soluble and non-peptidic polymer by reacting a
terminal hydroxyl of the polymer with di(l-benzotriazoyl)carbonate in an organic solvent.
The active ester is used to form conjugates with a biologically active agent such as a protein
or peptide.
WO 99/45964 describes a conjugate comprising a biologically active agent and an
activated water soluble polymer comprising a polymer backbone having at least one terminus
linked to the polymer backbone through a stable linkage, wherein at least one terminus
comprises a branching moiety having proximal reactive groups linked to the branching
moiety, in which the biologically active agent is linked to at least one of the proximal reactive
groups. Other branched poly(ethylene glycols) are described in WO 96/21469, U.S. Patent
No. 5,932,462 describes a conjugate formed with a branched PEG molecule that includes a
branched terminus that includes reactive functional groups. The free reactive groups arc
available to react with a biologically active species, such as a protein or peptide, forming
conjugates between the poly(ethylene glycol) and the biologically active species. U.S. Patent
No. 5,446,090 describes a Afunctional PEG linker and its use in forming conjugates having a
peptide at each of the PEG linker termini.
Conjugates that include degradable PEG linkages are described in WO 99/34833; and
WO 99/14259, as well as in U.S. Patent No. 6,348,558. Such degradable linkages are
applicable in the present invention.
Although both reactive PEG derivatives and conjugates formed using the derivatives
are known in the art, until the present invention, it was not recognized that a conjugate could
be formed between PEG (or other polymer) and another species, such as a peptide or
glycopeptide, through an intact glycosyl linking group.
Many water-soluble polymers are known to those of skill in the art and are useful in
practicing the present invention. The term water-soluble polymer encompasses species such
as saccharides (e.g.. dextran, amylosc, hyaluronic acid, poly (sialic acid), heparans, heparins,
etc.); poly (amino acids), e.g., poly(glutamic acid); nucleic acids; synthetic polymers (e.g.,
poly(acrylic acid), poly(ethers), e.g., poly(ethylene glycol); peptides, proteins, and the like.
The present invention may be practiced with any water-soluble polymer with the sole
limitation that the polymer must include a point at which the remainder of the conjugate can
be attached.
Methods for activation of polymers can also be found in WO 94/17039, U.S. Pat. No.
5.324,844, WO 94/18247, WO 94/04193, U.S. Pat. No. 5,219,564, U.S. Pat. No. 5,122,614.
WO 90/13540, U.S. Pat. No. 5,281,698, and more WO 93/15189, and for conjugation
between activated polymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625).
hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No. 4,412,989),
ribonuclease and superoxide dismutase (Veronese at ai. App. Biochem. Biotech. 11; 141-45
(1985)).
Preferred water-soluble polymers are those in which a substantial proportion of the
polymer molecules in a sample of the polymer are of approximately the same molecular
weight; such polymers are "homodisperse."
The present invention is further illustrated by reference to a poly(ethylene glycol)
conjugate. Several reviews and monographs on the functionalization and conjugation of PHG
are available. See, for example, Harris, Macronol. Chem. Phys. C25: 325-373 (1985);
Scouten, Methods in Enzymology 135: 30-65 (1987); Wong el a!.. Enzyme Microb. Techno!.
14: 866-874 (1992): Dclgado el al., Critical Reviews in Therapeutic Drug Carrier Systems 9:
249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al.,
Pharmazie, 57:5-29 (2002).
Poly(ethylene glycol) molecules suitable for use in the invention include, but are not
limited to, those described by the following Formula 3:

R= H, alkyl, benzyl, aryl, acetal, OHC-, H2N-CH2CH2-, HS-CH2CH2-,

X., Y, W, U (independently selected) = O, S, NH, N-R';
R', R'" (independently selected) = alkyl, benzyl, aryl, alkyl aryl, pyridyl, substituted aryl,
arylalkyl, acylaryl;
n = 1 to 2000;
m. q, p (independently selected) = 0 to 20
o "- 0 to 20;
Z = HO, NH2, halogen, S-R"", activated esters,

-sugar-nucleotide, protein, imidazole, HOBT, tetrazole, halide; and
V = HO. NH2, halogen, S-R"' activated esters, activated amides, -sugar-nucleotide. protein.

In preferred embodiments, the poly(ethylene glycol) molecule is selected from the
following:

The poly(ethylene glycol) useful in forming the conjugate of the invention is either linear or
branched. Branched poly(ethylene glycol) molecules suitable for use in the invention
include, but are not limited to, those described by the following Formula:

R'. R", R'" (independently selected) = H, alkyl, benzyl, aryl, acetal, OHC-, H2N-CH2CH2-.
HS-CH2CH2-. -(CH2)qCY-Z, -sugar-nucleotide, protein, methyl, ethyl, heteroaryl,
acylalkyl, acylaryl, acylalkylaryl;
X.Y. W, A, B (independently selected) = O, S, NH, N-R', (CH2),;
n, p (independently selected) = 1 to 2000;
m, q, o (independently selected) = 0 to 20;
Z - HO, NH2, halogen. S-R"', activated esters,

-sugar-nucleotidc. protein;
V = HO, NH2, halogen, S-R'", activated esters, activated amides,
-sugar-nucleotide. protein.
The in vivo half-life, area under the curve, and/or residence time of therapeutic
peptides can also be enhanced with water-soluble polymers such as polyethylene glycol
(PEG) and polypropylene glycol (PPG). For example, chemical modification of proteins with
PEG (PEGylation) increases their molecular size and decreases their surface- and functional
group-accessibility, each of which are dependent on the size of the PEG attached to the
protein. This results in an improvement of plasma half-lives and in proteolytic-stabilily. and
a decrease in immunogenicity and hepatic uptake (Chaffee et ah ./. Clin. Invest. 89: 1643-
1651 (1992); Pyatak et ah Res. Commun. Chew. Pathol Pharmacol. 29: 113-127 (1980)).
PEGylation of interleukin-2 has been reported to increase its antitumor potency in vivo (Kalre
et al. Prot: Natl. -lead. Sci. USA. 84: 1487-1491 (1987)) and PEGylation of a F(ab')2 derived
from the monoclonal antibody A7 has improved its tumor localization (Kitamura et al.,
Biochem. Biophys. Res. Commun. 28: 1387-1394(1990)).
In one preferred embodiment, the in vivo half-life of a peptide derivatized with a
water-soluble polymer by a method of the invention is increased relevant to the in vivo half-
life of the non-derivatized peptide. In another preferred embodiment, the area under the
curve of a peptide derivatized with a water-soluble polymer using a method of the invention
is increased relevant to the area under the curve of the non-derivatized peptide. In another
preferred embodiment, the residence time of a peptide derivatized with a water-soluble
polymer using a method of the invention is increased relevant to the residence time of the
non-derivatized peptide. Techniques to determine the in vivo half-life, the area under the
curve and the residence time are well known in the art. Descriptions of such techniques can
be found in J.G. Wagner, 1993, Pharmacokinetics for the Pharmaceutical Scientist.
Technomic Publishing Company. Inc. Lancaster PA.
The increase in peptide in vivo half-life is best expressed as a range of percent
increase in this quantity. The lower end of the range of percent increase is about 40%, about
60%. about 80%. about 100%. about 150% or about 200%. The upper end of the range is
about 60%, about 80%. about 100%. about 150%. or more than about 250%.
In an exemplary embodiment, the present invention provides a PEGylated follicle
stimulating hormone (Examples 23 and 24). In a further exemplary embodiment, the
invention provides a PEGylated transferrin (Example 42).
Other exemplary water-soluble polymers of use in the invention include, but are not
limited to linear or branched poly(alkylene oxides), poly(oxyethylated polyols), poly(olefinic
alcohols), and poly(acrylomorpholine), dextran, starch, poly(amino acids), etc.
b) Water-insoluble polymers
The conjugates of the invention may also include one or more water-insoluble
polymers. This embodiment of the invention is illustrated by the use of the conjugate as a
vehicle with which to deliver a therapeutic peptide in a controlled manner. Polymeric drug
delivery systems are known in the art. See, for example, Dunn et al, Eds. Polymeric
Drugs And Drug Delivery Systems, ACS Symposium Series Vol. 469, American
Chemical Society, Washington, D.C. 1991. Those of skill in the art will appreciate that
substantially any known drug delivery system is applicable to the conjugates of the present
invention.
Representative water-insoluble polymers include, but are not limited to,
polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates, polyalkylenes,
polyacry lam ides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates.
polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides,
polysiloxanes, polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),
poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate),
poly(isodecyl methacrylate). poly(lauryl methacrylate). poly(phenyl methacrylate).
poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate). poly(octadecyl
acrylate) polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly
(ethylene terephthalate), poly(vinyl acetate), polyvinyl chloride, polystyrene, polyvinyl
pyrrolidone, pluronics and polyvinylphenol and copolymers thereof.
Synthetically modified natural polymers of use in conjugates of the invention include,
but are not limited to. alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, and nitrocelluloses. Particularly preferred members of the broad classes of
synthetically modified natural polymers include, but are not limited to, methyl cellulose,
ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl
methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose
acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt,
and polymers of acrylic and methacrylic esters and alginic acid.
These and the other polymers discussed herein can be readily obtained from
commercial sources such as Sigma Chemical Co. (St. Louis, MO.), Polysciences (Warrenton,
PA.), Aldrich (Milwaukee, Wl.), Fluka (Ronkonkoma. NY), and BioRad (Richmond, CA), or
else synthesized from monomers obtained from these suppliers using standard techniques.
Representative biodegradable polymers of use in the conjugates of the invention
include, but are not limited to, polylactides, polyglycolides and copolymers thereof,
poly(ethylene terephthalate). poly(butyric acid), poly(valeric acid), poly(lactide-co-
caprolactone), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends and
copolymers thereof. Of particular use are compositions that form gels, such as those
including collagen, pluronics and the like.
The polymers of use in the invention include "hybrid' polymers that include water-
insoluble materials having within at least a portion of their structure, a bioresorbable
molecule. An example of such a polymer is one that includes a water-insoluble copolymer,
which has a bioresorbable region, a hydrophilic region and a plurality of crosslinkable
functional groups per polymer chain.
For purposes of the present invention, "water-insoluble materials" includes materials
that are substantially insoluble in water or water-containing environments. Thus, although
certain regions or segments of the copolymer may be hydrophilic or even water-soluble, the
polymer molecule, as a whole, does not to any substantial measure dissolve in water.
For purposes of the present invention, the term "bioresorbable molecule" includes a
region that is capable of being metabolized or broken down and resorbed and/or eliminated
through normal excretory routes by the body. Such metabolites or break down products are
preferably substantially non-toxic to the body.
The bioresorbable region may be either hydrophobic or hydrophilic, so long as the
copolymer composition as a whole is not rendered water-soluble. Thus, the bioresorbable
region is selected based on the preference that the polymer, as a whole, remains water-
insoluble. Accordingly, the relative properties, i.e., the kinds of functional groups contained
by, and the relative proportions of the bioresorbable region, and the hydrophilic region are
selected to ensure that useful bioresorbable compositions remain water-insoluble.
Exemplary resorbable polymers include, for example, synthetically produced
resorbable block copolymers of poly(a-hydroxy-carboxylic acid)/poly(oxyalkylene, (see.
Cohn et al, U.S. Patent No. 4,826,945). These copolymers are not crosslinked and are water-
soluble so that the body can excrete the degraded block copolymer compositions. See.
Younes et al. J Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al. J Biomed.
Mater. Res. 22: 993-1009 (1988).
Presently preferred bioresorbable polymers include one or more components selected
from poly(esters). poly(hydroxy acids), poly(lactones), poly(amides), poly(ester-amides).
poly (amino acids), poly(anhydrides), poly(orthoesters), poly(carbonates).
poly(phosphazines). poly(phosphoesters), poly(thioesters), polysaccharides and mixtures
thereof. More preferably still, the biosresorbable polymer includes a poly(hydroxy) acid
component. Of the poly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproic acid,
polybutyric acid, polyvaleric acid and copolymers and mixtures thereof are preferred.
In addition to forming fragments that are absorbed in vivo ("'bioresorbed"), preferred
polymeric coatings for use in the methods of the invention can also form an excretable and/or
metabolizable fragment.
Higher order copolymers can also be used in the present invention. For example,
Casey et al, U.S. Patent No. 4,438,253, which issued on March 20, 1984, discloses tri-block
copolymers produced from the transesterification of poly(glycolic acid) and an hydroxyl-
ended poly(alkylene glycol). Such compositions are disclosed for use as resorbable
monofilament sutures. The flexibility of such compositions is controlled by the incorporation
of an aromatic orthocarbonate, such as tetra-p-tolyl orthocarbonate into the copolymer
structure.
Other coatings based on lactic and/or glycolic acids can also be utilized. For example,
Spinu, U.S. Patent No. 5,202,413, which issued on April 13, 1993. discloses biodegradable
multi-block copolymers having sequentially ordered blocks of polylactide and/or
polyglycolide produced by ring-opening polymerization of lactide and/or glycolide onto
either an oligomeric diol or a diamine residue followed by chain extension with a di-
functional compound, such as, a diisocyanate, diacylchloride or dichlorosilane.
Bioresorbable regions of coatings useful in the present invention can be designed to
be hydrolytically and/or enzymatically cleavable. For purposes of the present invention,
"hydrolytically cleavable" refers to the susceptibility of the copolymer, especially the
bioresorbable region, to hydrolysis in water or a water-containing environment. Similarly,
"enzymatically cleavable" as used herein refers to the susceptibility of the copolymer,
especially the bioresorbable region, to cleavage by endogenous or exogenous enzymes.
When placed within the body, the hydrophilic region can be processed into excretable
and/or metabolizable fragments. Thus, the hydrophilic region can include, for example,
polyethers, polyalkylene oxides, polyols. polyvinyl pyrrolidine), polyvinyl alcohol),
poly(alkyl oxazolincs). polysaccharides, carbohydrates, peptides, proteins and copolymers
and mixtures thereof. Furthermore, the hydrophilic region can also be. for example, a
poly(alkylene) oxide. Such poly(alkylene) oxides can include, for example, poly(ethylene)
oxide, poly(propylene) oxide and mixtures and copolymers thereof.
Polymers that are components of hydrogels are also useful in the present invention.
Hydrogels are polymeric materials that are capable of absorbing relatively large quantities of
water. Examples of hydrogel forming compounds include, but are not limited to, polyacrylic
acids, sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine, gelatin,
carrageenan and other polysaccharides, hydroxyethylenemethacrylic acid (HEMA), as well as
derivatives thereof, and the like. Hydrogels can be produced that are stable, biodegradable
and bioresorbable. Moreover, hydrogel compositions can include subunits that exhibit one or
more of these properties.
Bio-compatible hydrogel compositions whose integrity can be controlled through
crosslinking are known and are presently preferred for use in the methods of the invention.
For example, Hubbell et al., U.S. Patent Nos. 5,410,016, which issued on April 25, 1995 and
5,529,914, which issued on June 25, 1996, disclose water-soluble systems, which are
crosslinked block copolymers having a water-soluble central block segment sandwiched
between two hydrolytically labile extensions. Such copolymers are further end-capped with
photopolymerizable acrylate functionalities. When crosslinked, these systems become
hydrogels. The water soluble central block of such copolymers can include poly(ethylene
glycol); whereas, the hydrolytically labile extensions can be a poly(a-hydroxy acid), such as
polyglycolic acid or polylactic acid. See, Sawhney el al., Macromolecules 26: 581-587
(1993).
In another preferred embodiment, the gel is a thermoreversible gel. Thermoreversible
gels including components, such as pluronics, collagen, gelatin, hyalouronic acid,
polysaccharides, polyurethane hydrogel, polyurethane-urea hydrogel and combinations
thereof are presently preferred.
In yet another exemplary embodiment, the conjugate of the invention includes a
component of a liposome. Liposomes can be prepared according to methods known to those
skilled in the art, for example, as described in Eppstein el a/., U.S. Patent No. 4,522.811,
which issued on June 11, 1985. For example, liposome formulations may be prepared by
dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl
phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic
solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the
container. An aqueous solution of the active compound or its pharmaceutically acceptable
salt is then introduced into the container. The container is then swirled by hand to free lipid
material from the sides of the container and to disperse lipid aggregates, thereby forming the
liposomal suspension.
The above-recited microparticles and methods of preparing the microparticles arc
offered by way of example and they are. not intended to define the scope of microparticles of
use in the present invention. It will be apparent to those of skill in the art that an array of
microparticles, fabricated by different methods, are of use in the present invention.
c) Biomolecules
In another preferred embodiment, the modified sugar bears a biomolecule. In still
further preferred embodiments, the biomolecule is a functional protein, enzyme, antigen,
antibody, peptide, nucleic acid (e.g., single nucleotides or nucleosides, oligonucleotides,
polynucleotides and single- and higher-stranded nucleic acids), lectin, receptor or a
combination thereof.
Some preferred biomolecules are essentially non-fluorescent, or emit such a minimal
amount of fluorescence that they are inappropriate for use as a fluorescent marker in an assay.
Other biomolecules may be fluorescent. The use of an otherwise naturally occurring sugar
that is modified by covalent attachment of another entity (e.g., PEG, biomolecule, therapeutic
moiety, diagnostic moiety, etc.) is appropriate. In an exemplary embodiment, a sugar moiety,
which is a biomolecule, is conjugated to a linker arm and the sugar-linker arm cassette is
subsequently conjugated to a peptide via a method of the invention.
Biomolecules useful in practicing the present invention can be derived from any
source. The biomolecules can be isolated from natural sources or they can be produced by
synthetic methods. Peptides can be natural peptides or mutated peptides. Mutations can be
effected by chemical mutagenesis, site-directed mutagenesis or other means of inducing
mutations known to those of skill in the art. Peptides useful in practicing the instant
invention include, for example, enzymes, antigens, antibodies and receptors. Antibodies can
be either polyclonal or monoclonal; either intact or fragments. The peptides are optionally
the products of a program of directed evolution.
Both naturally derived and synthetic peptides and nucleic acids are of use in
conjunction with the present invention; these molecules can be attached to a sugar residue
component or a crosslinking agent by any available reactive group. For example, peptides
can be attached through a reactive amine, carboxyl, sulfhydryl, or hydroxyl group. The
reactive group can reside at a peptide terminus or at a site internal to the peptide chain.
Nucleic acids can be attached through a reactive group on a base (e.g., exocyclic amine) or an
available hydroxyl group on a sugar moiety (e.g., 3'- or 5'-hydroxyl). The peptide and
nucleic acid chains can be further derivatized at one or more sites to allow for the attachment
of appropriate reactive groups onto the chain. See, Chrisey el al. Nucleic Acids Res. 24:
3031-3039(1996).
In a further preferred embodiment, the biomolecule is selected to direct the peptide
modified by the methods of the invention to a specific tissue, thereby enhancing the delivery
of the peptide to that tissue relative to the amount of underivatized peptide that is delivered to
the tissue. In a still further preferred embodiment, the amount of derivatized peptide
delivered to a specific tissue within a selected time period is enhanced by derivatization by at
least about 20%, more preferably, at least about 40%, and more preferably still, at least about
100%. Presently, preferred biomolecules for targeting applications include antibodies,
hormones and ligands for cell-surface receptors. Exemplary targeting biomolecules include,
but are not limited to. an antibody specific for the transferrin receptor for delivery of the
molecule to the brain (Penichet et al., 1999, J. Immunol. 163:4421-4426; Pardridge, 2002.
Adv. Exp. Med. Biol. 513:397-430), a peptide that recognizes the vasculature of the prostate
(Arap et al., 2002. PNAS 99:1527-1531), and an antibody specific for lung caveolae
(Mcintosh et al. 2002, PNAS 99:1996-2001).
In a presently preferred embodiment, the modifying group is a protein. In an
exemplary embodiment, the protein is an interferon. The interferons are antiviral
glycoproteins that, in humans, are secreted by human primary fibroblasts after induction with
virus or double-stranded RNA. Interferons are of interest as therapeutics, e.g., antivirals and
treatment of multiple sclerosis. For references discussing interferon-p, see, e.g., Yu, el al, J
Neuroimmunol, 64(1):91-100 (1996); Schmidt, J., J. Neurosci. Res., 65(l):59-67 (2001);
Wendcr. et al, Folia Neuropathol, 39(2):91-93 (2001); Martin, el al., Springer Semin.
lmmunopathol, 18(1): 1-24 (1996); Takane, et al, J. Pharmacol. Exp. Ther., 294(2):746-752
(2000); Sburlati, et al., Biotechnol. Prog., 14:189-192 (1998); Dodd, et al, Biochimica et
Biophysica Acta, 787:183-187 (1984); Edelbaum, et al, J. Interferon Res., 12:449-453
(1992); Conradt, et al.. J. Biol. Chem., 262(30): 14600-14605 (1987); Civas, et al, Eur. J.
Biochem., 173:311-316 (1988); Demolder. et al., J. Biotechnol., 32:179-189 (1994); Sedmak.
et al., J. Interferon Res.. 9(Suppl 1):S61-S65 (1989); Kagawa, et al, J. Biol. Chem.,
263(33): 17508-17515 (1988); Hershenson, et al., U.S. Patent No. 4,894,330; Jayaram, et al.,
J. Interferon Res., 3(2): 177-180 (1983); Menge, et al.. Develop. Biol. Standard, 66:391-401
(1987); Vonk, et al.,J. Interferon Res., 3(2):169-175 (1983); and Adolf, et al., J. Interferon
Res., 10:255-267 (1990). For references relevant to interferon-a, see, Asano, et al., Eur. J.
Cancer, 27(SuppI 4):S21-S25 (1991); Nagy, et al, Anticancer Research, 8(3):467-470
(1988); Dron, et al, J. Biol. Regul. Homeost. Agents, 3(1): 13-19 (1989); Habib, el al, Am.
Surg., 67(3):257-260 (3/2001); and Sugyiama, et al, Eur. J. Biochem., 217:921-927 (1993).
In an exemplary interferon conjugate, interferon ß is conjugated to a second peptide
via a linker arm. The linker arm includes an intact glycosyl linking group through which it is
attached to the second peptide via a method of the invention. The linker arm also optionally
includes a second intact glycosyl linking group, through which it is attached to the interferon.
In another exemplary embodiment, the invention provides a conjugate of follicle
stimulating hormone (FSH). FSH is a glycoprotein hormone. See, for example, Saneyoshi,
et al, Biol. Reprod, 65:1686-1690 (2001); Hakola, et al, J. Endocrinol, 158:441-448
(1998): Stanton, el al, Mol Cell. Endocrinol, 125:133-141 (1996); Walton, et al, J. Clin.
Endocrinol Metab., 86(8):3675-3685 (08/2001); Ulloa-Aguirre, et al. Endocrine, 11(3):205-
215 (12/1999); Castro-Fernandez, et all, J. Clin. Endocrinol Matab., 85(12):4603-4610
(2,000); Prevost, Rebecca R., Pharmacotherapy, 18(5): 1001-1010 (1998); Linskens, et al,
The FASEB Journal, 13:639-645 (04/1999); Butnev, et al, Biol. Reprod, 58:458-469 (1998):
Muyan, et al. Mot. Endo., 12(5):766-772 (1998); Min, et al, Endo../., 43(5):585-593 (1996);
Boime. et al Recent Progress in Hormone Research, 34:271 -289 (1999); and Rafferty, et al,
J. Endo.. 145:527-533 (1995). The ESH conjugate can be formed in a manner similar to thai
described for interferon.
In yet another exemplary embodiment, the conjugate includes erythropoietin (EPO).
EPO is known to mediate response to hypoxia and to stimulate the production of red blood
cells. For pertinent references, see, Cerami, et al, Seminars in Oncology, 28(2)(Suppl 8):66-
70 (04/2001). An exemplary EPO conjugate is formed analogously to the conjugate of
interferon.
In a further exemplary embodiment, the invention provides a conjugate of human
granulocyte colony stimulating factor (G-CSF). G-CSF is a glycoprotein that stimulates
proliferation, differentiation and activation of neutropoietic progenitor cells into functionally
mature neutrophils. Injected G-CSF is known to be rapidly cleared from the body. See, for
example, Nohynek, et al. Cancer Chemother. Pharmacol.. 39:259-266 (1997): Lord, et al.
Clinical Cancer Research, 7(7):2085-2090 (07/2001); Rotondaro, et al, Molecular
Biotechnology, 11(2): 117-128 (1999); and Bonig, et al, Bone Marrow Transplantation,
28:259-264 (2001). An exemplary conjugate of G-CSF is prepared as discussed above for
the conjugate of the interferons. One of skill in the art will appreciate that many other
proteins may be conjugated to interferon using the methods and compositions of the
invention, including but not limited to, the peptides listed in Tables 7 and 8 (presented
elsewhere herein) and Figure 28, and in Figures 29-57, where individual modification
schemes are presented.
In still a further exemplary embodiment, there is provided a conjugate with biotin.
Thus, for example, a selectively biotinylated peptide is elaborated by the attachment of an
avidin or streptavidin moiety bearing one or more modifying groups.
In a further preferred embodiment, the biomolecule is selected to direct the peptide
modified by the methods of the invention to a specific intracellular compartment, thereby
enhancing the delivery of the peptide to that intracellular compartment relative to the amount
of underivatized peptide that is delivered to the tissue. In a still further preferred
embodiment, the amount of derivatized peptide delivered to a specific intracellular
compartment within a selected time period is enhanced by derivatization by at least about
20%, more preferably, at least about 40%, and more preferably still, at least about 100%. In
another particularly preferred embodiment, the biomolecule is linked to the peptide by a
cleavable linker that can hydrolyze once internalized. Presently, preferred biomolecules for
intracellular targeting applications include transferrin, lactotransferrin (lactoferrin),
melanotransferrin (p97), cerulopIasmin,.and divalent cation transporter, as well as antibodies
directed against specific vascular targets. Contemplated linkages include, but are not limited
to, protein-sugar-linker-sugar-protein, protein-sugar-linker-protein and multivalent forms
thereof, and protein-sugar-linker-drug where the drug includes small molecules, peptides,
lipids, among others.
Site-specific and target-oriented delivery of therapeutic agents is desirable for the
purpose of treating a wide variety of human diseases, such as different types of malignancies
and certain neurological disorders. Such procedures are accompanied by fewer side effects
and a higher efficiacy of drug. Various principles have been relied on in designing these
del ivery systems. For a review, see Garnett, Advanced Drug Delivery Reviews 53:171 -216
(2001).
One important consideration in designing a drug delivery system to target tissues
specifically. The discovery of tumor surface antigens has made it possible to develop
therapeutic approaches where tumor cells displaying definable surface antigens are
specifically targeted and killed. There are three main classes of therapeutic monoclonal
antibodies (antibody) that have demonstrated effectiveness in human clinical trials in treating
malignancies: (1) unconjugated MAb, which either directly induces growth inhibition and/or
apoptosis, or indirectly activates host defense mechanisms to mediate antitumor cytotoxicity:
(2) drug-conjugated MAb, which preferentially delivers a potent cytotoxic toxin to the tumor
cells and therefore minimizes the systemic cytotoxicity commonly associated with
conventional chemotherapy; and (3) radioisotope-conjugated MAb, which delivers a
sterilizing dose of radiation to the tumor. See review by Reff et al., Cancer Control 9:152-
166(2002).
In order to arm MAbs with the power to kill malignant cells, the MAbs can be
connected to a toxin, which may be obtained from a plant, bacterial, or fungal source, to form
chimeric proteins called immunotoxins. Frequently used plant toxins are divided into two
classes: (1) holotoxins (or class II ribosome inactivating proteins), such as ricin, abrin.
mistletoe lectin, and modeccin, and (2) hemitoxins (class I ribosome inactivating proteins),
such as pokeweed antiviral protein (PAP), saporin, Bryodin 1, bouganin, and gelonin.
Commonly used bacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin
(PK). Kreitman, Current Pharmaceutical Biotechnology 2:313-325 (2001). Other toxins
contemplated for use with the present invention include, but are not limited to, those in Table
2.
Conventional immunotoxins contain an MAb chemically conjugated to a toxin that is
mutated or chemically modified to minimized binding to normal cells. Examples include
anti-B4-blocked ricin, targeting CD5; and RFB4-deglycosyIated ricin A chain, targeting
CD22. Recombinant immunotoxins developed more recently are chimeric proteins
consisting of the variable region of an antibody directed against a tumor antigen fused to a
protein toxin using recombinant DNA technology. The toxin is also frequently genetically
modified to remove normal tissue binding sites but retain its cytotoxicity. A large number of
differentiation antigens, overexpressed receptors, or cancer-specific antigens have been
identified as targets for immunotoxins, e.g., CD 19, CD22, CD20, IL-2 receptor (CD25),
CD33, IL-4 receptor. FGF receptor and its mutants, ErB2, Lewis carbohydrate, mesothelin,
transferrin receptor, GM-CSF receptor, Ras, Bcr-Abl, and c-Kit, for the treatment of a variety
of malignancies including hematopoietic cancers, glioma, and breast, colon, ovarian, bladder,
and gastrointestinal cancers. See e.g., Brinkmann et al., Expert Opin. Biol. Ther. 1:693-702
(2001); Perentesis and Sievers, Hematology/Oncology Clinics of North America 15:677-701
(2001).
MAbs conjugated with radioisotope are used as another means of treating human
malignancies, particularly hematopoietic malignancies, with a high level of specificity and
effectiveness. The most commonly used isotopes for therapy are the high-energy -emitters,
such as l3ll and 90Y. Recently, 213Bi-labeled anti-CD33 humanized MAb has also been tested
in phase I human clinical trials. Reff et al., supra.
A number of MAbs have been used for therapeutic purposes. For example, the use of
rituximab (Rituxan™), a recombinant chimeric anti-CD20 MAb, for treating certain
hematopoietic malignancies was approved by the FDA in 1997. Other MAbs that have since
been approved for therapeutic uses in treating human cancers include: alemtuzumab
(Campath-11 -I™), a humanized rat antibody against CD52; and gemtuzumab ozogamicin
(Mylotarg™), a calicheamicin-conjugated humanized mouse antCD33 MAb. The FDA is
also currently examining the safety and efficacy of several other MAbs for the purpose of
site-specific delivery of cytotoxic agents or radiation, e.g.. radiolabeled Zevalin™ and
Bexxar™. Reffet a I., supra.
A second important consideration in designing a drug delivery system is the
accessibility of a target tissue to a therapeutic agent. This is an issue of particular concern in
the case of treating a disease of the central nervous system (CNS), where the blood-brain
barrier prevents the diffusion of macromolecules. Several approaches have been developed
to bypass the blood-brain barrier for effective delivery of therapeutic agents to the CNS.
The understanding of iron transport mechanism from plasma to brain provides a
useful tool in bypassing the blood-brain barrier (BBB). Iron, transported in plasma by
transferrin, is an essential component of virtually all types of cells. The brain needs iron for
metabolic processes and receives iron through transferrin receptors located on brain capillary
endothelial cells via receptor-mediated transcytosis and endocytosis. Moos and Morgan,
Cellular and Molecular Neurobiology 20:77-95 (2000). Delivery systems based on
transferrin-transferrin receptor interaction have been established for the efficient delivery of
peptides, proteins, and liposomes into the brain. For example, peptides can be coupled with a
Mab directed against the transferrin receptor to achieve greater uptake by the brain, Moos and
Morgan, Supra. Similarly, when coupled with an MAb directed against the transferrin
receptor, the transportation of basic fibroblast growth factor (bFGF) across the blood-brain
barrier is enhanced. Song et al.. The Journal of Pharmacology and Experimental
Therapeutics 301:605-610 (2002); Wu et al., Journal of Drug Targeting 10:239-245 (2002).
In addition, a liposomal delivery system for effective transport of the chemotherapy drug,
doxorubicin, into C6 glioma has been reported, where transferrin was attached to the distal
ends of liposomal PEG chains. Eavarone et al., J. Biomed. Mater. Res. 51:10-14 (2000). A
number of US patents also relate to delivery methods bypassing the blood-brain barrier based
on transferrin-transferrin receptor interaction. See e.g., US Patent Nos. 5,154,924;
5.182.107; 5,527,527: 5,833,988; 6,015,555.
There are other suitable conjugation partners for a pharmaceutical agent to bypass the
blood-brain barrier. For example, US Patent Nos. 5,672,683, 5,977,307 and WO 95/02421
relate to a method of delivering a neuropharmaceutical agent across the blood-brain barrier,
where the agent is administered in the form of a fusion protein with a ligand that is reactive
with a brain capillary endothelial cell receptor; WO 99/00150 describes a drug delivery
system in which the transportation of a drug across the blood-brain barrier is facilitated by
conjugation with an MAb directed against human insulin receptor; WO 89/10134 describes a
chimeric peptide, which includes a peptide capable of crossing the blood brain barrier at a
relatively high rate and a hydrophilic neuropeptide incapable of transcytosis, as a means of
introducing hydrophilic neuropeptides into the brain; WO 01/60411 Al provides a
pharmaceutical composition that can easily transport a pharmaceutically active ingredient
into the brain. The active ingredient is bound to a hibernation-specific protein that is used as
a conjugate, and administered with a thyroid hormone or a substance promoting thyroid
hormone production. In addition, an alternative route of drug delivery for bypassing the
blood-brain barrier has been explored. For instance, intranasal delivery of therapeutic agents
without the need for conjugation has been shown to be a promising alternative delivery
method (Frey, 2002, Drug Delivery Technology, 2(5):46-49).
In addition to facilitating the transportation of drugs across the blood-brain barrier,
transferrin-transferrin receptor interaction is also useful for specific targeting of certain tumor
cells, as many tumor cells overexpress transferrin receptor on their surface. This strategy has
been used for delivering bioactive macromolecules into K562 cells via a transferrin conjugate
(Wellhoner et al., The Journal of Biological Chemistry 266:4309-4314 (1991)), and for
delivering insulin into enterocyte-like Caco-2 cells via a transferrin conjugate (Shah and
Shen, Journal of Pharmaceutical Sciences 85:1306-1311 (1996)).
Furthermore, as more becomes known about the functions of various iron transport
proteins, such as lactotransferrin receptor, melanotransferrin, ceruloplasmin, and Divalent
Cation Transporter and their expression pattern, some of the proteins involved in iron
transport mechanism(e.g., melanotransferrin), or their fragments, have been found to be
similarly effective in assisting therapeutic agents transport across the blood-brain barrier or
targeting specific tissues (WO 02/13843 A2, WO 02/13873 A2). For a review on the use of
transferrin and related proteins involved in iron uptake as conjugates in drug delivery, see Li
and Qian, Medical Research Reviews 22:225-250 (2002).
The concept of tissue-specific delivery of therapeutic agents goes beyond the
interaction between transferrin and transferrin receptor or their related proteins. For example,
a bone-specific delivery system has been described in which proteins are conjugated with a
bone-seeking aminobisphosphate for improved delivery of proteins to mineralized tissue.
Uludag and Yang, Biotechnol. Prog. 18:604-611 (2002). For a review on this topic, see Vyas
ct al., Critical Reviews in Therapeutic Drug Carrier System 18:1 -76 (2001).
A variety of linkers may be used in the process of generating bioconjugates for the
purpose of specific delivery of therapeutic agents,. Suitable linkers include homo- and
heterobifunctional cross-linking reagents, which may be cleavable by, e.g., acid-catalyzed
dissociation, or non-cleavable {see, e.g., Srinivasachar and Neville, Biochemistry 28:2501-
2509 (1989); Wellhoner et al., The Journal of Biological Chemistry 266:4309-4314 (1991)).
Interaction between many known binding partners, such as biotin and avidin/streptavidin, can
also be used as a means to join a therapeutic agent and a conjugate partner that ensures the
specific and effective delivery of the therapeutic agent. Using the methods of the invention,
proteins may be used to deliver molecules to intracellular compartments as conjugates.
Proteins, peptides, hormones, cytokines, small molecules or the like that bind to specific cell
surface receptors that are internalized after ligand binding may be used for intracellular
targeting of conjugated therapeutic compounds. Typically, the receptor-ligand complex is
internalized into intracellular vesicles that are delivered to specific cell compartments,
including, but not limited to, the nucleus, mitochondria, golgi, ER, lysosome, and endosome.
depending on the intracellular location targeted by the receptor. By conjugating the receptor
ligand with the desired molecule, the drug will be carried with the receptor-ligand complex
and be delivered to the intracellular compartments normally targeted by the receptor. The
drug can therefore be delivered to a specific intracellular location in the cell where it is
needed to treat a disease.
Many proteins may be used to target therapeutic agents to specific tissues and organs.
Targeting proteins include, but are not limited to, growth factors (EPO, HGH. HGF, nerve
growth factor, FGF, among others), cytokines (GM-CSF, G-CSF, the interferon family,
interleukins, among others), hormones (FSH, LH, the steroid families, estrogen.
corticosteroids, insulin, among others), serum proteins (albumin, lipoproteins, fetoprotein,
human serum proteins, antibodies and fragments of antibodies, among others), and vitamins
(folate, vitamin C, vitamin A, among others). Targeting agents are available that are specific
for receptors on most cells types.
Contemplated linkage configurations include, but are not limited to, protein-sugar-
linker-sugar-protein and multivalent forms thereof, protein-sugar-linker-protein and
multivalent forms thereof, protein-sugar-linker-therapeutic agent, where the therapeutic agent
includes, but are not limited to, small molecules, peptides and lipids. In some embodiments,
a hydrolysable linker is used that can be hydrolyzed once internalized. An acid labile linker
can be used to advantage where the protein conjugate is internalized into the endosomes or
lysosomes which have an acidic pH. Once internalized into the endosome or lysosome, the
linker is hydrolyzed and the therapeutic agent is released from the targeting agent.
In an exemplary embodiment, transferrin is conjugated via a linker to an enzyme or a
nucleic acid vector that encoded the enzyme desired to be targeted to a cell that presents
transferrin receptors in a patient. The patient could, for example, require enzyme
replacement therapy for that particular enzyme. In particularly preferred embodiments, the
enzyme is one that is lacking in a patient with a lysosomal storage disease (see Table 5).
Once in circulation, the transferrin-enzyme conjugate is linked to transferrin receptors and is
internalized in early endosomes (Xing et al., 1998, Biochem. J. 336:667; Li et al., 2002,
Trends in Pharmcol. Sci. 23:206; Suhaila et al., 1998, J. Biol. Chem. 273:14355). Other
contemplated targeting agents that are related to transferrin include, but are not limited to,
lactotransferrin (lactoferrin), melanotransferrin (p97), ceruloplasmin, and divalent cation
transporter.
In another exemplary embodiment, transferrin-dystrophin conjugates would enter
endosomes by the transferrin pathway. Once there, the dystrophin is released due to a
hydrolysable linker which can then be taken to the intracellular compartment where it is
required. This embodiment may be used to treat a patient with muscular dystrophy by
supplementing a genetically defective dystrophin gene and/or protein with the functional
dystrophin peptide connected to the transferrin.
E. Therapeutic Moieties
In another preferred embodiment, the modified sugar includes a therapeutic moiety.
Those of skill in the art will appreciate that there is overlap between the category of
therapeutic moieties and biomolecules; many biomolecules have therapeutic properties or
potential.
The therapeutic moieties can be agents already accepted for clinical use or they can be
drugs whose use is experimental, or whose activity or mechanism of action is under
investigation. The therapeutic moieties can have a proven action in a given disease state or
can be only hypothesized to show desirable action in a given disease state. In a preferred
embodiment, the therapeutic moieties are compounds, which are being screened for their
ability to interact with a tissue of choice. Therapeutic moieties, which are useful in practicing
the instant invention include drugs from a broad range of drug classes having a variety of
pharmacological activities. In some embodiments, it is preferred to use therapeutic moieties
that are not sugars. An exception to this preference is the use of a sugar that is modified by
covalent attachment of another entity, such as a PEG, biomolecule, therapeutic moiety,
diagnostic moiety and the like. In an exemplary embodiment, an antisense nucleic acid
moeity is conjugated to a linker arm which is attached to the targeting moiety. In another
exemplary embodiment, a therapeutic sugar moiety is conjugated to a linker arm and the
sugar-linker arm cassette is subsequently conjugated to a peptide via a method of the
invention.
Methods of conjugating therapeutic and diagnostic agents to various other species are
well known to those of skill in the art. See, for example Hermanson, Bioconjugate
Techniques, Academic Press, San Diego, 1996; and Dunn et al, Eds. Polymeric Drugs
And Drug Delivery Systems, ACS Symposium Series Vol. 469, American Chemical
Society. Washington, D.C. 1991.
In an exemplary embodiment, the therapeutic moiety is attached to the modified sugar
via a linkage that is cleaved under selected conditions. Exemplary conditions include, but are
not limited to, a selected pH (e.g., stomach, intestine, endocytotic vacuole), the presence of
an active enzyme (e.g., esterase, protease, reductase, oxidase), light, heat and the like. Many
cleavable groups are known in the art. See, for example, Jung et al, Biochem. Biophys. Acta,
761: 152-162 (1983); Joshie et al., J. Biol. Chem.,265: 14518-14525 (1990); Zarling et al, J.
Immunol., 124: 913-920 (1980); Bouizar et al, Eur. J. Biochem., 155: 141-147 (1986); Park
eial..J. Biol. Chem., 261: 205-210(1986); Browning etal,J. Immunol., 143: 1859-1867
(1989).
Classes of useful therapeutic moieties include, for example, non-steroidal anti-
inflammatory drugs (NSA1DS). The NSAIDS can. for example, be selected from the
following categories: (e.g., propionic acid derivatives, acetic acid derivatives, fenamic acid
derivatives, biphenylcarboxylic acid derivatives and oxicams); steroidal anti-inflammatory
drugs including hydrocortisone and the like; adjuvants; antihistaminic drugs (e.g.,
chlorpheniramine, triprolidine); antitussive drugs (e.g., dextromethorphan, codeine,
caramiphen and carbetapentane); antipruritic drugs (e.g., methdilazine and trimeprazine);
anticholinergic drugs (e.g., scopolamine, atropine, homatropine, levodopa); anti-emetic and
antinauseant drugs (e.g., cyclizine, meclizine, chlorpromazine, buclizine); anorexic drugs
(e.g., benzphetamine, phentermine, chlorphentermine, fenfluramine); central stimulant drugs
(e.g., amphetamine, methamphetamine, dextroamphetamine and methylphenidate);
antiarrhythmic drugs (e.g., propanolol, procainamide, disopyramide, quinidine, encainide); B-
adrenergic blocker drugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);
cardiotonic drugs (e.g., milrinone, amrinone and dobutamine); antihypertensive drugs (e.g.,
enalapril, clonidine, hydralazine, minoxidil, guanadrel, guanethidine);diuretic drugs (e.g.,
amiloride and hydrochlorothiazide); vasodilator drugs (e.g., diltiazem, amiodarone.
isoxsuprine, nylidrin. tolazoline and verapamil); vasoconstrictor drugs (e.g..
dihydroergotamine, ergotamine and methylsergide); antiulcer drugs (e.g., ranitidine and
cimetidine); anesthetic drugs (e.g., lidocaine, bupivacaine, chloroprocaine, dibucaine);
antidepressant drugs (e.g., imipramine, desipramine, amitryptiline, nortryptiline); tranquilizer
and sedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide, flurazepam,
hydroxyzine, loxapine and promazine); antipsychotic drugs (e.g., chlorprothixene,
fluphenazine, haloperidol, molindone, thioridazine and trifluoperazine); antimicrobial drugs
(antibacterial, antifungal, antiprotozoal and antiviral drugs).
Classes of useful therapeutic moieties include adjuvants. The adjuvants can, for
example, be selected from keyhole lymphet hemocyanin conjugates, monophosphoryl lipid
A. mycoplasma-derivcd lipopeptide MALP-2, cholera toxin B subunit. Escherichia coli heut-
labilc toxin, universal T helper epitope from tetanus toxoid, interleukin-12, CpG
oligodeoxynucleotides. dimethyldioctadecylammonium bromide, cyclodcxtrin. squalene,
aluminum salts, meningococcal outer membrane vesicle (OMV). montanide ISA. TiterMax™
(available from Sigma. St. Louis MO), nitrocellulose absorption, immune-stimulating
complexes such as Quil A. Gerbu™ adjuvant (Gerbu Biotechnik, Kirchwald, Germany),
threonyl muramyl dipeptide, thymosin alpha, bupivacaine, GM-CSF, Incomplete Freund's
Adjuvant, MTP-PE/MF59 (Ciba/Geigy, Basel, Switzerland), polyphosphazene, saponin
derived from the soapbark tree Quillaja saponaria, and Syntex adjuvant formulation
(Biocinc, Emeryville, CA), among others well known to those in the art.
Antimicrobial drugs which are preferred for incorporation into the present
composition include, for example, pharmaceutically acceptable salts of p-lactam drugs,
quinolone drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan,
doxycycline, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin,
ethambutol, hexamidine isothionate, metronidazole, pentamidine, gentamycin. kanamycin.
lineomycin, methacycline. methenamine, minocycline, neomycin, netilmycin, paromomycin,
streptomycin, tobramycin, miconazole and amantadine.
Other drug moieties of use in practicing the present invention include antineoplastic
drugs (e.g., antiandrogens (e.g., leuprolide or flutamide), cytocidal agents (e.g., adriamycin,
doxorubicin, taxol, cyclophosphamide, busulfan, cisplatin, (3-2-interferon) anti-estrogens
(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate, mercaptopurine,
thioguanine). Also included within this class are radioisotope-based agents for both
diagnosis and therapy, and conjugated toxins, such as ricin, geldanamycin, mytansin, CC-
1065. C-1027, the duocarmycins, calicheamycin and related structures and analogues thereof,
and the toxins listed in Table 2.
The therapeutic moiety can also be a hormone (e.g., medroxyprogesterone, estradiol,
leuprolide. megestrol, octreotide or somatostatin); muscle relaxant drugs (e.g., cinnamedrine,
cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine, idaverine, ritodrine.
diphenoxylate, dantrolene and azumolen); antispasmodic drugs; bone-active drugs (e.g.,
diphosphonate and phosphonoalkylphosphinate drug compounds); endocrine modulating
drugs (e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol, norethindrone, mestranol,
desogestrel, medroxyprogesterone), modulators of diabetes (e.g., glyburide or
chlorpropamide), anabolics, such as testolactone or stanozolol, androgens (e.g..
methyltestosterone, testosterone or fluoxymesterone), antidiuretics (e.g., desmopressin) and
calcitonins).
Also of use in the present invention are estrogens (e.g.. diethylstilbesterol).
glucocorticoids (e.g., triamcinolone, betamethasone, etc.) andprogesterones, such as
norethindrone, ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g., liothyroninc or
levothyroxine) or anti-thyroid agents (e.g., methimazole); antihyperprolactinemic drugs (e.g..
cabergoline); hormone suppressors (e.g., danazol or goserelin), oxytocics (e.g..
methylergonovine or oxytocin) and prostaglandins, such as mioprostol, alprostadil or
dinoprostone, can also be employed.
Other useful modifying groups include immunomodulating drugs (e.g.,
antihistamines, mast cell stabilizers, such as lodoxamide and/or cromolyn, steroids (e.g.,
triamcinolone, beclomethazone, cortisone, dexamethasone, prednisolone,
methylprednisolone, beclomethasone, or clobetasol), histamine H2 antagonists (e.g.,
famotidine, eimetidine, ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.
Groups with anti-inflammatory activity, such as sulindac, etodolac, ketoprofen and ketorolac,
are also of use. Other drugs of use in conjunction with the present invention will be apparent
to those of skill in the art.
Classes of useful therapeutic moieties include, for example, antisense drugs and also
naked DNA. The antisense drugs can be selected from for example Affinitak (ISIS,
Carlsbad, CA) and Gcnasense ™ (from.Gcnta, Berkeley Heights, NJ). Naked DNA can be
delivered as a gene therapy therapeutic for example with the DNA encoding for example
factors VIII and IX for treatment of hemophilia disorders.
F. Preparation of Modified Sugars
Modified sugars useful in forming the conjugates of the invention are discussed
herein. The discussion focuses on preparing a sugar modified with a water-soluble polymer
for clarity of illustration. In particular, the discussion focuses on the preparation of modified
sugars that include a poly(cthylene glycol) moiety. Those of skill will appreciate that the
methods set forth herein are broadly applicable to the preparation of modified sugars,
therefore, the discussion should not be interpreted as limiting the scope of the invention.
In general, the sugar moiety and the modifying group are linked together through the
use of reactive groups, which are typically transformed by the linking process into a new-
organic functional group or unreactive species. The sugar reactive functional group(s), is
located at any position on the sugar moiety. Reactive groups and classes of reactions useful
in practicing the present invention are generally those that are well known in the art of
bioconjugate chemistry. Currently favored classes of reactions available with reactive sugar
moieties are those, which proceed under relatively mild conditions. These include, but are
not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl
halides, active esters), electrophilic substitutions {e.g., enamine reactions) and additions to
carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder
addition). These and other useful reactions are discussed in, for example, Smith and March,
Advanced Organic Chemistry, 5th Ed., John Wiley & Sons, New York, 2001;
Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et
al. Modification of Proteins; Advances in Chemistry Series, Vol. 198, American
Chemical Society, Washington, D.C., 1982.
Useful reactive functional groups pendent from a sugar nucleus or modifying group
include, but are not limited to:
(a) carboxyl groups and various derivatives thereof including, but not limited to,
N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acyl imidazoles,
thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;
(b) hydroxyl groups, which can be converted to, e.g., esters, ethers, aldehydes, etc.
(c) haloalkyl groups, wherein the halide can be later displaced with a nucleophilic
group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an
alkoxide ion, thereby resulting in the covalent attachment of a new group at the functional
group of the halogen atom;
(d) dienophile groups, which are capable of participating in Diels-Alder reactions
such as, for example, maleimido groups;
(e) aldehyde or ketone groups, such that subsequent derivatization is possible via
formation of carbonyl derivatives such as, for example, imines, hydra/ones, semicarba/oncs
or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
(f) sulfonyl halide groups for subsequent reaction with amines, for example, to form
sulfonamides;
(g) thiol groups, which can be, for example, converted to disulfides or reacted with
alkyl and acyl halides:
(h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated or
oxidized;
(i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael
addition, etc: and
(j) epoxides, which can react with, for example, amines and hydroxy! compounds.
The reactive functional groups can be chosen such that they do not participate in. or
interfere with, the reactions necessary to assemble the reactive sugar nucleus or modifying
group. Alternatively, a reactive functional group can be protected from participating in the
reaction by the presence of a protecting group. Those of skill in the art understand how to
protect a particular functional group such that it does not interfere with a chosen set of
reaction conditions. For examples of useful protecting groups, see, for example, Greene el
al., Protectivf. Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.
In the discussion that follows, a number of specific examples of modified sugars that
are useful in practicing the present invention are set forth. In the exemplary embodiments, a
sialic acid derivative is utilized as the sugar nucleus to which the modifying group is
attached. The focus of the discussion on sialic acid derivatives is for clarity of illustration
only and should not be construed to limit the scope of the invention. Those of skill in the art
will appreciate that a variety of other sugar moieties can be activated and derivatized in a
manner analogous to that set forth using sialic acid as an example. For example, numerous
methods are available for modifying galactose, glucose, N-acetylgalactosamine and fucose to
name a few sugar substrates, which are readily modified by art recognized methods. See, for
example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schafer et al., J. Org. Chem.
65: 24 (2000).
In an exemplary embodiment, the peptide that is modified by a method of the
invention is a peptide that is produced in mammalian cells (e.g., CHO cells) or in a transgenic
animal and thus, contains N- and/or O-linked oligosaccharide chains, which are incompletely
sialylated. The oligosaccharide chains of the glycopeptide lacking a sialic acid and
containing a terminal galactose residue can be PEGylated, PPGylated or otherwise modified
with a modified sialic acid.
In Scheme 4. the mannosaminc glycoside 1, is treated with the active ester of a
protected amino acid (e.g., glycine) derivative, converting the sugar amine residue into the
corresponding protected amino acid amide adduct. The adduct is treated with an aldolase to
form the sialic acid 2. Compound 2 is converted to the corresponding CMP derivative by the
action of CMP-SA synthetase, followed by catalytic hydrogenation of the CMP derivative to
produce compound 3. The amine introduced via formation of the glycine adduct is utilized as
a locus of PEG or PPG attachment by reacting compound 3 with an activated PEG or PPG
derivative (e.g., PEG-C(0)NHS, PPG-C(O)NHS), producing 4 or 5, respectively.
Table 3 sets forth representative examples of sugar monophosphates that are
derivatized with a PEG or PPG moiety. Certain of the compounds of Table 3 are prepared by
the method of Scheme 1. Other derivatives are prepared by art-recognized methods. See. for
example. Keppler et al.. Glycobiology 11: 11R (2001); and Charter et al.. Glycobiology 10:
1049 (2000)). Other amine reactive PEG and PPG analogues are commercially available, or
they can be prepared by methods readily accessible to those of skill in the art.
Table 3. Examples of sugar monophosphates that are derivatized with a PEG or PPG
moiety

The modified sugar phosphates of use in practicing the present invention can be
substituted in other positions as well as those set forth above, "i" may be Na or another salt
and "i" may be interchangeable with Na. Presently preferred substitutions of sialic acid arc
set forth in Formula 5.

in which X is a linking group, which is preferably selected from -O-, -N(H)-, -S,
CH2-. and N(R)2, in which each R is a member independently selected from R'-R5. "i" may
be Na or another salt, and Na may be interchangeable with "i:The symbols Y, Z, A and B
each represent a group that is selected from the group set forth above for the identity of X. X.
Y, Z, A and B are each independently selected and, therefore, they can be the same or
different. The symbols R1, R2, R3, R4 and R5 represent H, polymers, a water-soluble polymer,
therapeutic moiety, biomolecule or other moiety. The symbol R6 represents H, OH, or a
polymer. Alternatively, these symbols represent a linker that is linked to a polymer, water-
soluble polymer, therapeutic moiety, biomolecule or other moiety.
In another exemplary embodiment, a mannosamine is simultaneously acylated and
activated for a nucleophilic substitution by the use of chloroacetic anhydride as set forth in
Scheme 5. In each of the schemes presented in this section, i-4 or Na+ can be interchangeable,
wherein the salt can be sodium, or can be any other suitable salt.

The resulting chloro-derivatized glycan is contacted with pyruvate in the presence of an
aldolase, forming a chloro-derivatized sialic acid. The corresponding nucleotide sugar is
prepared by contacted the sialic acid derivative with an appropriate nucleotide triphosphates
and a synthetase. The chloro group on the sialic acid moiety is then displaced with a
nucleophilic PFG derivative, such as thio-PEG.
In a further exemplary embodiment, as shown is Scheme 6, a mannosamine is
acylated with a bis-HOBT dicarboxylate, producing the corresponding amido-alkyl-
carboxylic acid, which is subsequently converted to a sialic acid derivative. The sialic acid
derivative is converted to a nucleotide sugar, and the carboxylic acid is activated and reacted
with a nucleophilic PEG derivative, such as amino-PEG.

In another exemplary embodiment, set forth in Seheme 7, amine- and carboxyl-
proteeted neuraminic acid is activated by converting the primary hydroxyl group to the
corresponding p-toluenesulfonate ester, and the methyl ester is cleaved. The activated
neuraminic acid is converted to the corresponding nucleotide sugar, and the activating group
is displaced by a nucleophilic PEG species, such as thio-PEG.

in yet a further exemplary embodiment, as set forth in Scheme 8, the primary
hydroxyl moiety of an amine- and carboxyl-protected neuraminic acid derivative is alkylated
using an electrophilic PEG, such as chloro-PEG. The methyl ester is subsequently cleaved
and the PEG-sugar is converted to a nucleotide sugar.

Glycans other than sialic acid can be derivatized with PEG using the methods set forth
herein. The derivatized glycans. themselves, are also within the scope of the invention.
Thus, Scheme 9 provides an exemplary synthetic route to a PEGylated galactose nucleotide
sugar. The primary hydroxy! group of galactose is activated as the corresponding
toluenesulfonate ester, which is subsequently converted to a nucleotide sugar.

Scheme 10 sets forth an exemplary route for preparing a galactose-PEG derivative
that is based upon a galactose-6-amine moiety. Thus, galactosamine is converted to a
nucleotide sugar, and the amine moiety of galactosamine is functional ized with an active
PliG derivative.

Scheme I 1 provides another exemplary route to galactose derivatives. The starting
point for Scheme 1 1 is galactose-2-amine. which is converted to a nucleotide sugar. The
amine moiety of the nucleotide sugar is the locus for attaching a PEG derivative, such as
Methoxy-PEG (mPEG) carboxylic acid.

Exemplary moieties attached to the conjugates disclosed herein include, but are not
limited to. PFG derivatives (e.g.. acyl-PEG, acyl-alkyl-PEG. alkyl-acyl-PEG carbamoyl-
Pl-G, aryl-PEG. alkyl-PEG), PPG derivatives (e.g., acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-
PPG carbamoyl-PPG. aryl-PPG). polyapartic acid, polyglutamate, polylysine, therapeutic
moieties, diagnostic moieties, mannose-6-phosphate, heparin, heparan, SLe mannose.
mannose-6-phosphate. Sialyl Lewis X, FGI", VFGI', proteins (e.g., transferrin), chondroitin,
keratan, dermatan, dextran, modified dextran, amylose, bisphosphate, poly-SA, hyaluronic
acid, keritan. albumin, integrins, antennary oligosaccharides, peptides and the like. Methods
of conjugating the various modifying groups to a saccharide moiety are readily accessible to
those of skill in the art (POLY (ETHYLENE GLYCOL CHEMISTRY : BlOTECllNlCAL and
Biomedical Applications. J. Milton Harris, Ed., Plenum Pub. Corp.. 1992; Poly
(Ethylene Glycol) Chemical and Biological Applications, J. Milton Harris, Ed., ACS
Symposium Series No. 680, American Chemical Society, 1997; Hermanson, Bioconjugate
Technioues. Academic Press. San Diego, 1996; and Dunn et al., Eds. Polymeric Drugs
And Drug Derivery Systems. ACS Symposium Series Vol. 469. American Chemical
Society. Washington, D.C. 1991).
Purification of sugars, nucleotide sugars and derivatives
The nucleotide sugars and derivatives produced by the above processes can be used
without purification. However. it is usually preferred to recover the product. Standard, well-
known techniques for recovery of glycosylated saccharides such as thin or thick layer
chromatography, column chromatography, ion exchange chromatography, or membrane
filtration can be used. It is preferred to use membrane filtration, more preferably utilizing a
reverse osmotic membrane, or one or more column chromatographic techniques for the
recovery as is discussed hereinafter and in the literature cited herein. For instance, membrane
filtration wherein the membranes have molecular weight cutoff of about 3000 to about 10.000
can be used to remove proteins for reagents having a molecular weight of less than 10,000
Da.. Membrane filtration or reverse osmosis can then be used to remove salts and/or purify
the product saccharides (see, e.g., WO 98/15581). Nanofilter membranes are a class of
reverse osmosis membranes that pass monovalent salts but retain polyvalent salts and
uncharged solutes larger than about 100 to about 2,000 Daltons, depending upon the
membrane used. Thus, in a typical application, saccharides prepared by the methods of the
present invention will be retained in the membrane and contaminating salts will pass through.
G. Cross-linking Groups
Preparation of the modified sugar for use in the methods of the present invention
includes attachment of a modifying group to a sugar residue and forming a stable adduct.
which is a substrate for a glycosyltransferase. Thus, it is often preferred to use a cross-
linking agent to conjugate the modifying group and the sugar. Exemplary Afunctional
compounds which can be used for attaching modifying groups to carbohydrate moieties
include, but are not limited to, Afunctional polyethylene glycols), polyamides, polyethcrs.
polyesters and the like. General approaches for linking carbohydrates to other molecules are
known in the literature. See, for example, Lee et al.. Biochemistry 28: 1856 (1989): Bhatia
et al.. Anal. Binchem. 178: 408 (1989): Janda et al.,./. Am. Chem. Soc: 112: 8886 (1990) and
Bcdnarski et al. WO 92/18135. In the discussion that follows, the reactive groups are treated
as benign on the sugar moiety of the nascent modified sugar. The focus of the discussion is
for clarity of illustration. Those of skill in the art will appreciate that the discussion is
relevant to reactive groups on the modifying group as well.
An exemplary strategy involves incorporation of a protected sulthydryl onto the sugar
using the heterobifunctional crosslinker SPDP (n-succinimidyl-3-(2-pyridyldithio)propionatc
and then deprotecting the sulfhydryl for formation of a disulfide bond with another sulfhydryl
on the modi lying group.
If SPDP detrimentally affects the ability of the modified sugar to act as a
glycosyltransferase substrate, one of an array of other crosslinkers such as 2-iminothiolane or
N-succinimidyl S-acetylthioacetate (SATA) is used to form a disulfide bond. 2-
iminothiolane reacts with primary amines, instantly incorporating an unprotected sulfhydryl
onto the amine-containing molecule. SATA also reacts with primary amines, but
incorporates a protected sulfhydryl. which is later deacetylated using hydroxylamine to
produce a free sulfhydryl. In each case, the incorporated sulfhydryl is free to react with other
sulfhydryls or protected sulfhydryl. like SPDP, forming the required disulfide bond.
The above-described strategy is exemplary, and not limiting, of linkers of use in the
invention. Other crosslinkers are available that can be used in different strategies for
crosslinking the modifying group to the peptide. For example, TPCH(S-(2-thiopyridyl)-L-
cysteine hydrazide and TPMPH ((S-(2-thiopyridyl) mercapto-propionohydrazide) react with
carbohydrate moieties that have been previously oxidized by mild periodate treatment, thus
forming a hydrazone bond between the hydrazide portion of the crosslinker and the periodate
generated aldehydes. TPCH and TPMPH introduce a 2-pyridylthione protected sulfhydryl
group onto the sugar, which can be deprotected with DTT and then subsequently used for
conjugation, such as forming disulfide bonds between components.
If disulfide bonding is found unsuitable for producing stable modified sugars, other
crosslinkers may be used that incorporate more stable bonds between components. The
heterobifunctional crosslinkers GMBS (N-gama-malimidobutyryloxy)succinimide) and
SMCC (succinimidyl 4-(N-rnaleirnido-methyl)cyclohexane) react with primary amines, thus
introducing a maleimide group onto the component. The maleimide group can subsequently
react with sulthydry is on the other component, which can be introduced by previously
mentioned crosslinkers. thus forming a stable thioether bond between the components. If
steric hindrance between components interferes with either component's activity or the ability
of the modified sugar to act as a glycosyltransferase substrate, crosslinkers can be used which
introduce long spacer arms between components and include derivatives of some of the
previously mentioned crosslinkers (i.e.. SPDP). Thus, there is an abundance of suitable
crosslinkers, which are useful; each of which is selected depending on the effects it has on
optimal peptide conjugate and modified sugar production.
A variety of reagents are used to modify the components of the modified sugar with
intramolecular chemical crosslinks (lor reviews of crosslinking reagents and crosslinking
procedures see: Wold. F.. Meth. Enzymol. 25: 623-651, 1972; Weetall, H. H., and Cooney. D.
A.. In: Fn/ymf.s as Drugs. (Holcenberg, and Roberts, eds.) pp. 395-442, Wiley, New York.
1981; Ji, T. H., Meth. Enzymol. 91: 580-609, 1983; Mattson el al., Mol. Biol. Rep. 17: 167-
183. 1993, all of which are incorporated herein by reference). Preferred crosslinking reagents
are derived from various zero-length, homo-bifunctional, and hetero-bifunctional crosslinking
reagents. Zero-length crosslinking reagents include direct conjugation of two intrinsic
chemical groups with no introduction of extrinsic material. Agents that catalyze formation of
a disulfide bond belong to this category. Another example is reagents that induce
condensation of a carboxyl and a primary amino group to form an amide bond such as
carbodiimides, ethylchloroformaie. Woodward's reagent K (2-ethyl-5-phenylisoxazolium-3'-
sulfonate), and carbonyldiimidazole. In addition to these chemical reagents, the enzyme
transglutaminase (glutamyl-peptide ?-glutamyltransferase; EC 2.3.2.13) may be used as zero-
length crosslinking reagent. This enzyme catalyzes acyl transfer reactions at carboxamide
groups of protein-linked glutaminyl residues, usually with a primary amino group as
substrate. Preferred homo- and hetero-bifunctional reagents contain two identical or two
dissimilar sites, respectively, which may be reactive for amino, sulfhydryl, guanidino, indole,
or nonspecific groups.
2. Preferred Specific Sites in Crosslinking Reagents
a. Amino-Reactive Groups
In one preferred embodiment, the sites on the cross-linker are amino-reactive groups.
Useful non-limiting examples of amino-reactive groups include N-hydroxysuccinimide
(NHS) esters, imidoestcrs. isocyanates, acylhalides, arylazides. p-nitrophenyl esters,
aldehydes, and sulfonyl chlorides.
NHS esters react preferentially with the primary (including aromatic) amino groups of
a modified sugar component. The imidazole groups of hislidincs are known to compete with
primary amines for reaction, but the reaction products are unstable and readily hydrolyzed.
The reaction involves the nucleophilic attack of an amine on the acid carboxyl of an NHS
ester to form an amide, releasing the N-hydroxysuccinimide. Thus, the positive charge of the
original amino group is lost.
Imidoesters are the most specific acylating reagents for reaction with the amine
groups of the modified sugar components. At a pH between 7 and 10, imidoesters react only
with primary amines. Primary amines attack imidates nucleophilically to produce an
intermediate that breaks down to amidine at high pH or to a new imidate at low pH. The new
imidate can react with another primary amine, thus crosslinking two amino groups, a case of
a putatively monofunctional imidate reacting bifunctionally. The principal product of
reaction with primary amines is an amidine that is a stronger base than the original amine.
The positive charge of the original amino group is therefore retained.
lsocyanates (and isothiocyanates) react with the primary amines of the modified sugar
components to form stable bonds. Their reactions with sulfhydryl, imidazole, and tyrosyl
groups give relatively unstable products.
Acylazides are also used as amino-specific reagents in which nucleophilic amines of
the affinity component attack acidic carboxyl groups under slightly alkaline conditions, e.g.
pH 8.5.
Arylhalides such as 1.5-difluoro-2,4-dinitrobenzene react preferentially with the
amino groups and tyrosine phenolic groups of modified sugar components, but also with
sulfhydryl and imidazole groups.
p-Nitrophenyl esters of mono- and dicarboxylic acids arc also useful amino-reactive
groups. Although the reagent specificity is not very high, a- and e-amino groups appear to
react most rapidly.
Aldehydes such as glutaraldehyde react with primary amines of modified sugar.
Although unstable Schiff bases are formed upon reaction of the amino groups with the
aldehydes of the aldehydes, glutaraldehyde is capable of modifying the modified sugar with
stable crosslinks. At pH 6-8, the pH of typical crosslinking conditions, the cyclic polymers
undergo a dehydration to form a-ß unsaturated aldehyde polymers. Schiff bases, however.
are stable, when conjugated to another double bond. The resonant interaction of both double
bonds prevents hydrolysis of the Schiff linkage. Furthermore, amines at high local
concentrations can attack the cthylcnic double bond to form a stable Michael addition
product.
Aromatic sullbnyl chlorides react with a variety oi'sites of the modified sugar
components, but reaction with the amino groups is the most important, resulting in a stable
sulfonamide linkage.
b. Sulfhydryl-Reactive Groups
In another preferred embodiment, the sites arc sulfhydryl-reactive groups. Useful,
non-limiting examples of sulfhydryl-reactive groups include maleimides, alkyl halides.
pyridyl disulfides, and thiophthalimides.
Maleimides react preferentially with the sulfhydryl group of the modified sugar
components to form stable thioether bonds. They also react at a much slower rate with
primary amino groups and the imidazole groups of histidines. However, at pH 7 the
maleimide group can be considered a sulfhydryl-specific group, since at this pH the reaction
rate of simple thiols is 1000-fold greater than that of the corresponding amine.
Alkyl halides react with sulfhydryl groups, sulfides, imidazoles, and amino groups.
At neutral to slightly alkaline pH, however, alkyl halides react primarily with sulfhydryl
groups to form stable thioether bonds. At higher pH, reaction with amino groups is favored.
Pyridyl disulfides react with free sulfhydryls via disulfide exchange to give mixed
disulfides. As a result, pyridyl disulfides are the most specific sulfhydryl-reactive groups.
Thiophthalimides react with free sulfhydryl groups to form disulfides.
c. Carboxyl-Reactive Residue
In another embodiment, carbodiimides soluble in both water and organic solvent, are
used as carboxyl-reactive reagents. These compounds react with free carboxyl groups
forming a pscudourea that can then coupled to available amines yielding an amide linkage.
Procedures to modify a carboxyl group with carbodiimide is well know in the art (see,
Yamada et al., Biochemistry 20: 4836-4842, 1981).
3. Preferred Nonspecific Sites in Crosslinking Reagents
In addition to the use of site-specific reactive moieties, the present invention
contemplates the use of non-specific reactive groups to link the sugar to the modifying group.
Exemplary non-specific cross-linkers include photoactivatable groups, completely
inert in the dark, which arc converted to reactive species upon absorption of a photon of
appropriate energy. In one preferred embodiment, photoactivatable groups are selected from
precursors of nitrcnes generated upon heating or photolysis of azides. Electron-deficient
nitrenes are extremely reactive and can react with a variety of chemical bonds including N-H,
O-H. C-H, and C-C. Although three types of azides (aryl, alkyl, and acyl derivatives) may
be employed, arylazidcs are presently preferred. The reactivity of arylazides upon photolysis
is better with N-H and O-H than C-H bonds. Electron-deficient arylnitrenes rapidly ring-
expand to form dehydroazepines, which tend to react with nucleophiles, rather than form C-l I
insertion products. The reactivity of arylazides can be increased by the presence of electron-
withdrawing substituents such as nitro or hydroxyl groups in the ring. Such substituents push
the absorption maximum of arylazides to longer wavelength. Unsubstituted arylazides have
an absorption maximum in the range of 260-280 nm, while hydroxy and nitroarylazides
absorb significant light beyond 305 nm. Therefore, hydroxy and nitroarylazides are most
preferable since they allow to employ less harmful photolysis conditions for the affinity
component than unsubstituted arylazides.
In another preferred embodiment, photoactivatable groups are selected from
Chlorinated arylazides. The photolysis products of fluorinated arylazides are arylnitrenes. all
of which undergo the characteristic reactions of this group, including C-H bond insertion,
with high efficiency (Keana eta!., J. Org. Chem. 55: 3640-3647, 1990).
In another embodiment, photoactivatable groups are selected from benzophenone
residues. Benzophenone reagents generally give higher crosslinking yields than arylazide
reagents.
In another embodiment, photoactivatable groups are selected from diazo compounds,
which form an electron-deficient carbene upon photolysis. These carbenes undergo a variety
of reactions including insertion into C-H bonds, addition to double bonds (including aromatic
systems), hydrogen attraction and coordination to nucleophilic centers to give carbon ions.
In still another embodiment, pholoactivatable groups are selected from
diazopyruvates. For example, the p-nitrophenyl ester of p-nitrophenyl diazopyruvate reacts
with aliphatic amines to give diazopyruvic acid amides that undergo ultraviolet photolysis to
form aldehydes. The photolyzed diazopyruvate-modified affinity component will react like
formaldehyde or glutaraldehyde forming crosslinks.
4. Homobifunctional Reagents
a. Homobifunctional crosslinkers reactive with primary amines
Synthesis, properties, and applications of amine-reactive cross-linkers are
commercially described in the literature (for reviews of crosslinking procedures and reagents,
see above). Many reagents are available (e.g., Pierce Chemical Company, Rockford, III.:
Sigma Chemical Company, St. Louis, Mo.; Molecular Probes, Inc., Eugene, OR.).
Preferred, non-limiting examples of homobifunctional NHS esters include
disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)
suberate (BS), disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate (sulfo-DST), bis-
2-(suceinimidoo\ycarbonyloxy)cthylsulfone (BSOCOES), bis-2-(sulfosuccinimidooxy-
carbonyloxy)ethylsulfone (sulfo-BSOCOES), ethylene glycolbis(succinimidylsuccinate)
(EOS), ethylene glycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS), dithiobis(succinimidyl-
propionate (DSP), and dithiobis(sulfosuccinimidylpropionate (sulfo-DSP). Preferred, non-
limiting examples of homobifunctional imidoesters include dimethyl malonimidate (DMM).
dimelhyl succinimidate (DMSC), dimethyl adipimidate (DMA), dimethyl pimelimidatc
(I)MP). dimethyl suberimidate (DMS). dimethyl-3,3'-oxydipropionimidate (DODP),
dimethyl-3,3'-(methylenedioxy)dipropionimidate (DMDP), dimethyl-,3'-
(dimethylenedioxy)dipropionimidate(DDDP), dimethyl-3,3'-(tetramethylenedioxy)-
dipropionimidate (DTDP). and dimethyl-3.3'-dithiobispropionimidate (DTBP).
Preferred, non-limiting examples of homobifunctional isothiocyanates include: p-
phenylenediisothiocyanate (DITC). and 4.4'-diisothiocyano-2,2'-disulfonic acid sttlbene
(DIDS).
Preferred, non-limiting examples of homobifunctional isocyanates include xylene-
diisocyanate, toluene-2,4-diisocyanate, toluene-2-isocyanate-4-isothiocyanate, 3-
methoxydiphenylmethane-4,4'-diisocyanate, 2,2'-dicarboxy-4.4'-azophenyldiisocyanate, and
hexamclhylencdi isocv anatc.
Preferred, non-limiting examples of homobifunctional arylhalides include 1,5-
difluoro-2.4-dinitroben/ene (DFDNB). and 4.4'-difluoro-3,3'-dinitrophenyl-sulfone.
Preferred, non-limiting examples of homobifunctional aliphatic aldehyde reagents
include glyoxal, malondialdehyde, and glutaraldehyde.
Preferred, non-limiting examples of homobifunctional acylating reagents include
nitrophenyl esters of dicarboxylic acids.
Preferred, non-limiting examples of homobifunctional aromatic sulfonyl chlorides
include phcnol-2.4-disulfonyl chloride, and a-naphthol-2,4-disulfonyl chloride.
Preferred, non-limiting examples of additional amino-rcactive homobifunctional
reagents include crythritolbiscarbonate which reacts with amines to give biscarbamates.
b. Homobifunctional Crosslinkers Reactive with Free Sulfhydryl
Groups
Synthesis, properties, and applications of such reagents are described in the literature
(for reviews of crosslinking procedures and reagents, see above). Many of the reagents are
commercially available (e.g.. Pierce Chemical Company, Rockford, 111.; Sigma Chemical
Company, St. Louis. Mo.; Molecular Probes, Inc., Eugene, OR).
Preferred, non-limiting examples of homobifunctional maleimides include
bismaleimidohexane (BMH), N,N'-(1,3-phenylene) bismaleimide, N,N'-(1,2-
phenylene)bismaleimide. azophenyldimaleimide, and bis(N-maleimidomethyl)ether.
Preferred, non-limiting examples of homobifunctional pyridyl disulfides include 1.4-
di-3'-(2'-pyridyldithio)propionamidobutane(DPDPB).
Preferred, non-limiting examples of homobifunctional alkyl halides include 2,2'-
dicarbo\y-4.4'-diiodoacetamidoazobenzene. a,a'-diiodo-p-xylenesulfonic acid. a.,a'-dibromo-
p-xylenesulfonic acid. N.N'-bis(b-bromoethyl)benzylaminc, N.N'-
di(bromoacetyl)phenylthydrazine, and 1,2-di(bromoacetyl)amino-3-phenylpropane.
c. Homobifunctional Photoactivatable Crosslinkers
Synthesis, properties, and applications of such reagents are described in the literature
(for reviews of crosslinking procedures and reagents, see above). Some of the reagents are
commercially available (e.g.. Pierce Chemical Company, Rockford, III.; Sigma Chemical
Company, St. Louis. Mo.; Molecular Probes, Inc., Eugene. OR).
Preferred, non-limiting examples of homobifunctional photoactivatable crosslinkcr
include bis-ß-(4-azidosalicylamido)ethyldisulfide (BASED), di-N-(2-nitro-4-azidophenyl)-
cystamine-S.S-dioxide (DNCO), and 4.4'-dithiobisphenylazide.
5. HeteroBifunctional Reagents
a. Amino-Reactive HeteroBifunctional Reagents with a Pyridyl
Disulfide Moiety
Synthesis, properties, and applications of such reagents are described in the literature
(for reviews of crosslinking procedures and reagents, see above). Many of the reagents are
commercially available (e.g.. Pierce Chemical Company. Rockford, III.: Sigma Chemical
Company. St. Louis. Mo.; Molecular Probes, Inc., Eugene, OR).
Preferred, non-limiting examples of hetero-bifunctional reagents with a pyridyl
disulfide moiety and an ami no-reactive NHS ester include N-succinimidyl-3-(2-
pyridyldithio)propionate (SPDP), succinimidyl 6-3-(2-pyridyldithio)propionamidohexanoate
(LC-SPDP), sulfosuccinimidyl 6-3-(2-pyridyldithio)propionamidohexanoate (sulfo-
I.CSPDP). 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluenc (SMPT). and
sulfosuccinimidyl 6-a-methyl-a-(2-pyridyldithio)toluamidohexanoate (sulfo-LC-SMPT).
b. Amino-Reactive HeteroBifunctional Reagents with a Maleimide
Moiety
Synthesis, properties, and applications of such reagents are described in the literature.
Preferred, non-limiting examples of hetero-bifunctional reagents with a maleimide moiety
and an amino-reactive NHS ester include succinimidyl maleimidylacetate (AMAS),
succinimidyl 3-maleimidylpropionate (BMPS), N- y-maleimidobutyryloxysuccinimide ester
(GMBS)N-y-maleimidobutyryloxysulfo succinimide ester (sulfo-GMBS) succinimidyl 6-
maleimidylhcxanoatc (EMCS). succinimidyl 3-maleimidylbenzoate (SMB),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). m-maleimidobenzoyl-N-
hydroxysulfosuccinimide ester (sulfo-MBS), succinimidyl 4-(N-maleimidomethyl)-
cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-
I-carboxylate (sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), and
sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).
c. Amino-Reactive HeteroBifunctional Reagents with an Alkyl Halide
Moiety
Synthesis, properties, and applications of such reagents are described in the literature.
Preferred, non-limiting examples of hetero-bifunctional reagents with an alky! halide moiety
and an amino-reactive NHS ester include N-succinimidyl-(4-iodoacetyl)aminobenzoate
(SIAB). sulfosuccinimidyl-(4-iodoacetyl)aminobenzoatc (sulfo-SIAB), succinimidyl-6-
(iodoacetyl)aminohexanoate (SIAX), succinimidyl-6-(6-((iodoacetyl)-
amino)hexanoylamino)hexanoate (SIAXX), succinimidyl-6-(((4-(iodoacetyl)-amino)-
methyl)-cyclohexane-l-carbonyl)aminohexanoate (SIACX), and succinimidyl-4((iodoacetyl)-
amino)methylcyclohexane-l -carboxylate (SIAC).
A preferred example of a hetero-bifunctional reagent with an amino-reactive NHS
ester and an alkyl dihalide moiety is N-hydroxysuccinimidyl 2,3-dibromopropionate (SDBP).
SDBP introduces intramolecular crosslinks to the affinity component by conjugating its
amino groups. The reactivity of the dibromopropionyl moiety towards primary amine groups
is controlled by the reaction temperature (McKenzie et al, Protein Chem. 7: 581-592
(1988)).
Preferred, non-limiting examples of hetero-bifunctional reagents with an alkyl halide
moiety and an amino-reactive p-nitrophenyl ester moiety include p-nitrophenyl iodoacetate
(NP1A).
Other cross-linking agents are known to those of skill in the art. See, for example,
Pomato et al. U.S. Patent No. 5,965,106. It is within the abilities of one of skill in the art to
choose an appropriate cross-linking agent for a particular application.
d. Cleavable Linker Groups
In yet a further embodiment, the linker group is provided with a group that can be
cleaved to release the modifying group from the sugar residue. Many cleavable groups are
known in the art. See. for example. Jung et al.. Biochem. Biophys. Acta 761: 152-162 (108.1):
Joshi et al.. J. Biol. Chem. 265: 14518-14525 (1990); Zarling et al.. J. Immunol. 124: 913-920
(1980): Houizar et al.. Eur. J. Biochem. 155: 141-147(1986); Park et al., J. Biol. Chem. 261:
205-210 (1986); Browning et al., J. Immunol. 143: 1859-1867(1989). Moreover a broad
range of cleavable, bifunctional (both homo- and hetero-bifunctional) linker groups is
commercially available from suppliers such as Pierce.
Exemplary cleavable moieties can be cleaved using light, heat or reagents such as
thiols, hydroxylamine, bases, periodate and the like. Moreover, certain preferred groups are
cleaved in vivo in response to being endocytosed (e.g., cis-aconityl; sec, Shen et al., Biochem.
Biophys. Res. Commun. 102: 1048 (1991)). Preferred cleavable groups comprise a cleavable
moiety which is a member selected from the group consisting of disulfide, ester, imide.
carbonate, nitrobenzyl, phenacyl and benzoin groups.
e. Conjugation of Modified Sugars to Peptides
The modified sugars are conjugated to a glycosylated or non-glycosylated peptide
using an appropriate enzyme to mediate the conjugation. Preferably, the concentrations of
the modified donor sugar(s), enzyme(s) and acceptor peptide(s) are selected such that
glycosylation proceeds until the acceptor is consumed. The considerations discussed below,
while set forth in the context of a sialyltransferase, are generally applicable to other
glycosyltransfcrase reactions.
A number of methods of using glycosyltransferases to synthesize desired
oligosaccharide structures are known and are generally applicable to the instant invention.
Exemplary methods are described, for instance, WO 96/32491. Ito et al.. Pure Appl. Chem.
65: 753 (1993), and U.S. Pat.Nos. 5,352,670, 5,374,541, and 5,545,553.
The present invention is practiced using a single glycosyltransferase or a combination
of glyeosyluansferases. For example, one can use a combination of a sialyltransferase and a
galactosyltransferase. In those embodiments using more than one enzyme, the enzymes and
substrates are preferably combined in an initial reaction mixture, or the enzymes and reagents
for a second enzymatic reaction are added to the reaction medium once the first enzymatic
reaction is complete or nearly complete. By conducting two enzymatic reactions in sequence
in a single vessel, overall yields are improved over procedures in which an intermediate
species is isolated. Moreover, cleanup and disposal of extra solvents and by-products is
reduced.
In a preferred embodiment, each of the first and second enzyme is a
glycosyltransferase. In another preferred embodiment, one enzyme is an endoglycosidase. In
another preferred embodiment, one enzyme is an exoglycosidase. In an additional preferred
embodiment, more than two enzymes are used to assemble the modified glycoprotein of the
invention. The enzymes are used to alter a saccharide structure on the peptide at any point
either before or alter the addition of the modified sugar to the peptide.
In another embodiment, at least two of the enzymes are glycosyltransferases and the
last sugar added to the saccharide structure of the peptide is a non-modified sugar. Instead,
the modified sugar is internal to the glycan structure and therefore need not be the ultimate
sugar on the glycan. In an exemplary embodiment, galactosyltransferase may catalyze the
transfer of Gal-PEG from UDP-Gal-PEG onto the glycan, followed by incubation in the
presence of ST3Gal3 and CMP-SA, which serves to add a "capping" unmodified sialic acid
onto the glycan (Figure 23A).
In another embodiment, at least two of the enzymes used are glycosyltransferases. and
at least two modified sugars are added to the glycan structures on the peptide. In this manner,
two or more different glycoconjugates may be added to one or more glycans on a peptide.
This process generates glycan structures having two or more functionally different modified
sugars. In an exemplary embodiment, incubation of the peptide with GnT-I. II and UDP-
GlcNAc-PFG serves to add a GlcNAc-PFG molecule to the glycan; incubation with
galactosyltransferase and UDP-Gal then serves to add a Gal residue thereto; and, incubation
with ST3Gal3 and CMP-SA-Man-6-Phosphate serves to add a SA-mannose-6-phosphate
molecule to the glycan. This series of reactions results in a glycan chain having the
functional characteristics of a PFGylated glycan as well as mannose-6-phosphate targeting
activity (Figure 23B).
In another embodiment, at least two of the enzymes used in the reaction are
glycosyltransferases, and again, different modified sugars are added to N-linked and O-
linked glycans on the peptide. This embodiment is useful when two different modified
sugars are to be added to the glycans of a peptide, but when it is important to spatially
separate the modified sugars on the peptide from each other. For example, if the modified
sugars comprise bulky molecules, including but not limited to, PEG and other molecules such
as a linker molecule, this method may be preferable. The modified sugars may be added
simultaneously to the glycan structures on a peptide, or they may be added sequentially. In
an exemplary embodiment, incubation with ST3Gal3 and CMP-SA-PEG serves to add sialic
acid-PEG to the N-linked glycans, while incubation with ST3Gall and CMP-SA-
bisPhosphonate serves to add sialic acid-BisPhosphonate to the O-linked glycans (Figure
23C).
In another embodiment, the method makes use of one or more exo- or
endogKcosidasc. The glycosidase is typically a mutant, which is engineered to form gl\cos\l
bonds rather than rupture them. The mutant glycanase, sometimes called a glycosynthase.
typically includes a substitution of an amino acid residue for an active site acidic amino acid
residue. For example, when the endoglycanase is endo-H, the substituted active site residues
will typically be Asp at position 130, Glu at position 132 or a combination thereof. The
amino acids are generally replaced with serine, alanine, asparagine, or glutamine.
Exoglycosidases such as transialylidase arc also useful.
The mutant enzyme catalyzes the reaction, usually by a synthesis step that is
analogous to the reverse reaction of the endoglycanase hydrolysis step. In these
embodiments, the glycosyl donor molecule (e.g., a desired oligo- or mono-saccharide
structure) contains a leaving group and the reaction proceeds with the addition of the donor
molecule to a GlcNAc residue on the protein. For example, the leaving group can be a
halogen, such as fluoride. In other embodiments, the leaving group is a Asn, or a Asn-
peptidc moiety. In yet further embodiments, the GlcNAc residue on the glycosyl donor
molecule is modified. For example, the GlcNAc residue may comprise a 1,2 oxazoline
moiety.
In a preferred embodiment, each of the enzymes utilized to produce a conjugate of the
invention are present in a catalytic amount. The catalytic amount of a particular enzyme
varies according to the concentration of that enzyme's substrate as well as to reaction
conditions such as temperature, time and pH value. Means for determining the catalytic
amount for a given enzyme under preselected substrate concentrations and reaction
conditions are well known to those of skill in the art.
The temperature at which an above-described process is carried out can range from
just above freezing to the temperature at which the most sensitive enzyme denatures.
Preferred temperature ranges are about 0 °C to about 55 °C, and more preferably about 20 ºC
to about 37 °C. In another exemplary embodiment, one or more components of the present
method are conducted at an elevated temperature using a thermophilic enzyme.
The reaction mixture is maintained for a period of time sufficient for the acceptor to
be glycosylated, thereby forming the desired conjugate. Some of the conjugate can often be
detected after a few hours, with recoverable amounts usually being obtained within 24 hours
or less. Those of skill in the art understand that the rate of reaction is dependent on a number
of variable factors (e.g. enzyme concentration, donor concentration, acceptor concentration,
temperature, solvent volume), which are optimized for a selected system.
The present invention also provides for the industrial-scale production of modified
peptides. As used herein, an industrial scale generally produces at least one gram of finished,
purified conjugate.
In the discussion that follows, the invention is exemplified by the conjugation of
modified sialic acid moieties to a glycosylated peptide. The exemplary modified sialic acid is
labeled with PEG. The focus of the following discussion on the use of PEG-modified sialic
acid and glycosylated peptides is for clarity of illustration and is not intended to imply that
the invention is limited to the conjugation of these two partners. One of skill understands that
the discussion is generally applicable to the additions of modified glycosyl moieties other
than sialic acid. Moreover, the discussion is equally applicable to the modification of a
glycosyl unit with agents other than PEG including other water-soluble polymers, therapeutic
moieties, and biomolecules.
An enzymatic approach can be used for the selective introduction of PEGylated or
PPGylated carbohydrates onto a peptide or glycopeptide. The method utilizes modified
sugars containing PEG, PPG. or a masked reactive functional group, and is combined with
the appropriate glycosyltransferase or glycosynthase. By selecting the glycosyltransferase
that will make the desired carbohydrate linkage and utilizing the modified sugar as the donor
substrate, the PEG or PPG can be introduced directly onto the peptide backbone, onto
existing sugar residues of a glycopeptide or onto sugar residues that have been added to a
peptide.
An acceptor for the sialyltransferase is present on the peptide to be modified by the
methods of the present invention either as a naturally occurring structure or one placed there
recombinantly. enzymaticalk or chemically. Suitable acceptors, include, for example,
galactosyl acceptors such as Ga1ß1,4GlcNAe, Ga1ß1,4GalNAc, Galß1,3GalNAc, lacto-N-
tetraose, Galpl,3GlcNAc, Galß1Ara, Galßl,6GlcNAc, Ga1ß1,4Glc (lactose), and other
acceptors known to those of skill in the art (.see, e.g., Paulson el ah, J. Biol. Chem. 253: 5617-
5624(1978)).
In one embodiment, an acceptor for the sialyltransferase is present on the peptide to
be modified upon in vivo synthesis of the peptide. Such peptides can be sialylated using the
claimed methods without prior modification of the glycosylation pattern of the peptide.
Alternatively, the methods of the invention can be used to sialylate a peptide that does not
include a suitable acceptor; one first modifies the peptide to include an acceptor by methods
known to those of skill in the art. In an exemplary embodiment, a GalNAc residue is added
by the action of a GalNAc transferase.
In an exemplary embodiment, the galactosyl acceptor is assembled by attaching a
galactose residue to an appropriate acceptor linked to the peptide, e.g., a GIcNAc. The
method includes incubating the peptide to be modified with a reaction mixture that contains a
suitable amount of a galactosyltransferase (e.g., galß1,3 or gaißi ,4). and a suitable galactosyl
donor (e.g.. UDP-galactose). The reaction is allowed to proceed substantially to completion
or. alternatively, the reaction is terminated when a preselected amount of the galactose
residue is added. Other methods of assembling a selected saccharide acceptor will be
apparent to those of skill in the art.
In yet another embodiment, peptide-linked oligosaccharides are first "trimmed,"
either in whole or in part, to expose either an acceptor for the sialyltransferase or a moiety to
which one or more appropriate residues can be added to obtain a suitable acceptor. Enzymes
such as glycosyltransferases and endoglycosidases (.see, for example U.S. Patent No.
5.716,812) are useful for the attaching and trimming reactions. A detailed discussion of
"trimming" and remodeling N-linked and O-linked glycans is provided elsewhere herein.
In the discussion that follows, the method of the invention is exemplified by the use of
modified sugars having a water-soluble polymer attached thereto. The focus of the
discussion is for clarity of illustration. Those of skill will appreciate that the discussion is
equally relevant to those embodiments in which the modified sugar bears a therapeutic
moiety, biomolecule or the like.
An exemplary embodiment of the invention in which a carbohydrate residue is
"trimmed'" prior to the addition of the modified sugar is set forth in Figure 14, which sets
forth a scheme in which high mannose is trimmed back to the first generation biantennary
structure. A modified sugar bearing a water-soluble polymer is conjugated to one or more of
the sugar residues exposed by the "trimming back." In one example, a water-soluble polymer
is added via a GlcNAc moiety conjugated to the water-soluble polymer. The modified
GlcNAc is attached to one or both of the terminal mannose residues of the biantennary
structure. Alternatively, an unmodified GlcNAc can be added to one or both of the termini of
the branched species.
In another exemplary embodiment, a water-soluble polymer is added to one or both of
the terminal mannose residues of the biantennary structure via a modified sugar having a
galactose residue, which is conjugated to a GlcNAc residue added onto the terminal mannose
residues. Alternatively, an unmodified Gal can be added to one or both terminal GlcNAc
residues.
In yet a further example, a water-soluble polymer is added onto a Gal residue using a
modified sialic acid.
Another exemplary embodiment is set forth in Figure 15, which displays a scheme
similar to that shown in Figure 14, in which the high mannose structure is "trimmed back"' to
the mannose from which the biantennary structure branches. In one example, a water-soluble
polymer is added via a GlcNAc modified with the polymer. Alternatively, an unmodified
GlcNAc is added to the mannose, followed by a Gal with an attached water-soluble polymer.
In yet another embodiment, unmodified GlcNAc and Gal residues are sequentially added to
the mannose. followed by a sialic acid moiety modified with a water-soluble polymer.
Figure 16 sets forth a further exemplary embodiment using a scheme similar to that
shown in Figure 14, in which high mannose is "trimmed back" to the GlcNAc to which the
first mannose is attached. The GlcNAc is conjugated to a Gal residue bearing a water-soluble
polymer. Alternatively, an unmodified Gal is added to the GlcNAc, followed by the addition
of a sialic acid modified with a water-soluble sugar. In yet a further example, the terminal
GlcNAc is conjugated with Gal and the GlcNAc is subsequently fucosylated with a modified
fucose bearing a water-soluble polymer.
Figure 17 is a scheme similar to that shown in Figure 14. in which high mannose is
trimmed back to the first GlcNAc attached to the Asn of the peptide. In one example, the
GlcNAc of the GlcNAc-(Fuc)a residue is conjugated with a GlcNAc bearing a water soluble
polymer. In another example, the GlcNAc of the GlcNAc-(Fuc)a residue is modified with
Gal. which bears a water soluble polymer. In a still further embodiment, the GlcNAc is
modified with Gal. followed by conjugation to the Gal of a sialic acid modified with a water-
soluble polymer.
Other exemplary embodiments are set forth in Figures 18-22. An illustration of the
array of reaction types with which the present invention may be practiced is provided in each
of the aforementioned figures.
The Examples set forth above provide an illustration of the power of the methods set
fort h herein. Using the methods of the invention, it is possible to "trim back" and build up a
carbohydrate residue of substantially any desired structure. The modified sugar can be added
to the termini of the carbohydrate moiety as set forth above, or it can be intermediate between
the peptide core and the terminus of the carbohydrate.
In an exemplary embodiment, an existing sialic acid is removed from a glycopeptide
using a sialidase, thereby unmasking all or most of the underlying galactosyl residues.
Alternatively, a peptide or glycopeptide is labeled with galactose residues, or an
oligosaccharide residue that terminates in a galactose unit. Following the exposure of or
addition of the galactose residues, an appropriate sialyltransferase is used to add a modified
sialic acid. The approach is summarized in Scheme 12.
In yet a further approach, summarized in Scheme 13, a masked reactive functionality is present on the sialic acid. The masked reactive group is preferably unaffected by the
conditions used to attach the modified sialic acid to the peptide. After the covalenl
attachment of the modified sialic acid to the peptide, the mask is removed and the peptide is
conjugated with an agent such as PEG, PPG, a therapeutic moiety, biomolecule or other
agent. The agent is conjugated to the peptide in a specific manner by its reaction with the unmasked reactive group on the modified sugar residue.
Any modified sugar can be used with its appropriate glycosyltransferase, depending
on the terminal sugars of the oligosaccharide side chains of the glycopcptidc (Table 4). As
discussed above, the terminal sugar of the glycopeptide required for introduction of the
PL-lGylated or PPGylated structure can be introduced naturally during expression or it can be
produced post expression using the appropriate glycosidase(s), glycosyltransferase(s) or mix
of glycosidase(s) and gKcosyltransferasc(s).
In a further exemplary embodiment, UDP-galactose-PEG is reacted with bovine milk
ß l.4-galactosyltransferase. thereby transferring the modified galactose to the appropriate
terminal N-acetylglucosamine structure. The terminal GlcNAc residues on the glycopeptide
may be produced during expression, as may occur in such expression systems as mammalian,
insect, plant or fungus, but also can be produced by treating the glycopeptide with a sialydase
and/or glycosidase and/or glycosyltransferase, as required.
In another exemplary embodiment, a GlcNAc transferase, such as GnT-I-I V, is
utilized to transfer PEGylated-GlcNc to a mannose residue on a glycopeptide. In a still
further exemplary embodiment, the N- and/or O-linked glycan structures are enzymaticaily
removed from a glycopeptide to expose an amino acid or a terminal glycosyl residue that is
subsequently conjugated with the modified sugar. For example, an endoglycanase is used to
remove the N-linked structures of a glycopeptide to expose a terminal GlcNAc as a GlcNAc-
linked-Asn on the glycopeptide. UDP-Gal-PEG and the appropriate galactosyltransferase is
used to introduce the PEG- or PPG-galactose functionality onto the exposed GlcNAc.
In an alternative embodiment, the modified sugar is added directly to the peptide
backbone using a glycosyltransferase known to transfer sugar residues to the peptide
backbone. This exemplary embodiment is set forth in Scheme 14. Exemplary
glycosyltransferascs useful in practicing the present invention include, but are not limited to,
GaINAc transferases (GalNAc Tl-14), GlcNAc transferases, fucosyltransferases.
glucosyltransferases, xylosyltransferases, mannosyltransferases and the like. Use of this
approach allows the direct addition of modified sugars onto peptides that lack any
carbohydrates or, alternatively, onto existing glycopeptides. In both cases, the addition of the
modified sugar occurs at specific positions on the peptide backbone as defined by the
substrate specificity of the glycosyltransferase and not in a random manner as occurs during
modification of a protein's peptide backbone using chemical methods. An array of agents
can be introduced into proteins or glycopeptides that lack the glycosyltransferase substrate
peptide sequence by engineering the appropriate amino acid sequence into the peptide chain.
In each of the exemplary embodiments set forth above, one or more additional
chemical or enzymatic modification steps can be utilized following the conjugation of the
modified sugar to the peptide. In an exemplary embodiment, an enzyme (e.g..
fucosyltransferase) is used to append a glycosyl unit (e.g., fucose) onto the terminal modified
sugar attached to the peptide. In another example, an enzymatic reaction is utilized to "cap"
sites to which the modified sugar failed to conjugate. Alternatively, a chemical reaction is
utilized to alter the structure of the conjugated modified sugar. For example, the conjugated
modified sugar is reacted with agents that stabilize or destabilize its linkage with the peptide
component to which the modified sugar is attached. In another example, a component of the
modified sugar is deprotected following its conjugation to the peptide. One of skill will
appreciate that there is an array of enzymatic and chemical procedures that are useful in the
methods of the invention at a stage after the modified sugar is conjugated to the peptide.
Further elaboration of the modified sugar-peptide conjugate is within the scope of the
invention.
Peptide Targeting With Mannose-6-Phosphate
In an exemplary embodiment the peptide is derivatized with at least one mannose-6-
phosphate moiety. The mannose-6-phosphate moiety targets the peptide to a lysosome of a
cell, and is useful, for example, to target therapeutic proteins to Iysosomcs for therapy of
lysosomal storage diseases.
Lysosomal storage diseases are a group of over 40 disorders which are the result of
defects in genes encoding enzymes that break down glycolipid or polysaccharide waste
products within the lysosomes of cells. The enzymatic products, e.g., sugars and lipids, are
then recycled into new products. Each of these disorders results from an inherited autosomal
or X-linked recessive trait which affects the levels of enzymes in the lysosome. Generally,
there is no biological or functional activity of the affected enzymes in the cells and tissues of
affected individuals. Table 5 provides a list of representative storage diseases and the
enzymatic defect associated with the diseases. In such diseases the deficiency in enzyme
function creates a progressive systemic deposition of lipid or carbohydrate substrate in
lysosomes in cells in the body, eventually causing loss of organ function and death. The
genetic etiology, clinical manifestations, molecular biology and possibility of the lysosomal
storage diseases are detailed in Scriver et al., eds., The Metabolic and Molecular Basis
of Inherited Disease, 7.sup.th Ed., Vol. II, McGraw Hill, (1995).
Table 5. Lysosomal storage diseases and associated enzymatic defects
De Duve first suggested that replacement of the missing lysosomal enzyme with
exogenous biologically active enzyme might be a viable approach to treatment of lysosomal
storage diseases (De Duve, Fed. Proc. 23: 1045 (1964). Since that time, various studies have
suggested that enzyme replacement therapy may be beneficial for treating various lysosomal
storage diseases. The best success has been shown with individuals with type 1 Gaucher
disease, who have been treated with exogenous enzyme (P-glucocerebrosidase). prepared
from placenta (Ceredase™) or, more recently, recombinantly (Cerezyme™). It has been
suggested that enzyme replacement may also be beneficial for treating Fabry's disease, as
well as other lysosomal storage diseases. See. for example, Dawson et al., Ped. Res. 7(8):
684-690 (1973) (in vitro) and Mapes et al., Science 169: 987 (1970) (in vivo). Clinical trials
of enzyme replacement therapy have been reported for Fabry patients using infusions of
normal plasma (Mapes et al., Science 169: 987-989 (1970)), a-galactosidase A purified from
placenta (Brady et al., N. Eng. J. Med. 279: 1163 (1973)); or a-galactosidase A purified from
spleen or plasma (Desnick et al., Proc. Natl. Acad. Sci., USA 76: 5326-5330 (1979)) and have
demonstrated the biochemical effectiveness of direct enzyme replacement for Fabry disease.
These studies indicate the potential for eliminating, or significantly reducing, the pathological
glycolipid storage by repeated enzyme replacement. For example, in one study (Desnick et al. supra), intravenous injection of purified enzyme resulted in a transient reduction in the
plasma levels of the stored lipid substrate, globotriasylceramide.
Accordingly, there exists a need in the art for methods for providing sufficient
quantities of biologically active lysosomal enzymes, such as human a-galactosidase A, to
deficient cells. Recently, recombinant approaches have attempted to address these needs, see.
e.g.. U.S. Pat. No. 5.658.567; 5.580.757: Bishop et al. Proc. Natl. Acad. Sci.. USA. 83: 4859-
4863 (1986); Medin et al. Proc. Natl. Acad. Sci., USA. 93: 7917-7922 (1996); Novo, P. J.,
dene Therapy. 4: 488-492 (1997): Ohshima et al. Proc. Natl. Acad. Sci.. USA. 94: 2540-
2544 (! 997); and Sugimoto el ai. Human Gene Therapy 6: 905-915,(1995). Through the
mannose-6-phosphate mediated targeting of therapeutic peptides to lysosomes, the present
invention provides compositions and methods for delivering sufficient quantities of
biologically active lysosomal peptides to deficient cells.
Thus, in an exemplary embodiment, the present invention provides a peptide
according to Table 7 that is dcrivatized with mannose-6-phosphate (Figure 24 and Figure 25).
The peptide may be recombinantly or chemically prepared. Moreover, the peptide can be the
full, natural sequence, or it may be modified by, for example, truncation, extension, or it may
include substitutions or deletions. Exemplary proteins that are remodeled using a method of
the present invention include glucocerebrosidase, [3-glucosidase, a-galactosidase A, acid-u-
glucosidase (acid maltase). Representative modified peptides that are in clinical use include,
but are not limited to, Ceredasc™, Cerezyme™, and Fabryzyme™. A glycosyl group on
modified and clinically relevant peptides may also be altered utilizing a method of the
invention. The mannose-6-phosphate is attached to the peptide via a glycosyl linking group.
In an exemplary embodiment, the glycosyl linking group is derived from sialic acid.
Exemplary sialic acid-derived glycosyl linking groups are set forth in Table 3, in which one
or more of the "R" moieties is mannose-6-phosphate or a spacer group having one or more
mannose-6-phosphate moieties attached thereto. The modified sialic acid moiety is
preferably the terminal residue of an oligosaccharide linked to the surface of the peptide
(Figure 26)
In addition to the mannose-6-phosphale. the peptides of the invention may be further
derivati/ed with a moiety such as a water-soluble polymer, a therapeutic moiety, or an
additional targeting moiety. Methods for attaching these and other groups are set forth
herein. In an exemplary embodiment, the group other than mannose-6-phosphate is attached
to the peptide via a derivatized sialic acid derivative according to Table 3, in which one or
more of the "R" moieties is a group other than mannose-6-phosphate.
In an exemplary embodiment, a sialic acid moiety modified with a Cbz-protected
glycine-based linker arm is prepared. The corresponding nucleotide sugar is prepared and the
Cbz group is removed by catalytic hydrogenation. The resulting nucleotide sugar has an
available, reactive amine that is contacted with an activated mannose-6-phosphate derivative,
providing a mannose-6-phosphate derivatized nucleotide sugar that is useful in practicing the
methods of the invention.
As shown in the scheme below (scheme 15), an exemplary activated mannose-6-
phosphate derivative is formed by converting a 2-bromo-benzyl-protected phosphotriester
into the corresponding triflate, in situ, and reacting the triflate with a linker having a reactive
oxygen-containing moiety, forming an ether linkage between the sugar and the linker. The
benzyl protecting groups are removed by catalytic hydrogenation, and the methyl ester of the
linker is hydrolyzed, providing the corresponding carboxylic acid. The carboxylic acid is
activated by any method known in the art. An exemplary activation procedure relies upon the
conversion of the carboxylic acid to the N-hydroxysuccinimide ester.
In another exemplary embodiment, as shown in the scheme below (scheme 16), a N-acetylated sialic acid is converted to an amine by manipulation of the pyruvyl
moiety. Thus, the primary hydroxyl is converted to a sulfonate ester and reacted with sodium
azide. The azide is catalytically reduced to the corresponding amine. The sugar is
subsequently converted to its nucleotide analogue and coupled, through the amine group, to
the linker arm-derivatized mannose-6-phosphate prepared as discussed above.

Peptides useful to treat lysosomal storage disease can be derivatized with other
targeting moieties including, but not limited to, transferrin (to deliver the peptide across the
blood-brain barrier, and to endosomes), carnitine (to deliver the peptide to muscle cells), and
phosphonates, e.g, bisphosphonate (to target the peptide to bone and other calciierous
tissues). The targeting moiety and therapeutic peptide are conjugated by any method
discussed herein or otherwise known in the art.
In an exemplary embodiment, the targeting agent and the therapeutic peptide are
coupled via a linker moiety. In this embodiment, at least one of the therapeutic peptide or the
targeting agent is coupled to the linker moiety via an intact glycosyl linking group according
to a method of the invention. In an exemplary embodiment, the linker moiety includes a
poly(ether) such as poly(ethylene glycol). In another exemplary embodiment, the linker
moiety includes at least one bond that is degraded in vivo, releasing the therapeutic peptide
from the targeting agent, following delivery of the conjugate to the targeted tissue or region
of the body.
In yet another exemplary embodiment, the in vivo distribution of the therapeutic
moiety is altered via altering a glycoform on the therapeutic moiety without conjugating the
therapeutic peptide to a targeting moiety. For example, the therapeutic peptide can be
shunted away from uptake by the reticuloendothelial system by capping a terminal galactose
moiety of a glycosyl group with sialic acid (or a derivative thereof) (Figures 24 and 27).
Sialylation to cover terminal Gal avoids uptake of the peptide by hepatic asialoglycoprotein
(ASGP) receptors, and may extend the half life of the peptide as compared with peptides
having only complex glycan chains, in the absence of sialylation.
11. Peptide/Glycopeptides of the Invention
In one embodiment, the present invention provides a composition comprising multiple
copies of a single peptide having an elemental trimannosyl core as the primary glycan
structure attached thereto. In preferred embodiments, the peptide may be a therapeutic
molecule. The natural form of the peptide may comprise complex N-linked glycans or may
be a high mannose glycan. The peptide may be a mammalian peptide, and is preferably a
human peptide. In some embodiments the peptide is selected from the group consisting of an
immunoglobulin, erythropoietin, tissue-type activator peptide, and others (See Figure 28).
Exemplary peptides whose glycans can be remodeled using the methods of the
invention are set forth in Figure 28.
Table 6. Preferred peptides for glycan remodeling
A more detailed list of peptides useful in the invention and their source is provided in
Figure 28.
Other exemplary peptides that are modified by the methods of the invention include
members of the immunoglobulin family (e.g., antibodies, MHC molecules, T cell receptors,
and the like), intercellular receptors (e.g.. integrins, receptors for hormones or growth factors
and the like) lectins, and cytokines (e.g., interleukins). Additional examples include
tissue-type plasminogen activator (TPA), renin, clotting factors such as Factor VIII and
factor IX. bombesin, thrombin, hematopoietic growth factor, colony stimulating factors, viral
antigens, complement peptides, a1-antitrypsin, erythropoietin, P-seleclin glycopeptide
ligand-1 (PSGL-1). granulocyte-macrophage colony stimulating factor, anti-thrombin 111.
interleukins, interferons, peptides A and C. fibrinogen, herceptin™, leptin. glycosidases.
among many others. This list of peptides is exemplary and should not be considered to be
exclusive. Rather, as is apparent from the disclosure provided herein, the methods of the
invention are applicable to any peptide in which a desired glycan structure can be fashioned.
The methods of the invention are also useful for modifying chimeric peptides,
including, but not limited to, chimeric peptides that include a moiety derived from an
immunoglobulin, such as IgG.
Peptides modified by the methods of the invention can be synthetic or wild-type
peptides or they can be mutated peptides, produced by methods known in the art, such as site-
directed mutagenesis. Glycosylation of peptides is typically cither N-linked or O-linked. An
exemplary N-linkage is the attachment of the modified sugar to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-
threonine, where X is any amino acid except proline, are the recognition sequences for
enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a peptide creates a potential glycosylation
site. As described elsewhere herein, O-linked glycosylation refers to the attachment of one
sugar (e.g., N-acetylgalactosamine, galactose, mannose, GlcMAc, glucose, fucose or xylose)
to a hydroxy side chain of a hydroxyamino acid, preferably serine or threonine, although 5-
hydroxyproline or 5-hydroxylysine may also be used.
Several exemplary embodiments of the invention are discussed below. While several
of these embodiments use peptides having names having trademarks, and other specific
peptides as the exemplary peptide, these examples are not confined to any specific peptide.
The following exemplary embodiments are contemplated to include all peptide equivalents
and variants of any peptide. Such variants include, but are not limited to, adding and deleting
N-linked and O-linked glycosylation sites, and fusion proteins with added glycosylation sites.
One of skill in the art will appreciate that the following embodiments and the basic methods
disclosed therein can be applied to many peptides with equal success.
In one exemplary embodiment, the present invention provides methods for modifying
Granulocyte Colony Stimulating Factor (G-CSF). Figures 29A to 29G set forth some
examples of how this is accomplished using the methodology disclosed herein. In Figure
29B. a G-CSF peptide that is expressed in a mammalian cell system is trimmed back using a
sialidase. The residues thus exposed are modified by the addition of a sialic acid-
poly(ethylene glycol) moiety (PEG moiety), using an appropriate donor therefor and
ST3Gal 1. Figure 29C sets forth an exemplary scheme for modifying a G-CSF peptide that is
expressed in an insect cell. The peptide is modified by adding a galactose moiety using an
appropriate donor thereof and a galactosyltransferase. The galactose residues arc
functionalized with PEG via a sialic acid-PEG derivative, through the action of ST3Gall. In
Figure 29D, bacterially expressed G-CSF is contacted with an N-acetylgalactosamine donor
and N-acetylgalactosamine transferase. The peptide is functionalized with PEG, using a
PFGylated sialic acid donor and a sialyltransferase. In Figure 29E, mammalian cell
expressed G-CSF is contacted with a sialic acid donor that is modified with levulinic acid,
adding a reactive ketone to the sialic acid donor. After addition to a glycosyl residue on the
glycan on the peptide, the ketone is derivatized with a moiety such as a hydrazine- or amine-
PEG. In Figure 29F, bacterially expressed G-CSF is remodeled by contacting the peptide
with an endo-GalNAc enzyme under conditions where it functions in a synthetic, rather than
a hydrolytic manner, thereby adding a PFG-Gal-GalNAc molecule from an activated
derivative thereof, figure 29G provides another route for remodeling bacterially expressed
G-CSF. The polypeptide is derivatized with a PEGylated N-acetylgalactosamine residue by
contacting the polypeptide with an N-acctylgalactosamine transferase and an appropriate
donor of PEGylated N-acetylgalactosamine.
In another exemplary embodiment, the invention provides methods for modifying
Interferon a-14C (IFNal4C). as shown in Figures 30A to 30N. The various forms of IFNa
are disclosed elsewhere herein. In Figure 30B, IFNal4C expressed in mammalian cells is
first treated with sialidase to trim back the sialic acid units thereon, and then the molecule is
PEGylated using ST3Gal3 and a PEGylated sialic acid donor. In Figure 30C, N-
acetylglucosamine is first added to IFNa14C which has been expressed in insect or fungal
cells, where the reaction is conducted via the action of GnT-I and/or II using an N-
acetylglucosaminc donor. The polypeptide is then PEGylated using a galactosyltransferase
and a donor of PEG-galactose. In Figure 30D, IFNal4C expressed in yeast is first treated
with Endo-H to trim back the glycosyl units thereon. The molecules is galactosylated using a
galactosyltransferase and a galactose donor, and it is then PEGylated using ST3Gal3 and a
donor of PEG-sialic acid. In Figure 30F, IFNal4C produced by mammalian cells is modified
to inched a PEG moiety using ST3Gal3 and a donor of PEG-sialic acid. In Figure 30G,
If Nal4C expressed in insect of fungal cells first has N-acetylglucosamine added using one or
more of GnT-I, II. IV. and V, and an N-acetylglucosamine donor. The protein is
subsequently galactosylated using an appropriate donor and a galactosyltransferase. Then.
lFNal4C is PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 30H, yeast
produced IFNal4C is first treated with mannosidases to trim back the mannosyl groups. N-
acctylglucosamine is then added using a donor of N-acetylglucosamine and one or more of
GnT-I. II. IV. and V. IFNal4C is further galactosylated using an appropriate donor and a
galactosyltransferase. Then, the polypeptide is PEGylated using ST3Gal3 and a donor of
PFG-sialic acid. In Figure 301. NSO cell expressed IFNal4C is modified by capping
appropriate terminal residues with a sialic acid donor that is modified with levulinic acid,
thereby adding a reactive ketone to the sialic acid donor. After addition to a glycosyl residue
of the peptide, the ketone is derivatized with a moiety such as a hydrazine- or amine- PEG.
In Figure 30J. IFNal4C expressed by mammalian cells is PEGylated using a donor of PEG-
sialic acid and a 2.8-sialyltransferasc. In Figure 30K, IFNal4C produced by mammalian
cells is first treated with sialidase to trim back the terminal sialic acid residues, and then the
molecule is PEGylated using trans-sialidase and PFGylated sialic acid-lactose complex. In
Figure 30E. IFNaHC expressed in a mammalian system is sialylated using a donor of sialic
acid and a 2.8-sialyllransferase. In Figure 30M, lFNal4C expressed in insect or fungal cells
first has N-acetylglucosamine added using an appropriate donor and GnT-I and/or II. The
molecule is then contacted with a galactosyltransferase and a galactose donor that is
derivatized with a reactive sialic acid via a linker, so that the polypeptide is attached to the
reactive sialic acid via the linker and the galactose residue. The polypeptide is then contacted
with ST3Gal3 and transferrin, and thus becomes connected with transferrin via the sialic acid
residue. In Figure 30N. lFNal4C expressed in either insect or fungal cells is first treated
with endoglycanase to trim back the glycosyl groups, and is then contacted with a
galactosyltransferase and a galactose donor that is derivatized with a reactive sialic acid via a
linker, so that the polypeptide is attached to the reactive sialic acid via the linker and the
galactose residue. I he molecule is then contacted with ST3Gal3 and transferrin, and thus
becomes connected with transferrin via the sialic acid residue.
In another exemplary embodiment, the invention provides methods for modifying
Interferon a-2a or 2b (IFNa), as shown in Figures 30O to 30EE. In Figure 30P, IFNa
produced in mammalian cells is first treated with sialidase to trim back the glycosyl units, and
is then PEGylated using ST3Gal3 and a PEGylated sialic acid donor. In Figure 30Q, IFNa
expressed in insect cells is first galactosylated using an appropriate donor and a
galactosyltransferase. and is then PEGylated using ST3Gall and a PEGylated sialic acid
donor. Figure 30R offers another method for remodeling IFNa expressed in bacteria:
PEGylated N-acetylgalactosamine is added to the protein using an appropriate donor and N-
acetylgalactosamine transferase. In Figure 30S, IFNa expressed in mammalian cells is
modified by capping appropriate terminal residues with a sialic acid donor that is modified
with levulinic acid, adding a reactive ketone to the sialic acid donor. After addition to a
glycosyl residue of the peptide, the ketone is derivatized with a moiety such as a hydrazinc-
or amine- PEG. In Figure 30T, IFNa expressed in bacteria is PEGylated using a modified
enzyme Endo-N-acetylgalactosamidase, which functions in a synthetic instead of a hydrolyiic
manner, and using a N-acetylgalactosamine donor derivatized with a PEG moiety. In Figure
301 J. N-aeetylgalactosamine is first added IFNa using an appropriate donor and N-
acctylgalactosamine transferase, and then is PEGylated using a sialyltransferasc and a
PEGylated sialic acid donor. In Figure 30V, IFNa expressed in a mammalian system is first
treated with sialidase to trim back the sialic acid residues, and is then PEGylated using a
suitable donor and ST3Gal I and/or ST3Gal3. In Figure 30W, IFNa expressed in mammalian
cells is first treated with sialidase to trim back the sialic acid residues. The polypeptide is
then contacted with ST3Gall and two reactive sialic acid residues that are connect via a
linker, so that the polypeptide is attached to one reactive sialic acid via the linker and the
second sialic acid residue. The polypeptide is subsequently contacted with ST3Gal3 and
transferrin, and thus becomes connected with transferrin via the sialic acid residue. In Figure
30Y, IFNa expressed in mammalian cells is first treated with sialidase to trim back the sialic
acid residues, and is then PEGylated using ST3Gall and a donor of PEG-sialic acid. In
Figure 30Z, IFNa produced by insect cells is PEGylated using a galactosyltransferase and a
donor of PEGylated galactose. In Figure 30AA. bacterially expressed IFNa first has N-
acctylgalactosamine added using a suitable donor and N-acetylgalactosamine transferase.
The protein is then PEGylated using a sialyltransferase and a donor of PEG-sialic acid. In
Figure 30CC, IFNa expressed in bacteria is modified in another procedure: PEGylated N-
acetylgalactosamine is added to the protein by N-acetylgalactosamine transferase using a
donor of PEGylated N-acetylgalactosamine. In Figure 30DD. IFNa expressed in bacteria is
remodeled in yet another scheme. The polypeptide is first contacted with N-
acetylgalactosaminc transferase and a donor of N-acetylgalactosamine that is derivatized with
a reactive sialic acid via a linker, so that IFNa is attached to the reactive sialic acid via the
linker and the N-acetylgalactosamine. IFNa is then contacted with ST3Gal3 and asialo-
transferrin so that it becomes connected with transferrin via the sialic acid residue. Then,
IFNa is capped with sialic acid residues using ST3Gal3 and a sialic acid donor. An
additional method for modifying bacterially expressed IFNa is disclosed in Figure 30EE.
where IFNa is first exposed to NHS-CO-linker-SA-CMP and is then connected to a reactive
sialic acid via the linker. It is subsequently conjugated with transferrin using ST3Gal3 and
transferrin.
The methods for remodeling INN omega are essentially identical to those presented
here lor IFN alpha except that the attachment of the glycan to the IFN omega peptide occurs
at amino acid residue 101 in SEQ ID NO:75. The nucleotide and amino acid sequences for
IFN omega are presented herein as SEQ ID NOS:74 and 75. Methods of making and using
IFN omega are found in U.S. Patent No. 4,917,887 and 5,317,089, and in EP Patent No.
0170204-A.
In another exemplary embodiment, the invention provides methods for modifying
Interferon ß (IFN-ß), as shown in Figures 31A to 31S. In Figure 31B, IFN-ß expressed in a
mammalian system is first treated with sialidase to trim back the terminal sialic acid residues.
The protein is then PEGylated using ST3Gal3 and a donor of PEGylated sialic acid. Figure
31C is a scheme for modifying IFN-ß produced by insect cells. First, N-acetylglucosamine is
added to IFN-ß using an appropriate donor and GnT-I and/or -II. The protein is then
galactosylated using a galactose donor and a galactosyltransferase. Finally, IFN-p is
PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 31D, IFN-ß expressed
in yeast is first treated with Endo-M to trim back its glycosyl chains, and is then
galactosylated using a galactose donor and a galactosyltransferase, and is then PEGylated
using ST3Gal3 and a donor of PEGylated sialic acid. In Figure 31E, IFN-ß produced by
mammalian cells is modified by PEGylation using ST3Gal3 and a donor of sialic acid already
derivatied with a PEG moiety. In Figure 31F. IFN-ß expressed in insect cells first has N-
acetylglueosamine added by one or more of GnT-I, II. IV, and V using a N-
acetylglucosaminc donor, and then is galactosylated using a galactose donor and a
galactosyltransferase. and is then PEGylated using ST3Gal3 and a donor of PEG-sialic acid.
In Figure 31G, IFN-ß expressed in yeast is first treated with mannosidases to trim back the
mannosyl units, then has N-acetylglucosamine added using a N-acetylglucosamine donor and
one or more of GnT-I. II. IV, and V. The protein is further galactosylated using a galactose
donor and a galactosyltransferase, and then PEGylated using ST3Gal3 and a PEG-sialic acid
donor. In Figure 3111, mammalian cell expressed IFN-ß is modified by capping appropriate
terminal residues with a sialic acid donor that is modified with levulinic acid, adding a
reactive ketone to the sialic acid donor. After addition to a glycosyl residue of the peptide,
the ketone is derivatized with a moiety such as a hydrazine- or amine- PEG. In Figure 3 11,
IFN-ß expressed in a mammalian system is PEGylated using a donor of PEG-sialic acid and a
2.8-sialyltransferase. In Figure 3IJ. IFN-ß expressed by mammalian cells is first treated with
sialidase to trim back its terminal sialic acid residues, and then PEGylated using trans-
sialidase and a donor of PFGylated sialic acid. In Figure 31K, IFN-ß expressed in
mammalian cells is first treated with sialidase to trim back terminal sialic acid residues, then
PFGylated using ST3Gal3 and a donor of PEG-sialic acid, and then sialylated using ST3Gal3
and a sialic acid donor. In Figure 31L, IFN-ß expressed in mammalian cells is first treated
with sialidase and galactosidase to trim back the glycosyl chains, then galactosylated using a
galactose donor and an a-galactosyltransferase, and then PEGylated using ST3Gal3 or a
sialyltransfcrase and a donor of PEG-sialic acid. In Figure 31M, IFN-ß expressed in
mammalian cells is first treated with sialidase to trim back the glycosyl units. It is then
PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and is then sialylated using
ST3Gal3 and a sialic acid donor. In Figure 3IN. IFN-ß expressed in mammalian cells is
modified by capping appropriate terminal residues with a sialic acid donor that is modified
with levulinic acid, adding a reactive ketone to the sialic acid donor. After addition to a
glycosyl residue of the peptide, the ketone is derivatized with a moiety such as a hydrazine-
or amine- PEG. In Figure 310, IFN-ß expressed in mammalian cells is sialylated using a
sialic acid donor and a 2,8-sialyltransferase. In Figure 31Q, IFN-ß produced by insect cells
first has N-acetylglucosamine added using a donor of N-acetylglucosamine and one or more
of GnT-I. II, IV, and V, and is further PEGylated using a donor of PEG-galactose and a
galactosyltransferase. In Figure 3IR, IFN-ß expressed in yeast is first treated with
endoglycanase to trim back the glycosyl groups, then galactosylated using a galactose donor
and a galactosyltransferase. and then PEGylated using ST3Gal3 and a donor of PEG-sialic
acid. In Figure 31S. IFN-ß expressed in a mammalian system is first contacted with ST3Gal3
and two reactive sialic acid residues connected via a linker, so that the polypeptide is attached
to one reactive sialic acid via the linker and the second sialic acid residue. The polypeptide is
then contacted with ST3Gal3 and desialylated transferrin, and thus becomes connected with
transferrin via the sialic acid residue. Then, IFN-ß is further sialylated using a sialic acid
donor and ST3Gal3.
In another exemplary embodiment, the invention provides methods for modifying
factor VII or VIIa, as shown in Figures 32 A to 32D. In Figure 32B, Factor VII or VIIa
produced by a mammalian system is first treated with sialidase to trim back the terminal
sialic acid residues, and then PEGylated using ST3Gal3 and a donor of PEGylated sialic acid.
Figure 32C. Factor VII or VIIa expressed by mammalian cells is first treated with sialidase to
trim back the terminal sialic acid residues, and then PEGylated using ST3Gal3 and a donor of
PEGylated sialic acid. Further, the polypeptide is sialylatcd with ST3Gal3 and a sialic acid
donor. Figure 32D offers another modification scheme for Factor VII or VIIa produced by
mammalian cells: the polypeptide is first treated with sialidase and galactosidasc to trim back
its sialic acid and galactose residues, then galactosylated using a galactosyltransferase and a
galactose donor, and then PEGylated using ST3Gal3 and a donor of PEGylated sialic acid.
In another exemplary embodiment, the invention provides methods for modifying
Factor IX, some examples of which are included in Figures 33A to 33G. In Figure 33B,
Factor IX produced by mammalian cells is first treated with sialidase to trim back the
terminal sialic acid residues, and is then PEGylated with ST3GaI3 using a PEG-sialic acid
donor. In Figure 33C, Factor IX expressed by mammalian cells is first treated with sialidase
to trim back the terminal sialic acid residues, it is then PEGylated using ST3Gal3 and a PEG-
sialic acid donor, and further sialylated using ST3Gall and a sialic acid donor. Another
scheme for remodeling mammalian cell produced Factor IX can be found in Figure 33D. The
polypeptide is first treated with sialidase to trim back the terminal sialic acid residues, then
galactosylated using a galactose donor and a galactosyltransferase, further sialylated using a
sialic acid donor and ST3Gal3, and then PEGylated using a donor of PEGylated sialic acid
and ST3Gall. In Figure 33E, Factor IX that is expressed in a mammalian system is
PEGylated through the process of sialylation catalyzed by ST3Gal3 using a donor of PEG-
sialic acid. In Figure 33F, Factor IX expressed in mammalian cells is modified by capping
appropriate terminal residues with a sialic acid donor that is modified with levulinic acid,
adding a reactive ketone to the sialic acid donor. After addition to a glycosyl residue of the
peptide, the ketone is derivatized with a moiety such as a hydrazine- or amine- PFXj. Figure
33G provides an additional method of modifying Factor IX. The polypeptide, produced by
mammalian cells, is PFXjylated using a donor of PEG-sialic acid and a 2.8-sialyltransferase.
In another exemplary embodiment, the invention provides methods for modification
of Follicle Stimulating Hormone (FSH). Figures 34A to 34J present some examples. In
Figure 34B, FSFI is expressed in a mammalian system and modified by treatment of sialidase
to trim back terminal sialic acid residues, followed by PEGylation using ST3Gal3 and a
donor of PEG-sialic acid. In Figure 34C, FSH expressed in mammalian cells is first treated
with sialidase to trim back terminal sialic acid residues, then PEGylated using ST3Gal3 and a
donor of PEG-sialic acid, and then sialylated using ST3Gal3 and a sialic acid donor. Figure
34D provides a scheme for modifying FSH expressed in a mammalian system. The
polypeptide is treated with sialidase and galactosidase to trim back its sialic acid and
galactose residues, then galactosylated using a galactose donor and a galactosyltransferase.
and then PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 34E. FS11
expressed in mammalian cells is modified in the following procedure: FSH is first treated
with sialidase to trim back the sialic acid residues, then PEGylated using ST3Gal3 and a
donor of PEG-sialic acid, and is then sialylated using ST3Gal3 and a sialic acid donor.
Figure 34F offers another example of modifying FSH produced by mammalian cells: The
polypeptide is modified by capping appropriate terminal residues with a sialic acid donor that
is modified with levulinic acid, adding a reactive ketone to the sialic acid donor. After
addition to a glycosyl residue of the peptide, the ketone is derivatized with a moiety such as a
hydrazine- or amine- PFG. In Figure 34G, FSH expressed in a mammalian system is
modified in another procedure: the polypeptide is remodeled with addition of sialic acid using
a sialic acid donor and an a 2.8-sialyltransferasc. In Figure 34H, FSH is expressed in insect
cells and modified in the following procedure: N-acetylglucosamine is first added to FSH
using an appropriate N-acetylglucosamine donor and one or more of GnT-I, II, IV. and V;
FSH is then PEGylated using a donor of PEG-galactose and a galactosyltransferase. Figure
341 depicts a scheme of modifying FSH produced by yeast. According to this scheme, FSH
is first treated with endoglycanase to trim back the glycosyl groups, galactosylated using a
galactose donor and a galactosyltransferase, and is then PEGylated with ST3Gal3 and a donor
of PEG-sialic acid. In Figure 34J, FSH expressed by mammalian cells is first contacted with
ST3Gal3 and two reactive sialic acid residues via a linker, so that the polypeptide is attached
to a reactive sialic acid via the linker and a second sialic acid residue. The polypeptide is
then contacted with ST3Gall and desialylated chorionic gonadotrophin (CG) produced in
CHO, and thus becomes connected with CG via the second sialic acid residue. Then, FSH is
sialylated using a sialic acid donor and ST3Gal3 and/or ST3Gall.
In another exemplary embodiment, the invention provides methods for modifying
erythropoietin (EPO), Figures 35A to 35AA set forth some examples which are relevant to
the remodeling of both wild-type and mutant-EPO-peptides. In Figure 35B, EPO expressed
in various mammalian systems is remodeled by contacting the expressed protein with a
sialidase to remove terminal sialic acid residues. The resulting peptide is contacted with a
sialyltransfcrase and a CMP-sialic acid that is derivatized with a PEG moiety. In Figure 35C.
EPO that is expressed in insect cells is remodeled with N-acetylglucosamine, using GnT-I
and/or GnT-M. Galactose is then added to the peptide, using galactosyltransferase. PEG
group is added to the remodeled peptide by contacting it with a sialyltransferase and a CMP-
sialic acid that is derivatized with a PEG moiety. In Figure 35D. EPO that is expressed in a
mammalian cell system is remodeled by removing terminal sialic acid moieties via the action
of a sialidase. The terminal galactose residues of the N-linked glycosyl units are "capped"
with sialic acid, using ST3Gal3 and a sialic acid donor. The terminal galactose residues on
the O-linked glycan are functionalized with a sialic acid bearing a PEG moiety, using an
appropriate sialic acid donor and ST3Gall. In Figure 35E, EPO that is expressed in a
mammalian cell system is remodeled by functionalizing the N-linked glycosyl residues with a
PHG-derivatized sialic acid moiety. The peptide is contacted with ST3Gal3 and an
appropriately modified sialic acid donor. In Figure 35F, EPO that is expressed in an insect
cell system, yeast or fungi, is remodeled by adding at least one N-acetylglucosamine residues
by contacting the peptide with a N-acetylglucosamine donor and one or more of GnT-I. GnT-
II. and GnT-V. The peptide is then PEGylated by contacting it with a PEGylated galactose
donor and a galactosyltransferase. In Figure 35G, EPO that is expressed in an insect cell
system, yeast or fungi, is remodeled by the addition of at least one N-acetylglucosamine
residues, using an appropriate N-acetylglucosamine donor and one or more of GnT-I, GnT-I I.
and GnT-V. A galactosidase that is altered to operate in a synthetic, rather than a hydrolytic
manner is used to add an activated PEGylated galactose donor to the N-acetylglucosamine
residues. In Figure 35H, EPO that is expressed in an insect cell system, yeast or fungi, is
remodeled by the addition of at least one terminal N-acetylglucosamine-PEG residue. The
peptide is contacted with GnT-I and an appropriate N-acetlyglucosamine donor that is
derivatized with a PEG moiety. In Figure 351, EPO that is expressed in an insect cell system.
yeast or fungi, is remodeled by adding one or more terminal galactose-PEG residues. The
peptide is contacted with GnT-I and an appropriate N-acetylglucosamine donor that is
derivatized with a PEG moiety. The peptide is then contacted with galactosyltransferase and
an appropriate galactose donor that is modified with a PEG moiety. In Figure 35J. EPO
expressed in an insect cell system, yeast or fungi, is remodeled by the addition of one more
terminal sialic acid-PEG residues. The peptide is contacted with an appropriate N-
acetylglueosamine donor and GnT-1. The peptide is further contacted with
galactosyltransferase and an appropriate galactose donor. The peptide is then contacted with
ST3Gal3 and an appropriate sialic acid donor that is derivatized with a PEG moiety. In
Figure 35K, EPO expressed in an insect cell system, yeast or fungi, is remodeled by the
addition of terminal sialic acid-PEG residues. The peptide is contacted with an appropriate
N-acetylglucosamine donor and one or more of GnT-1, GnT-11. and GnT-V. The peptide is
then contacted with galactosyltransferase and an appropriate galactose donor. The peptide is
further contacted with ST3Gal3 and an appropriate sialic acid donor that is derivatized with a
PEG moiety. In Figure 35L, EPO expressed in an insect cell system, yeast or fungi, is
remodeled by the addition of one or more terminal a2,6-sialic acid-PEG residues. The
peptide is contacted with an appropriate N-acetylglucosamine donor and one or more of GnT-
I. GnT-11. and GnT-V. The peptide is further contacted with galactosyltransferase and an
appropriate galactose donor. The peptide is then contacted with a2,6-sialyltransferase and an
appropriately modified sialic acid donor. In Figure 35M, EPO expressed in a mammalian cell
system is remodeled by addition of one or more terminal sialic acid-PEG residues. The
peptide is contacted with a sialidase to remove terminal sialic acid residues. The peptide is
further contacted with a sialyltransferase and an appropriate sialic acid donor. The peptide is
further contacted with a sialyltransferase and an appropriate sialic acid donor that is
derivatized with a PEG moiety. In Figure 35M, EPO expressed in a mammalian cell system
is remodeled by the addition of one or more terminal sialic acid-PEG residues. The peptide is
contacted with a sialyltransferase and an appropriate sialic acid donor that is derivatized with
a PEG moiety. In Figure 350, EPO expressed in a mammalian cell system is remodeled b>
the addition of one or more terminal a2,8-sialic acid-PEG residues to primarily O-linked
glycans. The peptide is contacted with a2,8-sialyltransferase and an appropriate sialic acid
donor that is derivatized with a PEG moiety. In Figure 35P, EPO expressed in a mammalian
cell is remodeled by the addition of one or more terminal a2,8-sialic acid-PEG residues to O-
linkewd and N-linked glycans. The peptide is contacted with a2,8-sialyltransferase and an
appropriate sialic acid donor that is derivatized with a PEG moiety. In Figure 35Q, EPO
expressed in yeast or fungi is remodeled by the addition of one or more terminal sialic acid-
PEG residues. The peptide is contacted with mannosidases to remove terminal mannose
residues. Next, the peptide is contacted with GnT-l and an appropriate N-acctylglucosaminc
donor. The peptide is further contacted with galactosyltransferase and an appropriate
galactose donor. The peptide is then contacted with a sialyltransferase and an appropriate
sialic acid donor that is derivatized with a PEG moiety. In Figure 35R, EPO expressed in
yeast or fungi is remodeled by the addition of at least one terminal N-acetylglucosamine-PEG
residues. The peptide is contacted with mannosidases to remove terminal mannose residue.
The peptide is then contacted with GnT-I and an appropriate N-acetylglucosamine donor that
is derivatized with a PEG moiety. In Figure 35S, EPO expressed in yeast or fungi is
remodeled by the additon of one mor more terminal sialic acid-PEG residues. The peptide is
contacted with mannosidase-I to remove a2 mannose residues. The peptide is further
contacted with GnT-I and an appropriate N-acetylglucosamine donor. The peptide is then
contacted with galactosyltransferase and an appropriate galacose donor. The peptide is then
contacted with a sialyltransferase and an appropriate sialic acid donor that is derivatized with
a PEG moiety. In Figure 35U, EPO expressed in yeast or fungi is remodeled by addition of
one or more galactose-PEG residues. The peptide is contacted with cndo-l I to trim back
glycosyl groups. The peptide is then contacted with galactosyltransferase and an appropriate
galactose donor that is derivatited with a PEG moiety. In Figure 35V. EPO expressed in
yeast or fungi is remodeled by the addition of one or more terminal sialic acid-PEG residues.
The peptide is contacted with endo-H to trim back glycosyl groups. The peptide is further
contacted with galactosyltransferase and an appropriate galactose donor. The peptide is then
contacted with a sialyltransferase and an appropriate sialic acid donor that is derivatized with
a PEG moiety. In Figure 35W. EPO expressed in an insect cell system is remodeled by the
addition of terminal galactose-PEG residues. The peptide is contacted with mannosidases to
remove terminal mannose residues. The peptide is then contacted with galactosyltransferase
and an appropriate galactose donor that is derivatized with a PEG moeity. In Figure 35Y. a
mutant EPO called "novel erythropoiesis-stimulating protein" or "NESP, expressed in NSO
murine myeloma cells is remodeled by capping appropriate terminal residues with a sialic
acid donor that is modified with Icvulinic acid, adding a reactive ketone to the sialic acid
donor. After addition to a glycosyl residue of the peptide, the ketone is derivatized with a
moiety such as a hydrazine- or amine-PEG. In Figure 35Z. mutant EPO. i.e. NESP.
expressed in a mammalian cell system is remodeled by addition of one or more terminal
sialic acid-PEG residues. PEG is added to the glycosyl residue on the glycan using a PEG-
modified sialic acid and an a 2,8-sialyltransferase. In Figure 35AA, NESP expressed in a
mammalian cell system is remodeled by the addition of terminal sialic acid residues. The
sialic acid is added to the glycosyl residue using a sialic acid donor and an ct2,8-
sialyltransferase.
In another exemplary embodiment, the invention provides methods for modifying
granulocyte-macrophage colony-stimulating factor (GM-CSF), as shown in Figures 36A to
36K. In Figure 36B. GM-CSF expressed in mammalian cells is first treated with sialidase to
trim back the sialic acid residues, and then PEGylatcd using ST3Gal3 and a donor of PEG-
sialic acid. In Figure 36C, GM-CSF expressed in mammalian cells is first treated with
sialidase to trim back the sialic acid residues, then PEGylated using ST3Gal3 and a donor of
PEG-sialic acid, and then is further sialylated using a sialic acid donor and ST3Gall and/or
ST3GaI3. In Figure 36D, GM-CSF expressed in NSO cells is first treated with sialidase and
a-galactosidase to trim back the glycosyl groups, then sialylated using a sialic acid donor and
ST3Gal3. and is then PEGylated using ST3Gall and a donor of PEG-sialic acid. In Figure
36E, GM-CSF expressed in mammalian cells is first treated with sialidase to trim back sialic
acid residues, then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and then is
further sialylated using ST3Gal3 and a sialic acid donor. In Figure 36F, GM-CSF expressed
in mammalian cells is modified by capping appropriate terminal residues with a sialic acid
donor that is modified with levulinic acid, adding a reactive ketone to the sialic acid donor.
After addition to a glycosyl residue of the peptide, the ketone is derivatized with a moiety
such as a hydrazine- or amine- PEG. In Figure 36G, GM-CSF expressed in mammalian cells
is sialylated using a sialic acid donor and a 2,8-sialyltransferase. In Figure 361, GM-CSF
expressed in insect cells is modified by addition of N-acetylglucosamine using a suitable
donor and one or more of GnT-I, II. IV. and V, followed by addition of PEGylated galactose
using a suitable donor and a galactosyltransferase. In Figure 36J, yeast expressed GM-CSF is
first treated with endoglycanase and/or mannosidase to trim back the glycosyl units, and
subsequently PEG) lated using a galactosyltransferase and a donor of PEG-galactose. In
figure 36K. GM-CSF expressed in mammalian cells is first treated with sialidase to trim
back sialic acid residues, and is subsequently sialylated using ST3Gal3 and a sialic acid
donor. The polypeptide is then contacted with ST3Gal1 and two reactive sialic acid residues
connected via a linker, so that the polypeptide is attached to one reactive sialic acid via the
linker and second sialic acid residue. The polypeptide is further contacted with ST3Gal3 and
transferrin, and thus becomes connected with transferrin.
In another exemplary embodiment, the invention provides methods for modification
of Interferon gamma (IFN?). Figures 37A to 37N contain some examples. In Figure 37B.
IFN? expressed in a variety of mammalian cells is first treated with sialidase to trim back
terminal sialic acid residues, and is subsequently PEGylated using ST3Gal3 and a donor of
PF.G-sialic acid. In Figure 37C, IFN? expressed in a mammalian system is first treated with
sialidase to trim back terminal sialic acid residues. The polypeptide is then PHGylated using
ST3Gal3 and a donor of PEG-sialic acid, and is further sialylated with ST3Gal3 and a donor
of sialic acid. In Figure 37D, mammalian cell expressed IFN? is first treated with sialidase
and a-galactosidase to trim back sialic acid and galactose residues. The polypeptide is then
galactosylated using a galactose donor and a galactosyltransferase. Then, IFN? is PEGylated
using a donor of PEG-sialic acid and ST3Gal3. In Figure 37E, IFN? that is expressed in a
mammalian system is first treated with sialidase to trim back terminal sialic acid residues.
The polypeptide is then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and is
further sialylated with ST3Gal3 and a sialic acid donor. Figure 37F describes another method
for modifying IFN? expressed in a mammalian system. The protein is modified by capping
appropriate terminal residues with a sialic acid donor that is modified with levulinic acid,
adding a reactive ketone to the sialic acid donor. After addition to a slvcosvl residue of the
peptide, the ketone is derivatized with a moiety such as a hydrazine- or amine- PEG. In
Figure 37G, IFN? expressed in mammalian cells is remodeled by addition of sialic acid using
a sialic acid donor and an a 2,8-sialyltransferase. In Figure 371, IFN? expressed in insect or
fungal cells is modified by addition of N-acetylglucosamine using an appropriate donor and
one or more of GnT-I, II, IV, and V. The protein is further modified by addition of PEG
moieties using a donor of PEGylated galactose and a galactosyltransferase. Figure 37.1 offers
a method for modifying IFN? expressed in yeast. The polypeptide is first treated with
endoglycanase to trim back the saccharide chains, and then galactosylated using a galactose
donor and a galactosyltransferase. Then, IFN? is PEGylated using a donor of PEGylated
sialic acid and ST3Gal3. In Figure 37K, IFN? produced by mammalian cells is modified as
follows: the polypeptide is first contacted with ST3Gal3 and a donor of sialic acid that is
derivatized with a reactive galactose via a linker, so that the polypeptide is attached to the
reactive galactose via the linker and sialic acid residue. The polypeptide is then contacted
with a galactosyltransferase and transferrin pre-treated with endoglycanase, and thus becomes
connected with transferrin via the galactose residue. In the scheme illustrated by Figure 37L.
IFN?. which is expressed in a mammalian system, is modified via the action of ST3Gal3:
PEGylated sialic acid is transferred from a suitable donor to IFN?. Figure 37M is an example
of modifying IFN? expressed in insect or fungal cells, where PEGylation of the polypeptide
is achieved by transferring PEGylated N-acetylglucosamine from a donor to IFN? using GnT-
I and/or II. In Figure 37N, IFN? expressed in a mammalian system is remodeled with
addition of PEGylated sialic acid using a suitable donor and an a 2,8-sialyltransferase.
In another exemplary embodiment, the invention provides methods for modifying a1
anti-trypsin (al-protease inhibitor). Some such examples can be found in Figures 38A to
38N. In Figure 383. a1 anti-trypsin expressed in a variety of mammalian cells is first treated
with sialidase to trim back sialic acid residues. PEGylated sialic acid residues are then added
using an appropriate donor, such as CMP-SA-PEG, and a sialyltransferase, such as ST3Gal3.
Figure 38C demonstrates another scheme of a1 anti-trypsin modification, a1 anti-trypsin
expressed in a mammalian system is first treated with sialidase to trim back sialic acid
residues. Sialic acid residues derivatized with PEG are then added using an appropriate
donor and a sialyltransferase, such as ST3Gal3. Subsequently, the molecule is further
modified by the addition of sialic acid residues using a sialic acid donor and ST3Gal3.
Optionally, mammalian cell expressed a1 anti-trypsin is first treated with sialidase and a-
galactosidase to trim back terminal sialic acid and a-linkage galactose residues. The
polypeptide is then galactosylated using galactosyltransferase and a suitable galactose donor.
Further, sialic acid derivatized with PEG is added by the action of ST3Gal3 using a
PEGylated sialic acid donor. In Figure 38D, a1 anti-trypsin expressed in a mammalian
system first has the terminal sialic acid residues trimmed back using sialidase. PEG is then
added to N-linked glycosyl residues via the action of ST3Gal3, which mediates the transfer of
PEGylated sialic acid from a donor, such as CMP-SA-PEG, to a1 anti-trypsin. More sialic
acid residues are subsequently attached using a sialic acid donor and ST3Gal3. Figure 38E
illustratcs another process through which a1 anti-trypsin is remodeled, a1 anti-trypsin
expressed in mammalian cells is modified by capping appropriate terminal residues with a
sialic acid donor that is modified with levulinic acid, adding a reactive ketone to the sialic
acid donor. After addition to a glycosyl residue of the peptide, the ketone is dcrivatized with
a moiety such as a hydrazine- or amine- PEG. In Figure 38F, yet another method of a1 anti-
trypsin modification is disclosed, a1 anti-trypsin obtained from a mammalian expression
system is remodeled with addition of sialic acid using a sialic acid donor and an a 2,8-
sialyltransferase. In Figure 38H, a1 anti-trypsin is expressed in insect or yeast cells, and
remodeled by the addition of terminal TM-acetylglucosamine residues by way of contacting the
polypeptide with UDP-N-acetylglucosamine and one or more of GnT-1, II, IV, or V. Then,
the polypeptide is modified with PEG moieties using a donor of PEGylated galactose and a
galactosyltransferase. In Figure 381, a1 anti-trypsin expressed in yeast cells is treated first
with endoglycanase to trim back glycosyl chains. It is then galactosylated with a
galactosyltransferase and a galactose donor. Then, the polypeptide is PEGylated using
ST3Gal3 and a donor of PEG-sialic acid. In Figure 38J, so anti-trypsin is expressed in a
mammalian system. The polypeptide is first contacted with ST3Gal3 and a donor of sialic
acid that is derivatized with a reactive galactose via a linker, so that the polypeptide is
attached to the reactive galactose via the linker and sialic acid residue. The polypeptide is
then contacted with a galactosyltransferase and transferrin pre-treated with endoglycanase,
and thus becomes connected with transferrin via the galactose residue. In Figure 38L. a1
anti-trypsin expressed in yeast is first treated with endoglycanase to trim back its glycosyl
groups. The protein is then PEGylated using a galactosyltransferase and a donor of galactose
with a PEG moiety. In Figure 38M, a1 anti-trypsin expressed in plant cells is treated with
hexosaminidase, mannosidase, and xylosidase to trim back its glycosyl chains, and
subsequently modified with N-acetylglucosamine derivatized with a PEG moiety, using N-
acctylglucosamine transferase and a suitable donor. In Figure 38N, a1 anti-trypsin expressed
in mammalian cells is modified by adding PEGylated sialic acid residues using ST3Gal3 and
a donor of sialic acid derivatized with PEG.
In another exemplary embodiment, the invention provides methods for modifying
glucocerebrosidase ((3-glucosidase. Cerezyme™ or Ceredase™), as shown in Figures 39A to
39K. In Figure 39B. Cerezyme™ expressed in a mammalian system is first treated with
sialidase to trim back terminal sialic acid residues, and is then PEGylated using ST3Gal3 and
a donor of PEG-sialic acid. In Figure 39C, Cerezyme™ expressed in mammalian cells is first
treated with sialidase to trim back the sialic acid residues, then has mannose-6-phosphate
group attached using ST3Gal3 and a reactive sialic acid derivatized with mannose-6-
phosphate, and then is sialylated using ST3Gal3 and a sialic acid donor. Optionally, NSO
cell expressed Cerezyme™ is first treated with sialidase and galactosidase to trim back the
glycosyl groups, and is then galactosylated using a galactose donor and an a-
galactosyltransferase. Then, mannose-6-phosphate moiety is added to the molecule using
ST3Gal3 and a reactive sialic acid derivatized with mannose-6-phosphate. In Figure 39D,
Cerezyme™ expressed in mammalian cells is first treated with sialidase to trim back the
sialic acid residues, it is then PEGylated using ST3Gal3 and a donor of PEG-sialic acid, and
is then sialylated using ST3Gal3 and a sialic acid donor. In Figure 39E, Cerezyme™
expressed in mammalian cells is modified by capping appropriate terminal residues with a
sialic acid donor that is modified with levulinic acid, adding a reactive ketone to the sialic
acid donor. After addition to a glycosyl residue of the peptide, the ketone is derivatized with
a moiety such as one or more mannose-6-phosphate groups. In Figure 39F, Cerezyme™
expressed in mammalian cells is sialylated using a sialic acid donor and a 2,8-
sialyltransferase. In Figure 39H, Cerezyme™ expressed in insect cells first has N-
acetylglucosamine added using a suitable donor and one or more of GnT-I. II, IV, and V, and
then is PEGylated using a galactosyltransferase and a donor of PEG-galactose. In Figure 391,
Cerezyme™ expressed in yeast is first treated with endoglycanase to trim back the glycosyl
groups, then galactosylated using a galactose donor and a galactosyltransferase, and then
PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 39JK, Cerezyme™
expressed in mammalian cells is first contacted with ST3Gal3 and two reactive sialic acid
residues connected via a linker, so that the polypeptide is attached to one reactive sialic acid
via the linker and the second sialic acid residue. The polypeptide is then contacted with
ST3Gal3 and desialylated transferrin, and thus becomes connected with transferrin. Then,
the polypeptide is sialylated using a sialic acid donor and ST3Gal3.
In another exemplary embodiment, the invention provides methods for modifying
Tissue-Type Plasminogen Activator (TPA) and its mutant. Several specific modification
schemes arc presented in Figures 40A lo 40W. Figure 40B illustrates one modification
procedure: after TPA is expressed by mammalian cells, it is treated with one or more of
mannosidase(s) and sialidase to trim back mannosyl and/or sialic acid residues. Terminal N-
acetylglucosamine is then added by contacting the polypeptide with a suitable donor of N-
acetylglucosamine and one or more of GnT-I, II, IV, and V. TPA is further galactosylated
using a galactose donor and a galactosyltransferase. Then, PEG is attached to the molecule
by way of sialylation catalyzed by ST3Gal3 and using a donor of sialic acid derivatized with
a PEG moiety. In Figure 40C. TPA is expressed in insect or fungal cells. The modification
includes the steps of addition of N-acetylglucosamine using an appropriate donor of N-
acetyiglucosamine and GnT-I and/or II; galactosylation using a galactose donor and a
galactosyltransferase: and attachment of PEG by way of sialylation using ST3Gal3 and a
donor of sialic acid derivatized with PEG. In Figure 40D, TPA is expressed in yeast and
subsequently treated with endoglycanase to trim back the saccharide chains. The polypeptide
is further PEGylated via the action of a galactosyltransferase. which catalyzes the transfer of
a PEG-galactose from a donor to TPA. In Figure 40E, TPA is expressed in insect or yeast
cells. The polypeptide is then treated with a- and ß- mannosidases to trim back terminal
mannosyl residues. Further, PEG moieties are attached to the molecule via transfer of PKG-
galactose from a suitable donor to TPA, which is mediated by a galactosyltransferase. Figure
40F provides a different method for modification of TPA obtained from an insect or yeast
system: the polypeptide is remodeled by addition of N-acetylglucosamine using a donor of N-
acetylglucosamine and GnT-I and/or II, followed by PEGylation using a galactosyltransferase
and a donor of PEGylated galactose. Figure 40G offers another scheme for remodeling TPA
expressed in insect or yeast cells. Terminal N-acetylglucosamine is added using a donor of
N-acetylglucosamine and GnT-I and/or II. A galactosidase that is modified to operate in a
synthetic, rather than a hydrolytic manner, is utilized to add PEGylated galactose from a
proper donor to the N-acetylglucosamine residues. In Figure 401, TPA expressed in a
mammalian system is first treated with sialidase and galactosidase to trim back sialic acid and
galactose residues. The polypeptide is further modified by capping appropriate terminal
residues with a sialic acid donor that is modified with levulinic acid, adding a reactive ketone
to the sialic acid donor. After addition to a glycosyl residue of the peptide, the ketone is
derivatized with a moiety such as a hydrazine- or amine- PEG. In Figure 40J, TPA, which is
expressed in a mammalian system, is remodeled following this scheme: first, the polypeptide
is treated with a- and ß- mannosidases to trim back the terminal mannosyl residues; sialic
acid residues arc then attached to terminal galactosyl residues using a sialic acid donor and
ST3Gal3; further. TPA is PEGylated via the transfer of PEGylated galactose from a donor to
a N-acctylglucosaminyl residue catalyzed by a galactosyltransferase. In Figure 40K, TPA is
expressed in a plant system. The modification procedure in this example is as follows: TPA
is first treated with hexosaminidase, mannosidase, and xylosidase to trim back its glycosyl
groups: PEGylated N-acctylglucosamine is then added to TPA using a proper donor and N-
acetylglucosamine transferase. In Figure 40M, a TPA mutant (TNK TPA), expressed in
mammalian cells, is remodeled. Terminal sialic acid residues are first trimmed back using
sialidase: ST3Gal3 is then used to transfer PEGylated sialic acid from a donor to TNK TPA,
such that the polypeptide is PEGylated. In Figure 40N. TNK TPA expressed in a mammalian
system is first treated with sialidase to trim back terminal sialic acid residues. The protein is
then PEGylated using CMP-SA-PEG as a donor and ST3Gal3, and further sialylatcd using a
sialic acid donor and ST3Gal3. In Figure 40O, NSO cell expressed TNK TPA is first treated
with sialidase and u-galactosidase to trim back terminal sialic acid and galactose residues.
'INK TPA is then galactosylated using a galactose donor and a galactosyltransferase. The
last step in this remodeling scheme is transfer of sialic acid derivatized with PEG moiety
from a donor to TNK TPA using a sialyltransferase such as ST3Gal3. In Figure 40Q, TNK
TPA is expressed in a mammalian system and is first treated with sialidase to trim back
terminal sialic acid residues. The protein is then PEGylated using ST3Gal3 and a donor of
PEGylated sialic acid. Then, the protein is sialylated using a sialic acid donor and ST3Gal3.
In Figure 40R, TNK TPA expressed in a mammalian system is modified by capping
appropriate terminal residues with a sialic acid donor that is modified with levulinic acid,
adding a reactive ketone to the sialic acid donor. After addition to a glycosyl residue of the
peptide, the ketone is derivatized with a moiety such as a hydrazine- or amine- PEG. In
Figure 40S, TNK TPA expressed in mammalian cells is modified via a different method: the
polypeptide is remodeled with addition of sialic acid using a sialic acid donor and a 2,8-
sialyllransferase. In Figure 40U. TNK TPA expressed in insect cells is remodeled by
addition of N-acctylglucosamine using an appropriate donor and one or more of GnT-I, II,
IV. and V. The protein is further modified by addition of PEG moieties using a donor of
PEGylated galactose and a galactosyltransf'erase. In Figure 40V, TNK TPA is expressed in
yeast. The polypeptide is first treated with endoglycanase to trim back its glycosyl chains
and then PEGylated using a galactose donor derivatized with PEG and a
galactosyltransferase. In Figure 40W, TNK TPA is produced in a mammalian system. The
polypeptide is first contacted with ST3Gal3 and a donor of sialic acid that is derivatized with
a reactive galactose via a linker, so that the polypeptide is attached to the reactive galactose
via the linker and sialic acid residue. The polypeptide is then contacted with a
galactosyltransferase and anti-TNF IG chimera produced in CHO, and thus becomes
connected with the chimera via the galactose residue.
In another exemplary embodiment, the invention provides methods for modifying
Interleukin-2 (II.-2). Figures 41A to 41G provide some examples. Figure 41B provides a
two-step modification scheme: IL-2 produced by mammalian cells is first treated with
sialidase to trim back its terminal sialic acid residues, and is then PEGylated using ST3Gal3
and a donor of PEGylated sialic acid. In Figure 41C, insect cell expressed IL-2 is modified
first by galactosylation using a galactose donor and a galactosyltransferase. Subsequently.
IL-2 is PEGylated using ST3Gal3 and a donor of PEGylated sialic acid. In Figure 41D, IL-2
expressed in bacteria is modified with N-acetylgalactosamine using a proper donor and N-
acetylgalactosamine transferase, followed by a step of PEGylation with a PEG-sialic acid
donor and a sialyltransferase. Figure 41E offers another scheme of modifying IL-2 produced
by a mammalian system. The polypeptide is modified by capping appropriate terminal
residues with a sialic acid donor that is modified with levulinic acid, adding a reactive ketone
to the sialic acid donor. After addition to a glycosyl residue of the peptide, the ketone is
derivatized with a moiety such as a hydrazine- or amine- PEG. Figure 41F illustrates an
example of remodeling IL-2 expressed by E. coli. The polypeptide is PEGylated using a
reactive N-acetylgalactosamine complex derivatized with a PEG group and an enzyme that is
modified so that it functions as a synthetic enzyme rather than a hydrolytic one. In Figure
41G. IL-2 expressed by bacteria is modified by addition of PEGylated N-acetylgalactosam inc-
using a proper donor and N-acetylgalactosamine transferase.
In another exemplary embodiment, the invention provides methods for modifying
Factor VIII, as shown in Figures 42A to 42N. In Figure 42B, Factor VIII expressed in
mammalian cells is first treated with sialidase to trim back the sialic acid residues, and is then
PEGylated using ST3Gal3 and a donor of PKG-sialic acid. In figure 42C. factor VIII
expressed in mammalian cells is first treated with sialidase to trim back the sialic acid
residues, then PEGylated using ST3GaI3 and a proper donor, and is then further sialylated
using ST3Gall and a sialic acid donor.
In figure 42b. mammalian cell produced factor V1I1 is modified by the single step of
PEGylation, using ST3Gal3 and a donor of PEGylated sialic acid, figure 42F offers another
example of modification of Factor VIII that is expressed by mammalian cells. The protein is
PEGylated using ST3Gall and a donor of PEGylated sialic acid. In Figure 42G, mammalian
cell expressed Factor VIII is remodeled following another scheme: it is PEGylated using a
2,8-sialyltransferase and a donor of PEG-sialic acid. In Figure 42 I, Factor VIII produce by
mammalian cells is modified by capping appropriate terminal residues with a sialic acid
donor that is modified with levulinic acid, adding a reactive ketone to the sialic acid donor.
After addition to a glycosyl residue of the peptide, the ketone is derivatized with a moiety
such as a hydrazine- or amine- PEG. In Figure 42J, Factor VIII expressed by mammalian
cells is first treated with Endo-H to trim back glycosyl groups. It is then PEGylated using a
galactosyltransferase and a donor of PEG-galactose. In Figure 42K, Factor VIII expressed in
a mammalian system is first sialylated using ST3Gal3 and a sialic acid donor, then treated
with Endo-H to trim back the glycosyl groups, and then PEGylated with a
galactosyltransferase and a donor of PEG-galactose. In Figure 42L, Factor VIII expressed in
a mammalian system is first treated with mannosidases to trim back terminal mannosyl
residues, then has an N-acetylglucosamine group added using a suitable donor and GnT-I
and/or II. and then is PEGylated using a galactosyltransferase and a donor of PEG-galactose.
In figure 42M. Factor VIII expressed in mammalian cells is first treated with mannosidases
to trim back mannosyl units, then has N-acetylglucosamine group added using N-
acety(glucosamine transferase and a suitable donor. It is further galactosylated using a
galactosyltransferase and a galactose donor, and then sialylated using ST3Gal3 and a sialic
acid donor. In Figure 42N, Factor VIII is produced by mammalian cells and modified as
follows: it is first treated with mannosidases to trim back the terminal mannosyl groups. A
PEGylated N-acetylglucosamine group is then added using GnT-I and a suitable donor of
PEGylated N-acetylglucosamine.
In another exemplary embodiment, the invention provides methods for modifying
urokinase, as shown in Figures 43A to 43M. In Figure 43B, urokinase expressed in
mammalian cells is first treated with sialidase to trim back sialic acid residues, and is then
PEGylated using ST3Gal3 and a donor of PEGylated sialic acid. In Figure 43C. urokinase
expressed in mammalian cells is first treated with sialidase to trim back sialic acid residues,
then PHGylated using ST3Gal3 and a donor of PEGylated sialic acid, and then sialylated
using ST3Gal3 and a sialic acid donor. Optionally, urokinase expressed in a mammalian
system is first treated with sialidase and galactosidase to trim back glycosyl chains, then
galactosylated using a galactose donor and an a-galactosyltransferase. and then PEGylated
using ST3Gal3 or sialyltransferase and a donor of PEG-sialic acid. In Figure 43D, urokinase
expressed in mammalian cells is first treated with sialidase to trim back sialic acid residues,
then PHGylated using ST3Gal3 and a donor of PEG-sialic acid, and then further sialylated
using ST3Gal3 and a sialic acid donor. In Figure 43E, urokinase expressed in mammalian
cells is modified by capping appropriate terminal residues with a sialic acid donor that is
modified with levulinic acid, adding a reactive ketone to the sialic acid donor. After addition
to a glycosyl residue of the peptide, the ketone is derivatized with a moiety such as a
hydrazine- or amine- PEG. In Figure 43F, urokinase expressed in mammalian cells is
sialylated using a sialic acid donor and a 2,8-sialyltransferase. In Figure 43H, urokinase
expressed in insect cells is modified in the following steps: first, N-acetylglucosamine is
added to the polypeptide using a suitable donor of N-acetylglucosamine and one or more of
GnT-1, II, IV, and V; then PEGylated galactose is added, using a galactosyltransferase and a
donor of PEG-galactose. In Figure 431. urokinase expressed in yeast is first treated with
cndoglycanase to trim back glycosyl groups, then galactosylated using a galactose donor and
a galactosyltransferase, and then PEGylated using ST3Gal3 and a donor of PEG-sialic acid.
In Figure 43J. urokinase expressed in mammalian cells is first contacted with ST3Gal3 and
two reactive sialic acid residues that are connected via a linker, so that the polypeptide is
attached to one reactive sialic acid via the linker and second sialic acid residue. The
polypeptide is then contacted with ST3Gal 1 and desialylated urokinase produced in
mammalian cells, and thus becomes connected with a second molecule of urokinase. Then,
the whole molecule is further sialylated using a sialic donor and ST3Gal 1 and/or ST3Gal3.
In Figure 43K, isolated urokinase is first treated with sulfohydrolase to remove sulfate
groups, and is then PEGylated using a sialyltransferase and a donor of PEG-sialic acid. In
Figure 43LM. isolated urokinase is first treated with sulfohydrolase and hexosaminidase to
remove sulfate groups and hexosamine groups, and then PEGylatcd using a
galaetosyltransferase and a donor of PEG-galactose.
In another exemplary embodiment, the invention provides methods for modifying
DNase I, as shown in Figures 44A to 44J. In Figure 44B, DNase I is expressed in a
mammalian system and modified in the following steps: first, the protein is treated with
sialidase to trim baek the sialic acid residues; then the protein is PEGylated with ST3Gal3
using a donor of PEG-sialic acid. In Figure 44C, DNase I expressed in mammalian cells is
first treated with sialidase to trim back the sialic acid residues, then PEGylated with ST3Gal3
using a PEG-sialic acid donor, and is then sialylated using ST3Gal3 and a sialic acid donor.
Optionally. DNase I expressed in a mammalian system is first exposed to sialidase and
galactosidase to trim back the glycosyl groups, then galactosylated using a galactose donor
and an a-galactosyltransferase, and then PEGylated using ST3Gal3 or sialyltransferase and a
donor of PEG-sialic acid. In Figure 44D, DNase I expressed in mammalian cells is first
treated with sialidase to trim back the sialic acid residues, then PEGylated using ST3Gal3 and
a PEG-sialic acid donor, and then sialylated with ST3Gal3 using a sialic acid donor. In
Figure 44E. DNase I expressed in mammalian cells is modified by capping appropriate
terminal residues with a sialic acid donor that is modified with levulinic acid, adding a
reactive ketone to the sialic acid donor. After addition to a glycosyl residue of the peptide,
the ketone is derivatized with a moiety such as a hydrazine- or amine- PEG. In Figure 44F,
DNase I expressed in mammalian cells is sialylated using a sialic acid donor and a 2,8-
sialytransferase. In Figure 44H, DNase I expressed in insect cells first has 1M-
acetylglucosamine added using a suitable donor and one or more of GnT-I, II, IV, and V.
The protein is then PEGylated using a galaetosyltransferase and a donor of PEG-galactose.
In Figure 441, DNase I expressed in yeast is first treated with endoglycanase to trim back the
glycosyl units, then galactosylated using a galactose donor and a galaetosyltransferase, and
then PEGylatcd using ST3Gal3 and a donor of PEG-sialic acid. In Figure 44JK. DNase I
expressed in mammalian cells is first contacted with ST3Gal3 and two reactive sialic acid
residues connected via a linker, so that the polypeptide is attached to one reactive sialic acid
via the linker and the second sialic acid residue. The polypeptide is then contacted with
SI 3Gall and desialylaled ix-1-protease inhibitor, and thus becomes connected with the
inhibitor via the sialic acid residue. Then, the polypeptide is further sialylated using a
suitable donor and ST3Gall and/or ST3Gal3.
In another exemplary embodiment, the invention provides methods for modifying
insulin that is mutated to contain an N-glycosylation site, as shown in Figures 45A to 45L. In
Figure 45B, insulin expressed in a mammalian system is first treated with sialidase to trim
back the sialic acid residues, and then PEGylated using ST3Gal3 and a PEG-sialic acid
donor. In Figure 45C. insulin expressed in insect cells is modified by addition of PEGylated
N-acetylglucosamine using an appropriate donor and GnT-I and/or II. In Figure 45D. insulin
expressed in yeast is first treated with Endo-H to trim back the glycosyl groups, and then
PEGylated using a galactosyltransferase and a donor of PEG-galactose. In Figure 45F,
insulin expressed in mammalian cells is first treated with sialidase to trim back the sialic acid
residues and then PEGylated using ST3Gall and a donor of PEG-sialic acid. In Figure 45G.
insulin expressed in insect cells is modified by means of addition of PEGylated galactose
using a suitable donor and a galactosyltransferase. In Figure 45H, insulin expressed in
bacteria first has N-acetylgalactosamine added using a proper donor and N-
acetylgalactosamine transferase. The polypeptide is then PEGylated using a sialyltransferase
and a donor of PEG-sialic acid. In Figure 45J, insulin expressed in bacteria is modified
through a different method: PEGylated N-acetylgalactosamine is added to the protein using a
suitable donor and N-acetylgalactosamine transferase. In Figure 45K, insulin expressed in
bacteria is modified following another scheme: the polypeptide is first contacted with N-
acetylgalactosamine transferase and a reactive N-acetylgalactosamine that is derivatized with
a reactive sialic acid via a linker, so that the polypeptide is attached to the reactive sialic acid
via the linker and N-acetylgalactosamine. The polypeptide is then contacted with ST3Gal3
and asialo-transferrin. and therefore becomes connected with transferrin. Then, the
polypeptide is sialylated using ST3Gal3 and a sialic acid donor. In Figure 45L, insulin
expressed in bacteria is modified using yet another method: the polypeptide is first exposed to
NHS-CO-linker-SA-CMP and becomes connected to the reactive sialic acid residue via the
linker. The polypeptide is then conjugated to transferrin using ST3Gal3 and asialo-
transferrin. Then, the polypeptide is further sialylated using ST3Gal3 and a sialic acid donor.
In another exemplary embodiment, the invention provides methods for modifying
Hepatitis B antigen (M antigen-preS2 and S), as shown in Figures 46A to 46K. In Figure
46B. M-antigen is expressed in a mammalian system and modified by initial treatment of
sialidase to trim baek the sialic acid residues and subsequent conjugation with lipid A, using
ST3Gal3 and a reactive sialic acid linked to lipid A via a linker. In Figure 46C. M-antigen
expressed in mammalian cells is first treated with sialidase to trim back the terminal sialic
acid residues, then conjugated with tetanus toxin via a linker using ST3Gall and a reactive
sialic acid residue linked to the toxin via the linker, and then sialylated using ST3Gal3 and a
sialic acid donor. In Figure 46D, M-antigen expressed in a mammalian system is first treated
with a galactosidase to trim back galactosyl residues, and then sialylated using ST3Gal3 and
a sialic acid donor. The polypeptide then has sialic acid derivatized with KLH added using
S'llGal 1 and a suitable donor. In Figure 46E, yeast expressed M-antigen is first treated with
a mannosidase to trim back the mannosyl residues, and then conjugated to a diphtheria toxin
using GnT-I and a donor of N-acetylglucosamine linked to the diphtheria toxin. In Figure
46F, mammalian cell expressed M-antigen is modified by capping appropriate terminal
residues with a sialic acid donor that is modified with levulinic acid, adding a reactive ketone
to the sialic acid donor. After addition to a glycosyl residue of the peptide, the ketone is
derivatized with a moiety such as a hydrazine- or amine- PEG. In Figure 46G, M-antigen
obtained from a mammalian system is remodeled by sialylation using a sialic acid donor and
poly a 2,8-sialyltransferase. In Figure 461, M-antigen expressed in insect cells is conjugated
to a Neisseria protein by using GnT-II and a suitable donor of N-acetylglucosamine linked to
the Neisseria protein. In Figure 46J, yeast expressed M-antigen is first treated with
cndoglycanase to trim back its glycosyl chains, and then conjugated to a Neisseria protein
using a galactosyltransferase and a proper donor of galactose linked to the Neisseria protein.
Figure 46K is another example of modification of M-antigen expressed in yeast. The
polypeptide is first treated with mannosidases to trim back terminal mannosyl residues, and
then has N-acetylglucosamine added using GnT-I and/or II. Subsequently, the polypeptide is
galactosylated using a galactose donor and a galactosyltransferase, and then capped with
sialic acid residues using a sialyltransferase and a sialic acid donor.
In another exemplary embodiment, the invention provides methods for modifying
human growth hormone (N, V, and variants thereof), as shown in Figures 47A to 47K. In
Figure 47B. human growth hormone either mutated to contain a N-linked site, or a naturally
occurring isoform that has an N-linkcd side (i.e., the placental enzyme) produced by
mammalian cells is first treated with sialidase to trim back terminal sialic acid residues and
subsequently PEGylated with ST3Gal3 and using a donor of PEGylated sialic acid. In Figure
47C human growth hormone expressed in insect cells is modified by addition of PEGylated
N-acctylglucosaminc using GnT-I and/or II and a proper donor of PEGylated N-
acetylglucosamine. In Figure 47D. human growth hormone is expressed in yeast, treated
with Endo-H to trim back glycosyl groups, and further PEGylated with a
galactosyltransferase using a donor of PEGylated galactose. In Figure 47F. human growth
hormone-mucin fusion protein expressed in a mammalian system is modified by initial
treatment of sialidase to trim back sialic acid residues and subsequent PEGylation using a
donor of PEG-sialic acid and ST3Gall. In Figure 47G, human growth hormone-mucin fusion
protein expressed in insect cells is remodeled by PEGylation with a galactosyltransferase and
using a donor of PEGylated galactose. In Figure 47H, human growth hormone-mucin fusion
protein is produced in bacteria. N-acetylgalactosamine is first added to the fusion protein bthe action of N-acetylgalactosamine transferase using a donor of N-acetylgalactosaminc,
followed by PEGylation of the fusion protein using a donor of PEG-sialic acid and a
sialyltransferase. Figure 471 describes another scheme of modifying bacterially expressed
human growth hormone-mucin fusion protein: the fusion protein is PEGylated through the
action of N-acetylgalactosamine transferase using a donor of PEGylated N-
acetylgalactosamine. Figure 47J provides a further remodeling scheme for human growth
hormone-mucin fusion protein. The fusion protein is first contacted with N-
acelylgalactosamine transferase and a donor of N-acetylgalactosamine that is derivatized with
a reactive sialic acid via a linker, so that the fusion protein is attached to the reactive sialic
acid via the linker and N-acetylgalactosamine. The fusion protein is then contacted with a
sialyltransferase and asialo-transfcrrin. and thus becomes connected with transferrin via the
sialic acid residue. Then, the fusion protein is capped with sialic acid residues using
ST3Gal3 and a sialic acid donor. In Figure 47K, yet another scheme is given for
modification of human growth hormone(N) produced in bacteria. The polypeptide is first
contacted with NHS-CO-linker-SA-CMP and becomes coupled with the reactive sialic acid
through the linker. The polypeptide is then contacted with ST3Gal3 and asialo-transferrin
and becomes linked to transferrin via the sialic acid residue. Then, the polypeptide is
sialylated using ST3Gal3 and a sialic acid donor.
In another exemplary embodiment, the invention provides methods for remodeling
TNI-' receptor IgG fusion protein (TNFR-IgG, or Enbrel™), as shown in Figures 48A to 48G.
Figure 48B illustrates a modification procedure in which TNFR-IgG, expressed in a
mammalian system is first sialylated with a sialic acid donor and a sialyltransferase,
ST3Gal 1: the fusion protein is then galactosylated with a galactose donor and a
galactosyltransferase; then, the fusion protein is PEGylated via the action of ST3Gal3 and a
donor of sialic acid derivatized with PEG. In Figure 48C, TNFR-IgG expressed in
mammalian cells is initially treated with sialidase to trim back sialic acid residues. PEG
moieties are subsequently attached to TNFR-IgG by way of transferring PEGylated sialic
acid from a donor to the fusion protein in a reaction catalyzed by ST3Gal 1. In Figure 48D.
TNFR-IgG is expressed in a mammalian system and modified by addition of PEG through
the galactosylation process, which is mediated by a galactosyltransferase using a PEG-
galactose donor. In Figure 48E, TNFR-IgG is expressed in a mammalian system. The first
step in remodeling of the fusion protein is adding O-linked sialic acid residues using a sialic
acid donor and a sialyltransferase, ST3Gal 1. Subsequently. PEGylated galactose is added to
the fusion protein using a galactosyltransferase and a suitable donor of galactose with a PEG
moiety. In Figure 48I-. TNFR-IgG expressed in mammalian cells is modified first by capping
appropriate terminal residues with a sialic acid donor that is modified with levulinic acid,
adding a reactive ketone to the sialic acid donor. After addition to a glycosyl residue of the
fusion protein, the ketone is derivatized with a moiety such as a hydrazine- or amine- PEG.
In Figure 48G, TNFR-IgG expressed in mammalian cells is remodeled by 2,8-
sialyltransferase, which catalyzes the reaction in which PEGylated sialic acid is transferred to
the fusion protein from a donor of sialic acid with a PEG moiety.
In another exemplary embodiment, the invention provides methods for generating
I lerceptin™ conjugates, as shown in Figures 49A to 49D. In Figure 49B, Herceptin™ is
expressed in a mammalian system and is first galactosylated using a galactose donor and a
galactosyltransferase. I lerceptin™ is then conjugated with a toxin via a sialic acid through
the action of ST3Gal3 using a reactive sialic acid-toxin complex. In Figure 49C. I lerceptin™
produced in either mammalian cells or fungi is conjugated to a toxin through the process of
galactosylation. using a galactosyltransfcrase and a reactive galactose-toxin complex. Figure
49D contains another scheme of making Herceptin™ conjugates: Herceptin™ produced in
fungi is first treated with Endo-H to trim back glycosyl groups, then galactosylated using a
galactose donor and a galactosyltransferase, and then conjugated with a radioisotope by way
of sialylation. by using ST3Gal3 and a reactive sialic acid-radioisotopc complex.
Alternatively, the reactive sialic acid moiety may have attached only the chelating moiety can
then be loaded with radioisotope at a subsequent stage.
In another exemplary embodiment, the invention provides methods for making
Synagis™ conjugates, as shown in Figures 50A to 50D. In Figure 50B, Synagis™ expressed
in mammalian cells is first galactosylated using a galactose donor and a galactosyltransfcrase.
and then PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In Figure 50C.
Synagis™ expressed in mammalian or fungal cells is PEGylated using a galactosyltransferase
and a donor of PEG-galactosc. In Figure 50D, Synagis™ expressed in First treated with
Fndo-H to trim back the glycosyl groups, then galactosylated using a galactose donor and a
galactosyltransfcrase. and is then PEGylated using ST3GaI3 and a donor of PKG-sialic acid.
In another exemplary embodiment, the invention provides methods for generating
Remicade™ conjugates, as shown in Figures 51A to 51D. In Figure 51B, Remicade™
expressed in a mammalian system is first galactosylated using a galactose donor and a
galactosyltransferase, and then PEGylated using ST3Gal3 and a donor of PEG-sialic acid. In
Figure 51C. Remicade™ expressed in a mammalian system is modified by addition of
PEGylated galactose using a suitable donor and a galactosyltransferase. In Figure 51D,
Remicade™ expressed in fungi is first treated with Endo-H to trim back the glycosyl chains,
then galactosylated using a galactose donor and a galactosyltransferase, and then conjugated
to a radioisotope using ST3Gal3 and a reactive sialic acid derivatized with the radioisotope.
In another exemplary embodiment, the invention provides methods for modifying
Reopro. which is mutated to contain an N glycosylation site. Figures 52A to 52L contain
such examples. In Figure 52B, Reopro expressed in a mammalian system is first treated with
sialidase to trim back the sialic acid residues, and then PEGylated using ST3Gal3 and a donor
of PF.G-sialic acid. In Figure 52C Reopro expressed in insect cells is modified by addition
of PEGylated N-acetylglucosamine using an appropriate donor and GnT-I and/or II. In
Figure 52D. Reopro expressed in yeast is first treated with Endo-H to trim back the glycosyl
groups. Subsequently, the protein is PEGylated using a galactosyltransferase and a donor of
PEG-galactose. In Figure 52F, Reopro expressed in mammalian cells is first treated with
sialidase to trim back the sialic acid residues and then PEGylated with ST3Gall using a
donor of PEGylated sialic acid. In Figure 52G. Reopro expressed in insect cells is modified
by PEGylation using a galactosyltransferase and a donor of PEG-galactose. In Figure 5211,
Reopro expressed in bacterial first has N-acetylgalactosamine added using N-
acetylgalactosaminc transferase and a suitable donor. The protein is then PEGylated using a
sialyltransferase and a donor of PEG-sialic acid. In Figure 52J, Reopro expressed in bacteria
is modified in a different scheme: it is PEGylated via the action of N-acetylgalactosaminc
transferase, using a donor of PEGylated N-acetylgalactosamine. In Figure 52K. bacterially
expressed Reopro is modified in yet another method: first, the polypeptide is contacted with
N-acetylgalactosaminc transferase and a donor of N-acetylgalactosamine that is derivatized
with a reactive sialic acid via a linker, so that the polypeptide is attached to the reactive sialic
acid via the linker and N-acetylgalactosaminc. The polypeptide is then contacted with
ST3Gal3 and asialo-transferrin and thus becomes connected with transferrin via the sialic
acid residue. Then, the polypeptide is capped with sialic acid residues using a proper donor
and ST3Gal3. Figure 52L offers an additional scheme of modifying bacterially expressed
Reopro. The polypeptide is first exposed to NHS-CO-linker-SA-CMP and becomes
connected with the reactive sialic acid through the linker. The polypeptide is then contacted
with ST3Gal3 and asialo-transferrin and thus becomes connected with transferrin via the
sialic acid residue. Then, the polypeptide is capped with sialic acid residues using a proper
donor and ST3Gal3.
In another exemplary embodiment, the invention provides methods for producing
Rituxan™ conjugates. Figures 53A to 53G presents some examples. In Figure 53B.
Riluxan™ expressed in various mammalian systems is first galactosylated using a proper
galactose donor and a galactosyltransferase. The peptide is then functionalized with a sialic
acid derivatized with a toxin moiety, using a sialic acid donor and ST3Gal3. In Figure 53C,
Rituxan™ expressed in mammalian cells or fungal cells is galactosylated using a
galactosyltransferase and a galactose donor, which provides the peptide galactose containing
a drug moiety. Figure 53D provides another example of remodeling Rituxan™ expressed in
a fungal system. The polypeptide's glycosyl groups are first trimmed back using Endo-M.
Galactose is then added using a galactosyltransferase and a galactose donor. Subsequently, a
radioisotope is conjugated to the molecule through a radioisotope-complexed sialic acid
donor and a sialyltransferase, ST3Gal3. In Figure 53F, Rituxan™ is expressed in a
mammalian system and first galactosylated using a galactosyltransferase and a proper
galactose donor; sialic acid with a PEG moiety is then attached to the molecule using
ST3Gal3 and a PF.Gylated sialic acid donor. As shown in Figure 53G. Rituxan™ expressed
in fungi, yeast, or mammalian cells can also be modified in the following process: first, the
polypeptide is treated with a- and ß- mannosidases to remove terminal mannosyl residues:
GlcNAc is then attached to the molecule using GnT-I, II and a GlcNAc donor, radioisotope is
then attached by way of galactosylation using a galactosyltransferase and a donor of
galactose that is coupled to a chelating moiety capable of binding a radioisotope.
In another exemplar) embodiment, the invention provides methods for modifying
anti-thrombin III (AT III). Figures 54A to 540 present some examples. In Figure 54B, anti-
thrombin III expressed in various mammalian systems is remodeled by the addition of one or
more terminal sialic acid-PEG moieties. The AT III molecule is first contacted with sialidase
to remove terminal sialic acid moieties. Then, the molecule is contacted with a
sialyltransferase and an appropriate sialic acid donor that has been derivatized with a PEG
moiety. In Figure 54C. AT III expressed in various mammalian systems is remodeled by the
addition of sialic acid-PEG moieties. The AT III molecule is contacted with sialidase to
remove terminal sialic acid moieties. The molecule is then contacted with a ST3Gal3 and an
appropriate sialic acid donor that has been derivatized with a PEG moiety at 1.2 mol eq. The
molecule is then contacted with a ST3Gal3 and an appropriate sialic acid donor to cap
remaining terminal galactose moieties. In Figure 54D, AT III is expressed in NSO murine
myeloma cells is remodeled to have complex glycan molecules with terminal sialic acid-PEG
moieties. The AT 111 molecule is contacted with sialidase and cx-galactosidase to remove
terminal sialic acid and galactose moieties. The molecule is then contacted with
galactosyltransferase and an appropriated galactose donor. The molecule is then contacted
with a ST3Gal3 and an appropriate sialic acid donor that has been derivatized with a PEG
moiety. In Figure 54E. AT III expressed in various mammalian systems is remodeled to have
nearly complete terminal sialic acid-PEG moieties. The AT III molecule is contacted with
sialidase to remove terminal sialic acid moieties. The molecule is then contacted with a
ST3Gal3 and an appropriate sialic acid donor that has been derivatized with a PEG moiety at
16 mol cq. The molecule is then contacted with ST3Gal3 and an appropriate sialic acid
donor to cap remaining terminal galactose moieties. In Figure 54F, AT III expressed in
various mammalian systems is remodeled by the addition of one or more terminal sialic acid
PEG moieties. The AT III molecule is contacted with ST3Gal3 and an appropriate sialic acid
donor that has been derivatized with a levulinatc moiety. The molecule is then contacted
with hydrazine-PEG. In Figure 54G, AT III expressed in various mammalian systems is
remodeled by the addition of one or more terminal poly-a2,8-linked sialic acid moieties. The
AT III molecule is contacted with poly-a2,8-sialyltransferase and an appropriate sialic acid
donor. In Figure 541. AT III expressed in insect, yeast or fungi cells is remodeled by the
addition of branching N- N-acetylglucosamine -PEG moieties. The AT III molecule is
contacted with GnT-I and an appropriate N-acetylglucosamine donor that has been
derivatized with PEG. In Figure 54.1, AT III expressed in yeast is remodeled by removing
high mannose glycan structures and the addition of terminal sialic acid-PEG moieties. The
AT III molecule is contacted with endoglycanase to trim back glycosyl groups. The molecule
is then contacted with galaetosyltransferase and an appropriate galactose donor. The
molecule is then contacted with ST3Gal3 and an appropriate sialic acid donor that has been
derivatized with a PEG moiety. In Figure 54K, AT III expressed in various mammalian
systems is remodeled by the addition of glycoconjugated transferrin. The AT III molecule is
contacted with ST3Gal3 and an appropriate sialic acid donor that has been derivatized with a
linker-galactose donor moiety. The molecule is then contacted with galaetosyltransferase and
endoglycanase-treated transferrin. In Figure 54M, AT III expressed in yeast is remodeled by
the removal of mannose glycan structures and the addition of terminal galactose-PEG
moieties. The molecule is contacted with endoglycanase to trim back glycosyl groups. The
molecule is further contacted with galaetosyltransferase and an appropriate galactose donor
that has been derivatized with a PEG moiety. In Figure 54N, AT III expressed in plant cells
is remodeled by converting the glycan structures into mammalian-type complex glycans and
then adding one or more terminal galactose-PEG moieties. The AT III molecule is contacted
with xylosidase to remove xylose residues. The molecule is then contacted with
galaetosyltransferase and an appropriate galactose donor that has been derivatized with a
PEG moiety. In Figure 540, AT III expressed in various mammalian systems is remodeled
by the addition of one or more terminal sialic acid-PEG moieties to terminal galactose
moieties. The AT 111 molecule is contacted with ST3Gal3 and an appropriate sialic acid PEG
donor that has been derivatized with PEG.
In another exemplary embodiment, the invention provides methods for modifying the
a and ß subunits of human Chorionic Gonadotropin (hCG). Figures 55 A to 55.1 present some
examples. In Figure 55B, hCG expressed in various mammalian and insect systems is
remodeled by the addition of terminal sialic acid-PEG moieties. The hCG molecule is
contacted with sialidase to remove terminal sialic acid moieties. The molecule is then
contacted with ST3Gal3 and an appropriate sialic acid donor molecule that has been
derivatized with a PEG moiety. In Figure 55C, hCG expressed in insect cell, yeast or fungi
systems is remodeled by building out the N-linked glycans and the addition of terminal sialic
acid-PEG moieties. The hCG molecule is contacted with GnT-I and GnT-II, and an
appropriated N-acetylglucosamine donor. The molecule is then contacted with
galactosyltransfcrasc and an appropriate galactose donor. The molecule is further contacted
with ST3Gal3 and an appropriate sialic acid donor that has been derivatized with a PEG
moiety. In Figure 55D. hCG expressed in various mammalian and insect systems is
remodeled by the addition of one or more terminal sialic acid-PEG moieties on O-linked
glycan structures. The hCG molecule is contacted with sialidase to remove terminal sialic
acid moieties. The molecule is then contacted with ST3Gal3 and an appropriate sialic acid
donor to cap the glycan structures with sialic acid moieties. The molecule is then contacted
with ST3Gal 1 and an appropriate sialic acid donor that has been derivatized with PEG. In
Figure 55E, hCG expressed in various mammalian and insect systems is remodeled by the
addition of sialic acid-PEG moieties to N-linked glycan structures. The hCG molecule is
contacted with ST3Gal3 and an appropriate sialic acid donor that has been derivatized with
PEG. In Figure 55F, hCG expressed in insect cells, yeast or fungi, is remodeled by the
addition of terminal N-acetylglucosamine-PEG molecules. The hCG molecule is contacted
with GnT-I and GnT-II. and an appropriate N-acetylglucosamine donor that has been
derivatized with PEG. In Figure 55G, hCG expressed in insect cells, yeast or fungi, is
remodeled by the addition of not more than one N-acetylglucosamine-PEG moiety per N-
linked glycan structure. The hCG molecule is contacted with GnT-I and an appropriate N-
acetylglucosamine donor that has been derivatized with a PEG moiety. In Figure 55H, hCG
expressed in various mammalian systems is remodeled by the addition of one or more
terminal sialic acid-PEG moiety to O-linked glycan structures. The hCG molecule is
contacted with ST3Gal3 and an appropriate sialic acid donor that has been derivatized with
PEG. In Figure 551. hCG expressed in various mammalian systems is remodeled by the
addition of terminal sialic acid-PEG moieties. The hCG molecule is contacted with a2.8-SA
and an appropriate sialic acid donor that has been derivatized with a PEG moiety. In Figure
55J. hCG expressed in various mammalian systems is remodeled by the addition of terminal
sialic acid moieties. The hCG molecule is contacted with poly-alpha2,8-ST and an
appropriate sialic acid donor that has been derivatized with a PEG moiety.
In another exemplary embodiment, the invention provides methods for modifying
alpha-galactosidase A (Fabrazyme™). Figures 56A to 56J present some examples. In Figure
56B. alpha-galactosidase A expressed in and secreted from various mammalian and insect
systems is remodeled by the addition of one or more terminal galactose-PEG-transferrin
moieties. The alpha-galactosidase A molecule is contacted with Endo-H to trim back
glycosyl groups. The molecule is then contacted with galactosyltransferase and an
appropriate galactose donor that has been derivatized with PEG and transferrin. In Figure
56C, alpha-galactosidase A expressed in and secreted from various mammal and insect cell
systems is remodeled by the addition of one or more terminal sialic acid-linker-mannose-6-
phosphate moieties. The alpha-galactosidase A molecule is contacted with sialidase to
remove terminal sialic acid moieties. The molecule is further contacted with ST3Gal3 and an
appropriate sialic acid donor that has been conjugated via a linker to mannose-6-phosphate.
In Figure 56D, alpha-galactosidase A expressed in NSO murine myeloma cells is remodeled
by the addition of terminal sialic acid-linker-mannose-6-phosphate moieties. The alpha-
galactosidase A molecule is contacted with sialidase and ct-galactosidase to remove terminal
sialic acid and galactose moieties. The molecule is then contacted with galactosyltransferase
and an appropriate galactose donor. The molecule is then contacted with sialyltransferase
and an appropriate sialic acid donor that has been conjugated via a linker to mannose-6-
phosphate. In Figure 56E, alpha-galactosidase A expressed in and secreted from various
mammalian and insect cell systems is remodeled by the addition of one or more terminal
sialic acid-PEG moieties. The alpha-galactosidase A molecule is contacted with sialidase to
remove terminal sialic acid moieties. The molecule is then contacted with sialyltransferase
and an appropriate sialic acid donor that has been derivatized with a PEG moiety. In Figure
56F. alpha-galactosidase A expressed in mammalian, insect, yeast or fungi systems, is
remodeled by the addition of one or more terminal mannose-linker-ApoE moieties. The
alpha-galactosidase A molecule is contacted with mannosyltransferase and an appropriate
mannose donor that has been conjugated via a linker to ApoE. In Figure 56G, alpha-
galactosidase A expressed in mammalian, insect, yeast or fungal systems is remodeled by the
addition of galactose-linker-alpha2-macroglobulin moieties. The alpha-galactosidase A
molecule is contacted with Endo-H to trim back glycosyl groups. The molecule is then
contacted with galactosyltransferase and an appropriate galactose donor that has been
conjugated via a linker to alpha2-macroglobulin. In Figure 56H, alpha-galactosidase A
expressed in insect, yeast and fungal systems, is remodeled by the addition of one or more N-
acetylglucosamine-PEG-mannose-6-phosphate moieties. The alpha-galactosidase molecule
is contacted with GnT-I and an appropriate N-acetyl-glucosamine donor that has been
derivatized with PEG and mannose-6-phosphate. In Figure 561, alpha-galactosidase A
expressed in insect, yeast or fungal systems, is remodeled by the addition of one or more
terminal galactosc-PEG-transferrin moieties. The alpha-galactosidase A molecule is
contacted with GnT-I and an appropriate N-acetyl-glucosamine donor. The molecule is then
contacted with galactosyltransferase and an appropriate galactose donor that has been
derivatized with PEG and transferrin. In Figure 56J. alpha-galactosidase A expressed in
insect, yeast or fungi systems is remodeled by the addition of one or more terminal sialic
acid-PEG-melanotransferrin moieties. The alpha-galactosidase A molecule is contacted with
GnT-I and GnT-I 1 and an appropriate N-acetyl-glucosaminc donor. The molecule is then
contacted with galactosyltransferase and an appropriate galactose donor. The molecule is
then contacted with sialytransferase and an appropriate sialic acid donor that has been
derivatized with PEG and melanotransferrin.
In another exemplary embodiment, the invention provides methods for modifying
alpha-iduronidase (Aldurazyme™). Figures 57A to 57J present some examples. In Figure
57B, alpha-iduronidase expressed in and secreted from various mammalian and insect
systems is remodeled by the addition of one or more terminal galactose-PEG-transferrin
moieties. The alpha-iduronidase molecule is contacted with Endo-H to trim back glycosyl
groups. The molecule is then contacted with galactosyltransferase and an appropriate
galactose donor that has been derivatized with PEG and transferrin. In Figure 57C, alpha-
iduronidase expressed in and secreted from various mammal and insect cell systems is
remodeled by the addition of terminal sialic acid-linker-mannose-6-phosphate moieties. The
alpha-iduronidase molecule is contacted with sialidase to remove terminal sialic acid
moieties. The molecule is then contacted with ST3Gal3 and an appropriate sialic acid donor
that has been conjugated via a linker to mannose-6-phosphate. In Figure 57D, alpha-
iduronidase expressed in NSO murine myeloma cells is remodeled by the addition of one or
more terminal sialic acid-linker-mannose-6-phosphatc moieties. The alpha-iduronidase
molecule is contacted with sialidase and a-galactosidase to remove terminal sialic acid and
galactose moieties. The molecule is then contacted with galactosyltransferase and an
appropriate galactose donor. The molecule is further contacted with sialyltransferase and an
appropriate sialic acid donor that has been conjugated via a linker to mannose-6-phosphate.
In Figure 57E, alpha-iduronidase expressed in and secreted from various mammalian and
insect cell systems is remodeled by the addition of one or more terminal sialic acid-PEG
moieties. The alpha-iduronidase molecule is contacted with sialidase to remove terminal
sialic acid moieties. The molecule is further contacted with sialyltransferase and an
appropriate sialic acid donor that has been derivatized with a PEG moiety. In Figure 57F,
alpha-iduronidase expressed in mammalian, insect, yeast or fungi systems is remodeled by
the addition of one or more terminal mannose-linker-ApoE moieties. The alpha-iduronidase
molecule is contacted with mannosyltransferase and an appropriate mannose donor that has
been conjugated via a linker to ApoE. In Figure 57G, alpha-iduronidase expressed in
mammalian, insect, yeast or fungal systems is remodeled by the addition of one or more
galactose-linker-alpha2-macroglobulin moieties. The alpha-iduronidase molecule is
contacted with Endo-H to trim back glycosyl groups. The molecule is then contacted with
galactosyltransferase and an appropriate galactose donor that has been conjugated via a linker
to alpha2-macroglobulin. In Figure 57H, alpha-iduronidase expressed in insect, yeast and
fungal systems, is remodeled by the addition of one or more N-acetylglucosamine-PEG-
mannose-6-phosphate moieties. The alpha-galactosidase molecule is contacted with Gn'f-I
and an appropriate N-acetyl-glucosamine donor that has been derivatized with PEG and
mannose-6-phosphate. In Figure 571. alpha-iduronidase expressed in insect, yeast or fungal
systems, is remodeled by the addition of one or more terminal galactose-PEG-transferrin
moieties. The alpha-iduronidasc molecule is contacted with GnT-1 and an appropriate N-
acetyl-glucosamine donor. The molecule is then contacted with galactosyltransferase and an
appropriate galactose donor that has been derivatized with PEG and transferrin. In Figure
57.1, alpha-iduronidase expressed in insect, yeast or fungi systems, is remodeled by the
addition of one or more terminal sialic acid-PEG-melanotransferrin moieties. The alpha-
iduronidase molecule is contacted with GnT-I and GnT-II and an appropriate N-acetyl-
glucosamine donor. The molecule is then contacted with galactosyltransferase and an
appropriate galactose donor. The molecule is further contacted with sialyltransferase and an
appropriate sialic acid donor that has been derivatized with PEG and melanotransferrin.
A. Creation or elimination of N-linked glycosylation sites
The present invention contemplates the use of peptides in which the site of the glycan
chain(s) on the peptide have been altered from that of the native peptide. Typically, N-linked
gKcan chains are linked to the primary peptide structure at asparaginc residues where the
asparagine residue is within an amino acid sequence that is recognized by a membrane-bound
glycosyltransferase in the endoplasmic reticulum (ER). Typically, the recognition site on the
primary peptide structure is the sequence asparagine-X-serine/threonine where X can be any
amino acid except proline and aspartic acid. While this recognition site is typical, the
invention further encompasses peptides that have N-linked glycan chains at other recognition
sites where the N-linked chains are added using natural or recombinant glycosyltransferases.
Since the recognition site for N-linked glycosylation of a peptide is known, it is
within the skill of persons in the art to create mutated primary peptide sequences wherein a
native N-linked glycosylation recognition site is removed, or alternatively or in addition, one
or more additional N-glycosylation recognition sites are created. Most simply, an asparagine
residue can be removed from the primary sequence of the peptide thereby removing the
attachment site for a glycan. thus removing one glycan from the mature peptide. For
example, a native recognition site with the sequence of asparaaine-serine-serine can be
genetically engineered to have the sequence leucine-serine-serine, thus eliminating a N-
linkcd glycosylation site at this position.
Further, an N-linked glycosylation site can be removed by altering the residues in the
recognition site so that even though the asparagine residue is present, one or more of the
additional recognition residues are absent. For example, a native sequence of asparagine-
scrine-serine can be mutated to asparagine-serine-lysine, thus eliminating an N-glycosylation
site at that position. In the case of N-linked glycosylation sites comprising residues other
than the typical recognition sites described above, the skilled artisan can determine the
sequence and residues required for recognition by the appropriate glycosyltransferase, and
then mutate at least one residue so the appropriate glycosyltransferase no longer recognizes
that site. In other words, it is well within the skill of the artisan to manipulate the primary
sequence of a peptide such that glycosylation sites are either created or are removed, or both,
thereby generating a peptide having an altered glycosylation pattern. The invention should
therefore not be construed to be limited to any primary peptide sequence provided herein as
the sole sequence for glycan remodeling, but rather should be construed to include any and all
peptide sequences suitable for glycan remodeling.
To create a mutant peptide, the nucleic acid sequence encoding the primary sequence
of the peptide is altered so that native codons encoding native amino acid residues arc
mutated to generate a codon encoding another amino acid residue. Techniques for altering
nucleic acid sequence are common in the art and are described for example in any well-
known molecular biology manual.
In addition, the nucleic acid encoding a primary peptide structure can be synthesized
in vitro, using standard techniques. For example, a nucleic acid molecule can be synthesized
in a "gene machine" using protocols such as the phosphoramidite method. If chemically-
synthesized double stranded DNA is required for an application such as the synthesis of a
nucleic acid or a fragment thereof, then each complementary strand is synthesized
separately. The production of short nucleic acids (60 to 80 base pairs) is technically
straightforward and can be accomplished by synthesizing the complementary strands and
then annealing them. For the production of longer nucleic acids (>300 base pairs), special
strategies may be required, because the coupling efficiency of each cycle during chemical
DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-
stranded) are assembled in modular form from single-stranded fragments that are froM 20 to
100 nucleotides in length. For reviews on polynucleotide synthesis, see, for example. Click
and Pasternak (Molecular Biotechnology, Principles and Applications of Recombinant DNA.
1994. ASM Press). Itakura et al. (1984. Annu. Rev. Biochem. 53:323), and Climie et al.
(1990. Proc. Nat'l Acad. Sci. USA 87:633).
Additionally, changes in the nucleic acid sequence encoding the peptide can be made
bv silo-directed mutagenesis. As will be appreciated, this technique typically employs a
phage vector which exists in both a single stranded and double stranded form. Typical
vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These
phage are readily available and their use is generally well known to those skilled in the art.
Double stranded plasmids are also routinely employed in site-directed mutagenesis which
eliminates the step of transferring the nucleic acid of interest from a plasmid to a phage.
In general, site-directed mutagenesis is performed by first obtaining a single-stranded
vector or melting the two strands of a double stranded vector which includes within its
sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer
bearing the desired mutated sequence is prepared generally synthetically. This primer is then
annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such
as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-
bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-
mutated sequence and the second strand bears the desired mutation. This heteroduplex vector
is then used to transform or transfect appropriate cells, such as E. coli cells, and clones are
selected which include recombinant vectors bearing the mutated sequence arrangement. A
genetic selection scheme was devised by Kunkel et al. (1987, Kunkel et al.. Methods
Enzymol. 154:367-382) to enrich for clones incorporating the mutagenic oligonucleotide.
Alternatively, the use of PCR™ with commercially available thermostable enzymes such as
Taq polymerase may be used to incorporate a mutagenic oligonucleotide primer into an
amplified DNA fragment that can then be cloned into an appropriate cloning or expression
vector. The PCR™-mediated mutagenesis procedures of Tomic et al. (1990, Nucl. Acids
Res., 12:1656) and Upenderet al. (1995, Biotechniques, 18:29-31) provide two examples of
such protocols. A PCR™ employing a thermostable ligase in addition to a thermostable
polymerase may also be used to incorporate a phosphorylated mutagenic oligonucleotide into
an amplified DNA fragment that may then be cloned into an appropriate cloning or
expression vector. The mutagenesis procedure described by Michael (1994. Biotechniques
16:410-412) provides an example of one such protocol.
Not all Asn-X-Ser/Thr sequences are N-glycosylated suggesting the context in which
the motif is presented is important. In another approach, libraries of mutant peptides having
novel N-linked consensus sites are created in order to identify novel N-linked sites that are
glycosylated in vivo and arc beneficial to the activity, stability or other characteristics of the
peptide.
As noted previously, the consensus sequence for the addition of "N-linked glycan
chains in glycoproteins is Asn-X-Ser/Thr where X can be any amino acid. The nucleotide
sequence encoding the amino acid two positions to the carboxyl terminal side of the Asn may
be mutated to encode a Ser and/or Thr residue using standard procedures known to those of
ordinary skill in the art. As stated above not all Asn-X-Ser/Thr sites are modified by the
addition of glycans. Therefore, each recombinant mutated glycoprotein must be expressed in
a fungal, yeast or animal or mammalian expression system and analyzed for the addition of
an N-linked glycan chain. The techniques for the characterization of glycosylation sites are
well known to one skilled in the art. Further, the biological function of the mutated
recombinant glycoprotein can be determined using assays standard for the particular protein
being examined. Thus, it becomes a simple matter to manipulate the primary sequence of a
peptide and identify novel glycosylation sites contained therein, and further determine the
effect of the novel site on the biological activity of the peptide.
In an alternative embodiment, the nucleotide sequence encoding the amino acid two
positions to the amino terminal side of Ser/Thr residues may be mutated to encode an Asn
using standard procedures known to those of ordinary skill in the art. The procedures to
determine whether a novel glycosylation site has been created and the effect of this site on the
biological activity of the peptide are described above.
B. Creation or elimination of O-linked glycosylation sites
The addition of an O-linked glycosylation site to a peptide is conveniently
accomplished by altering the primary amino acid sequence of the peptide such that it contains
one or more additional O-linked glycosylation sites compared with the beginning primary
amino acid sequence of the peptide. The addition of an O-linked glycosylation site to the
peptide may also be accomplished by incorporation of one or more amino acid species into
the peptide which comprises an -OH group, preferably serine or threonine residues, within
the sequence of the peptide, such that the OH group is accessible and available for O-linked
glycosylation. Similar to the discussion of alteration of N-linked glycosylation sites in a
peptide, the primary amino acid sequence of the peptide is preferably altered at the nucleotide
level. Specific nucleotides in the DNA sequence encoding the peptide may be altered such
that a desired amino acid is encoded by the sequence. Mutation(s) in DNA arc preferably
made using methods known in the art. such as the techniques of phosphoramidite method
DNA synthesis and site-directed mutagenesis described above.
Alternatively, the nucleotide sequence encoding a putative site for O-linked glycan
addition can be added to the DNA molecule in one or several copies to either 5' or the 3' end
of the molecule. The altered DNA sequence is then expressed in any one of a fungal, yeast,
or animal or mammalian expression system and analyzed for the addition of the sequence to
the peptide and whether or not this sequence is a functional O-linked glycosylation site.
Briefly, a synthetic peptide acceptor sequence is introduced at either the 5' or 3' end of the
nucleotide molecule. In principle, the addition of this type of sequence is less disruptive to
the resulting glycoprotein when expressed in a suitable expression system. The altered DNA
is then expressed in CHO cells or other suitable expression system and the proteins expressed
thereby are examined for the presence of an O-linked glycosylation site. In addition, the
presence or absence of glycan chains can be determined.
In yet another approach, advantageous sites for new O-linked sites may be found in a
peptide by creating libraries of the peptide containing various new O-linked sites. For
example, the consensus amino acid sequence for N-acetylgalactosamine addition by an N-
acetylgalactosaminyltransferase depends on the specific transferase used. The amino acid
sequence of a peptide may be scanned to identify contiguous groups of amino acids that can
be mutated to generate potential sites for addition of O-linked glycan chains. These
mutations can be generated using standard procedures known to those of ordinary skill in the
art as described previously. In order to determine if any discovered glycosylation site is
actually glycosylated, each recombinant mutated peptide is then expressed in a suitable
expression system and is subsequently analyzed for the addition of the site and/or the
presence of an O-linked glycan chain.
C. Chemical synthesis of peptides
While the primary structure of peptides useful in the invention can be generated most
efficiently in a cell-based expression system, it is within the scope of the present invention

that the peptides may be generated synthetically. Chemical synthesis of peptides is well
known in the art and include, without limitation, stepwise solid phase synthesis, and fragment
condensation either in solution or on solid phase. A classic stepwise solid phase synthesis of
involves covalcntly linking an amino acid corresponding to the carboxy-terminal amino acid
of the desired peptide chain to a solid support and extending the peptide chain toward the
amino end by stepwise coupling of activated amino acid derivatives having activated
carboxyl groups. After completion of the assembly of the fully protected solid phase bound
peptide chain, the peptide-solid phase covalent attachment is cleaved by suitable chemistry
and the protecting groups are removed to yield the product peptide. See, R. Merrifield, Solid
Phase Peptide Synthesis: The Synthesis of a Tetrapeptide, J. Am. Chem. Soc, 85:2149-2154
(1963). The longer the peptide chain, the more challenging it is to obtain high-purity well-
defined products. Due to the production of complex mixtures, the stepwise solid phase
synthesis approach has size limitations. In general, well-defined peptides of 100 contiguous
amino acid residues or more are not routinely prepared via stepwise solid phase synthesis.
The segment condensation method involves preparation of several peptide segments
by the solid phase stepwise method, followed by cleavage from the solid phase and
purification of these maximally protected segments. The protected segments are condensed
one-by-one to the first segment, which is bound to the solid phase.
The peptides useful in the present invention may be synthesized by exclusive solid
phase synthesis, partial solid phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill in the art (see. for
example. Merrifield. J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.. "Solid Phase Peptide
Synthesis" (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot. 3:3
(1986). Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach (1RL Press
1989), Fields and Colowick. "Solid-Phase Peptide Synthesis," Methods in Enzymology
Volume 289 (Academic Press 1997), and Lloyd-Williams et al., Chemical Approaches to the
Synthesis of Peptides and Peptides (CRC Press, Inc. 1997)). Variations in total chemical
synthesis strategies, such as "native chemical ligation" and "expressed peptide ligation" are
also standard (see, for example. Dawson et al., Science 266:776 (1994), Hackeng et al., Proc.
Nat'l Acad. Sci. USA 94:7845 (1997), Dawson, Methods Enzymol. 287: 34 (1997). Muir et
al. Proc. Nat'l Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol. Chem.
273:16205 (1998)). Also useful arc the solid phase peptide synthesis methods developed bGryphon Sciences. South San Francisco, CA. See, U.S. Patent Nos. 6,326.468, 6,217.873.
6.174.530. and 6,001,364. all of which are incorporated in their entirety by reference herein.
P. Post-translational modifications
It will be appreciated to one of ordinary skill in the art that peptides may undergo
post-translational modification besides the addition of N-linked and/or O-linked glycans
thereto. It is contemplated that peptides having post-translational modifications other than
glycosylation can be used as peptides in the invention, as long as the desired biological
activity or function of the peptide is maintained or improved. Such post-translational
modifications may be natural modifications usually carried out in vivo, or engineered
modifications of the peptide carried out in vitro. Contemplated known modifications include,
but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization. disulfide bond formation,
demethylation, formation of covalent crosslinks, formation of cysteine, formation of
pyroglutamate. formylation. gamma carboxylation, glycosylation, GP1 anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to peptides such as arginylation, and ubiquitination. Enzymes that
may be used to carry out many of these modifications are well known in the art, and available
commercially from companies such as Boehringer Mannheim (Indianapolis, IN) and Sigma
Chemical Company (St. Louis, MO), among others.
Such modifications are well known to those of skill in the art and have been described
in great detail in the scientific literature. Several particularly common modifications,
glycosylation. lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues,
hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as
Peptides-Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993). Many detailed reviews are available on this subject, such as by
Wold. F., Post-translational Covalent Modification of Peptides, B. C. Johnson, Ed., Academic
Press. New York 1 -12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and
Rattan etal. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
Covalent modifications of a peptide may also be introduced into the molecule in vitro
by reacting targeted amino-acid residues of the peptide with an organic derivatizing agent
that is capable of reacting with selected side chains or terminal amino-acid residues. Most
commonly derivatized residues are cysteinyl, histidyl, lysinyl, arginyl, tyrosyl, glutaminyl.
asparaginyl and amino terminal residues. Hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl and threonyl residues, methylation of the alpha-
amino groups of lysine, histidine, and histidine side chains, acetylation of the N-terminal
amine and amidation of the C-terminal carboxylic groups. Such derivatized moieties may
improve the solubility, absorption, biological half life and the like. The moieties may also
eliminate or attenuate any undesirable side effect of the peptide and the like.
In addition, derivatization with Afunctional agents is useful for cross-linking the
peptide to water insoluble support matrices or to other macromolecular carriers. Commonly
used cross-linking agents include glutaraldehyde, N-hydroxysuccinimide esters,
homobifunctional imidoesters, l,l-bis(-diazoloacetyl)-2-phenylethane, and Afunctional
maleimides. Derivatizing agents such as methyl-3-[9p-azidophenyl)]dithiopropioimidate
yield photoactivatable intermediates that are capable of forming crosslinks in the presence of
light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide activated
carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287 and 3,691,016
may be employed for peptide immobilization.
E. Fusion peptides/peptides
Peptides useful in the present invention may comprise fusion peptides. Fusion
peptides are particularly advantageous where biological and/or functional characteristics of
two peptides are desired to be combined in one peptide molecule. Such fusion peptides can
present combinations of biological activity and function that are not found in nature to create
novel and useful molecules of therapeutic and industrial applications. Biological activities of
interest include, but are not limited to, enzymatic activity, receptor and/or ligand activity,
immunogenic motifs, and structural domains.
Such fusion peptides are well known in the art, and the methods of creation will be
well-known to those in the art. For example, a human a-interferon—human albumin fusion
peptide has been made wherein the resulting peptide has the therapeutic benefits of a-
interferon combined with the long circulating life of albumin, thereby creating a therapeutic
composition that allows reduced dosing frequency and potentially reduced side effects in
patients. See. Albuferon™ from Human Genome Sciences, Inc. and U.S. Patent No.
5.766,883. Other fusion peptides include antibody molecules that are described elsewhere
herein.
F. Generation of smaller "biologically active' molecules
The peptides used in the invention may be variants of native peptides, wherein a
fragment of the native peptide is used in place of the full length native peptide. In addition,
pre-pro-, and pre-peptides are contemplated. Variant peptides may be smaller in size that the
native peptide, and may comprise one or more domains of a larger peptide. Selection of
specific peptide domains can be advantageous when the biological activity of certain domains
in the peptide is desired, but the biological activity of other domains in the peptide is not
desired. Also included are truncations of the peptide and internal deletions which may
enhance the desired therapeutic effect of the peptide. Any such forms of a peptide is
contemplated to be useful in the present invention provided that the desired biological
activity of the peptide is preserved.
Shorter versions of peptides may have unique advantages not found in the native
peptide. In the case of human albumin, it has been found that a truncated form comprising as
little as 63% of the native albumin peptide is advantageous as a plasma volume expander.
The truncated albumin peptide is considered to be better than the native peptide for this
therapeutic purpose because an individual peptide dose of only one-half to two-thirds that of
natural-human serum albumin, or recombinant human serum albumin is required for the
equivalent colloid osmotic effect. See U.S. Patent No. 5.380,712, the entirely of which is
incorporated by reference herein.
Smaller "biologically active" peptides have also been found to have enhanced
therapeutic activity as compared to the native peptide. The therapeutic potential of IL-2 is
limited by various side effects dominated by the vascular leak syndrome. A shorter
chemically synthesized version of the peptide consisting of residues 1-30 corresponding to
the entire a-helix was found to fold properly and contain the natural IL-2 biological activity
with out the attending side effects.
G. Generation of novel peptides
The peptide of the invention may be derived from a primary sequence of a
native peptide, or may be engineered using any of the many means known to those of skill in
the art. Such engineered peptides can be designed and/or selected because of enhanced or
novel properties as compared with the native peptide. For example, peptides may be
engineered to have increased enzyme reaction rates, increased or decreased binding affiniu
to a substrate or ligand. increased or decreased binding affinity to a receptor, altered
specificity for a substrate, ligand. receptor or other binding partner, increased or decreased
stability in vitro and/or in vivo, or increased or decreased immunogenicity in an animal.
H. Mutations
1. Rational design mutation
The peptides useful in the methods of the invention may be mutated to enhance a
desired biological activity or function, to diminish an undesirable property of the peptide,
and/or to add novel activities or functions to the peptide. "Rational peptide design" may be
used to generate such altered peptides. Once the amino acid sequence and structure of the
peptide is known and a desired mutation planned, the mutations can be made most
conveniently to the corresponding nucleic acid codon which encodes the amino acid residue
that is desired to be mutated. One of skill in the art can easily determine how the nucleic
acid sequence should be altered based on the universal genetic code, and knowledge of codon
preferences in the expression system of choice. A mutation in a codon may be made to
change the amino acid residue that will be polymerized into the peptide during translation.
Alternatively, a codon may be mutated so that the corresponding encoded amino acid residue
is the same, but the codon choice is better suited to the desired peptide expression system.
For example, cys-residues may be replaced with other amino acids to remove disulfide bonds
from the mature peptide, catalytic domains may be mutated to alter biological activity, and in
general, isoforms of the peptide can be engineered. Such mutations can be point mutations,
deletions, insertions and truncations, among others.
Techniques to mutate specific amino acids in a peptide are well known in the art. The
technique of site-directed mutagenesis, discussed above, is well suited for the directed
mutation of codons. The oligonucleotide-mediated mutagenesis method is also discussed in
detail in Sambrook et al. (2001. Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory. New York, starting at page 15.51). Systematic deletions, insertions and
truncations can be made using linker insertion mutagenesis, digestion with nuclease Bal 31.
and linker-scanning mutagenesis, among other method well known to those in the art
(Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory. New York).
Rational peptide design has been successfully used to increase the stability of
enzymes with respect to thermoinactivation and oxidation. For example, the stability of an
enzyme was improved by removal of asparagine residues in a-amylase (Declerck et al.. 2000.
J. Mol. Biol. 301:1041-1057), the introduction of more rigid structural elements such as
proline into a-amylase (Igarashi et al., 1999, Biosci. Biotechnol. Biochem. 63:1535-1540)
and D-xylose isomerase (Zhu et al., 1999, Peptide Eng. 12:635-638). Further, the
introduction of additional hydrophobic contacts stabilized 3-isopropylmalate dehydrogenase
(Akanuma et al.. 1999. F.ur. J. Biochem. 260:499-504) and formate dehydrogenase obtained
from Pseudomonas sp. (Rojkova et al., 1999. FEBS Lett. 445:183-188). The mechanisms
behind the stabilizing effect of these mutations is generally applicable to many peptides.
These and similar mutations are contemplated to be useful with respect to the peptides
remodeled in the methods of the present invention.
2. Random mutagenesis techniques
Novel peptides useful in the methods of the invention may be generated using
techniques that introduce random mutations in the coding sequence of the nucleic acid. The
nucleic acid is then expressed in a desired expression system, and the resulting peptide is
assessed for properties of interest. Techniques to introduce random mutations into DNA
sequences are well known in the art, and include PCR mutagenesis, saturation mutagenesis,
and degenerate oligonucleotide approaches. See Sambrook and Russell (2001, Molecular
Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY) and
Ausubel et al. (2002, Current Protocols in Molecular Biology, John Wiley & Sons, NY).
In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random
mutations into a cloned fragment of DNA (Leung et al., 1989. Technique 1:11-15). This is a
very powerful and relatively rapid method of introducing random mutations into a DNA
sequence. The DNA region to be mutagenized is amplified using the polymerase chain
reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA
polymerase, e.g., by using an altered dGTP/dATP ratio and by adding Mn2+ to the PCR
reaction. The pool of amplified DNA fragments are inserted into appropriate cloning vectors
to provide random mutant libraries.
Saturation mutagenesis allows for the rapid introduction of a large number of single
base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This
technique includes generation of mutations, e.g., by chemical treatment or irradiation of
single-stranded DNA in vitro, and synthesis of a complementary DNA strand. The mutation
frequency can be modulated by modulating the severity of the treatment, and essentially all
possible base substitutions can be obtained. Because this procedure does not involve a
genetic selection for mutant fragments, both neutral substitutions as well as those that alter
function, are obtained. The distribution of point mutations is not biased toward conserved
sequence elements.
A library of nucleic acid homologs can also be generated from a set of degenerate
oligonucleotide sequences. Chemical synthesis of a degenerate oligonucleotide sequences
can be carried out in an automatic DNA synthesizer, and the synthetic genes may then be
ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is
known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981)
Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,
Amsterdam: Elsevier pp. 273-289: Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura
et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques
have been employed in the directed evolution of other peptides (see. for example. Scott et al.
(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990)
Science 249: 404-406: Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos.
5,223,409, 5.198,346. and 5,096,815).
a. Directed evolution
Peptides useful in the methods of the invention may also be generated using "directed
evolution" techniques. In contrast to site directed mutagenesis techniques where knowledge
of the structure of the peptide is required, there now exist strategies to generate libraries of
mutations from which to obtain peptides with improved properties without knowledge of the
structural features of the peptide. These strategies are generally known as "directed
evolution" technologies and are different from traditional random mutagenesis procedures in
that they involve subjecting the nucleic acid sequence encoding the peptide of interest to
recursive rounds of mutation, screening and amplification.
In some "directed evolution" techniques, the diversity in the nucleic acids obtained is
generated by mutation methods that randomly create point mutations in the nucleic acid
sequence. The point mutation techniques include, but are not limited to, "error-prone
PCRTM- (Caldwell and Joyce, 1994; PCR Methods Appl. 2: 28-33; and Ke and Madison,
1997. Nucleic Acids Res. 25: 3371-3372), repeated oligonucleotide-directed mutagenesis
(Reidhaar-Olson et al., 1991. Methods Enzymol. 208:564-586), and any of the
aforementioned methods of random mutagenesis.
Another method of creating diversity upon which directed evolution can act is the use
of mutator genes. The nucleic acid of interest is cultured in a mutator cell strain the genome
of which typically encodes defective DNA repair genes (U.S. Patent No. 6,365,410:
Selifonova et al.. 2001. Appl. Environ. Microbiol. 67:3645-3649; Long-McGie et al., 2000.
Biotech. Bioeng. 68:121-125: sec, Genencor International Inc. Palo Alto CA).
Achieving diversity using directed evolution techniques may also be accomplished
using saturation mutagenesis along with degenerate primers (Gene Site Saturation
Mutagenesis™. Diversa Corp., San Diego, CA). In this type of saturation mutagenesis,
degenerate primers designed to cover the length of the nucleic acid sequence to be diversified
are used to prime the polymerase in PCR reactions. In this manner, each codon of a coding
sequence for an amino acid may be mutated to encode each of the remaining common
nineteen amino acids. This technique may also be used to introduce mutations, deletions and
insertions to specific regions of a nucleic acid coding sequence while leaving the rest of the
nucleic acid molecule untouched. Procedures for the gene saturation technique are well
known in the art, and can be found in U.S. Patent 6.171,820.
b. DNA shuffling
Novel peptides useful in the methods of the invention may also be generated using the
techniques of gene-shuffling, motif-shuffling, exon-shuffling. and/or codon-shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling techniques are may be
employed to modulate the activities of peptides useful in the invention and may be used to
generate peptides having altered activity. See, generally, U.S. Pat. Nos. 5.605.793;
5.81 1,238; 5.830.721: 5.834,252; and 5,837.458, and Stemmer et al. (1994. Nature
370(6488):389-391): Crameri et al. (1998, Nature 391 (6664):288-291); Zhang et al. (1997.
Proc. Natl. Acad. Sci. USA 94(9):4504-4509); Stemmer et al. (1994. Proc. Natl. Acad. Sci
USA 91(22): 10747-10751), Patten et al. (1997, Curr. Opinion Biotechnol. 8:724-33);
Harayama, (1998, Trends Biotechnol. 16(2):76-82); Hansson, et al., (1999, J. Mol. Biol.
287:265-76); and Lorenzo and Blasco (1998, Biotechniques 24(2):308-13) (each of these
patents are hereby incorporated by reference in its entirety).
DNA shuffling involves the assembly of two or more DNA segments by homologous
or site-specific recombination to generate variation in the polynucleotide sequence. DNA
shuffling has been used to generate novel variations of human immunodeficiency virustype-1
proteins (Pekrun et al., 2002, J. Virol. 76(6):2924-35), triazine hydrolases (Raillard£t al.
2001. Chem Biol 8(9):891-898), murine leukemia virus (MLV) proteins (Powell et al. 2000,
Nat Biotechnol 18(12): 1279-1282), and indoleglycerol phosphate synthase (Merz et al. 2000.
Biochemistry 39(5):880-889).
The technique of DNA shuffling was developed to generate biomolecular diversity by
mimicking natural recombination by allowing in vitro homologous recombination of DNA
(Stemmler, 1994. Nature 370: 389-391; and Stemmler. 1994, PNAS91: 10747-10751).
Generally, in this method a population of related genes is fragmented and subjected to
recursive cycles of denaturation, rehybridization, followed by the extension of the 5'
overhangs by Taq polymerase. With each cycle, the length of the fragments increases, and
DNA recombination occurs when fragments originating from different genes hybridize lo
each other. The initial fragmentation of the DNA is usually accomplished by nuclease
digestion, typically using DNase (see Stemmler references, above), but may also be
accomplished by interrupted PCR synthesis (U.S. Patent 5.965,408, incorporated herein by
reference in its entirety; see. Diversa Corp., San Diego, CA). DNA shuffling methods have
advantages over random point mutation methods in that direct recombination of beneficial
mutations generated by each round of shuffling is achieved and there is therefore a self
selection for improved phenotypes of peptides.
The techniques of DNA shuffling are well known to those in art. Detailed
explanations of such technology is found in Stemmler, 1994, Nature 370: 389-391 and
Stemmler. 1994. PNAS 91: 10747-10751. The DNA shuffling technique is also described in
U.S. Patents 6,180,406. 6,165.793, 6,132,970, 6,117,679. 6,096,548, 5.837,458, 5,834,252,
5.830,721. 5.811.238. and 5.605,793 (all of which are incorporated by reference herein in
their entirety) .
The art also provides even more recent modifications of the basic technique of DNA
shuffling. In one example, exon shuffling, exons or combinations of exons that encode
specific domains of peptides are amplified using chimeric oligonucleotides. The amplified
molecules are then recombined by self-priming PCR assembly (Kolkman and Stemmler.
2001. Nat. Biotech. 19:423-428). In another example, using the technique of random
chimeragenesis on transient templates (RACHITT) library construction, single stranded
parental DNA fragments are annealed onto a full-length single-stranded template (Coco et al.,
2001, Nat. Biotechnol. 19:354-359). In yet another example, staggered extension process
(StEP). thermocycling with very abbreviated annealing/extension cycles is employed to
repeatedly interrupt DNA polymerization from flanking primers (Zhao et al., 1998, Nat.
Biotechnol. 16: 258-261). In the technique known as CLERY. in vitro family shuffling is
combined with in vivo homologous recombination in yeast (Abecassis et al.. 2000, Nucleic
Acids Res. 28:E88:). To maximize intergenic recombination, single stranded DNA from
complementary strands of each of the nucleic acids are digested with DNase and annealed
(Kikuchi et al., 2000, Gene 243:133-137). The blunt ends of two truncated nucleic acids of
variable lengths that are linked by a cleavablc sequence are then ligated to generate gene
fusion without homologous recombination (Sieber et al., 2001. Nat Biotechnol. 19:456-460;
Lutz et al., 2001, Nucleic Acids Res. 29:E16; Ostermeier et al., 1999, Nat. Biotechnol.
17:1205-1209; Lutz and Benkovic, 2000, Curr. Opin. Biotechnol. 11:319-324).
Recombination between nucleic acids with little sequence homology in common has also
been enhanced using exonuclease-mediated blunt-ending of DNA fragments and ligating the
fragments together to recombine them (U.S. Patent No. 6,361,974, incorporated herein by
reference in its entirety). The invention contemplates the use of each and every variation
described above as a means of enhancing the biological properties of any of the peptides
and/or enzymes useful in the methods of the invention.
In addition to published protocols detailing directed evolution and gene shuffling
techniques, commercial services are now available that will undertake the gene shuffling and
selection procedures on peptides of choice. Maxygen (Redwood City, CA) offers
commercial services to generate custom DNA shuffled libraries. In addition, this company
will perform customized directed evolution procedures including gene shuffling and selection
on a peptide family of choice.
Optigenix, Inc. (Newark, DE) offers the related service of plasmid shuffling.
Optigenix uses families of genes to obtain mutants therein having new properties. The
nucleic acid of interest is cloned into a plasmid in an Aspergillus expression system. The
DNA of the related family is then introduced into the expression system and recombination in
conserved regions of the family occurs in the host. Resulting mutant DNAs are then
expressed and the peptide produced therefrom are screened for the presence of desired
properties and the absence of undesired properties.
c. Screening procedures
Following each recursive round of "evolution," the desired peptides expressed by
mutated genes are screened for characteristics of interest. The "candidate" genes are then
amplified and pooled for the next round of DNA shuffling. The screening procedure used is
highly dependant on the peptide that is being "evolved" and the characteristic of interest.
Characteristics such as peptide stability, biological activity, antigenicity, among others can be
selected using procedures that are well known in the art. Individual assays for the biological
activity of preferred peptides useful in the methods of the invention are described elsewhere
herein.
d. Combinations of techniques
It will be appreciated by the skilled artisan that the above techniques of mutation and
selection can be combined with each other and with additional procedures to generate the best
possible peptide molecule useful in the methods of the invention. Thus, the invention is not
limited to any one method for the generation of peptides, and should be construed to
encompass any and all of the methodology described herein. For example, a procedure for
introducing point mutations into a nucleic acid sequence may be performed initially, followed
by recursive rounds oI'DNA shuffling, selection and amplification. The initial introduction
of point mutations may be used to introduce diversity into a gene population where it is
lacking, and the following round of DNA shuffling and screening will select and recombinc
advantageous point mutations.
111. Glycosidases and Glycotransferases
A. Glycosidases
Glycosidases are glycosyltransferases that use water as an acceptor molecule, and as
such, arc typically glycoside-hydrolytic enzymes. Glycosidases can be used for the formation
of glycosidic bonds in vitro by controlling the thermodynamics or kinetics of the reaction
mixture. Even with modified reaction conditions, though, glycosidase reactions can be
difficult to work with, and glycosidases tend to give low synthetic yields as a result of the
reversible transglycosylase reaction and the competing hydrolytic reaction.
A glycosidase can function by retaining the stereochemistry at the bond being broken
during hydrolysis or by inverting the stereochemistry at the bond being broken during
hydrolysis, classifying the glycosidase as either a "retaining" glycosidase or an "inverting"'
glycosidase, respectively. Retaining glycosidases have two critical carboxylic acid moieties
present in the active site, with one carboxylate acting as an acid/base catalyst and the other as
a nucleophile. whereas with the inverting glycosidases. one carboxylic acid functions as an
acid and the other functions as a base.
Methods to determine the activity and linkage specificity of any glycosidase are well
known in the art, including a simplified HPLC protocol (Jacob and Scudder, 1994, Methods
in linzymol. 230: 280-300). A general discussion of glycosidases and glycosidase treatment
is found in Glycobiology. A Practical Approach, (1993, Fukuda and Kobata eds.. Oxford
University Press Inc., New York).
Glycosidases useful in the invention include, but are not limited to, sialidase, galactosidase,
endoglycanase. mannosidase (i.e., a and ß Manl, Manll and ManIII,) xylosidase, fucosidase,
Agrobaclerium sp. P-glucosidase, Cellulomonas flmi mannosidase 2A, Humicola insolens
glycosidase. Sulfolohus solfularicus glycosidase and Bacillus licheniformis glycosidase.
The choice of fucosidases for use in the invention depends on the linkage of the
fucose to other molecules. The specificities of many a-fucosidases useful in the methods of
the invention are well known to those in the art, and many varieties of fucosidase are also
commercially available (Glyko, Novato, CA; PROzyme, San Leandro. CA; Calbiochem-
Novabiochem Corp.. San Diego. CA; among others). a-Fucosidases of interest include, but
are not limited to, a-fucosidases from Turbo cornuhts, Charonia lampas. Bacillus fulminans.
Aspergillus niger. Clostridium perfringens. Bovine kidney (Glyko), chicken liver (Tyagarajan
et al., 1996, Glycobiology 6:83-93) and a-fucosidase II from Xanthomonas maniholis (Glyko.
PROzyme). Chicken liver fucosidase is particularly useful for removal of core fucosc from
N-linked glycans.
B. Glycosyltransfcrases
Glycosyltransferases catalyze the addition of activated sugars (donor NDP-sugars). in
a step-wise fashion, to a protein, glycopeptide, lipid or glycolipid or to the non-reducing end
of a growing oligosaccharide. N-linked glycopeptides are synthesized via a transferase and a
lipid-linked oligosaccharide donor Dol-PP-NAG2Glc3Man9 in an en block transfer followed
by trimming of the core. In this case the nature of the "core" saccharide is somewhat
different from subsequent attachments. A very large number of glycosyltransferases arc
known in the art.
The glycosyltransfcrase to be used in the present invention may be any as long as it
can utilize the modified sugar as a sugar donor. Examples of such enzymes include Leloir
pathway glycosyltransferases, such as galactosyltransferase, N-
acetylglucosaminyltransferase, N-acetylgalactosaminyltransferase, fucosyltransferase,
sialyltransferasc. mannosyltransferase. xylosyltransferase. glucurononyltransferase and the
like.
For enzymatic saccharide syntheses that involve glycosyltransferase reactions,
glycosyltransferases can be cloned, or isolated from any source. Many cloned
glycosyltransferases arc known, as are their polynucleotide sequences. See, e.g., Taniguchi
et al.. 2002, Handbook of glycosyltransferases and related genes, Springer. Tokyo.
Glycosyltransferase amino acid sequences and nucleotide sequences encoding
glycosyltransferases from which the amino acid sequences can be deduced are also found in
various publicly available databases, including GenBank, Swiss-Prot, EMBL, and others.
Glycosyltransferases that can be employed in the methods of the invention include,
but are not limited to. galactosyltransfcrases, fucosyltransfcrases, glucosyltransferases. N-
acetylgalactosaminyltransferases. N-acetylglucosaminyltransferases, glucurony (transferases,
sialyltransferases. mannosyltransferases. glucuronic acid transferases, galacturonic acid
transferases, and oligosaccharyltransferases. Suitable glycosyltransferases include those
obtained from eukaryotes. as well as from prokaryotes.
DNA encoding glycosyltransferases may be obtained by chemical synthesis, by
screening reverse transcripts of mRNA from appropriate cells or cell line cultures, by
screening genomic libraries from appropriate cells, or by combinations of these procedures.
Screening of mRNA or genomic DNA may be carried out using oligonucleotide probes
generated from the glycosyltransferases nucleic acid sequence. Probes may be labeled with a
detectable label, such as, but not limited to, a fluorescent group, a radioactive atom or a
chemiluminescent group in accordance with known procedures and used in conventional
hybridization assays. In the alternative, glycosyltransferases nucleic acid sequences may be
obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR
oligonucleotide primers being produced from the glycosyltransferases nucleic acid sequence.
See, U.S. Pat. No. 4.683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis.
A glycosyltransferases enzyme may be synthesized in a host cell transformed with a
vector containing DNA encoding the glycosyltransferases enzyme. A vector is a replicablc
DNA construct. Vectors are used either to amplify DNA encoding the glycosyltransferases
enzyme and/or to express DNA which encodes the glycosyltransferases enzyme. An
expression vector is a replicable DNA construct in which a DNA sequence encoding the
glycosyltransferases enzyme is operably linked to suitable control sequences capable of
effecting the expression of the glycosyltransferases enzyme in a suitable host. The need for
such control sequences will vary depending upon the host selected and the transformation
method chosen. Generally, control sequences include a transcriptional promoter, an optional
operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal
binding sites, and sequences which control the termination of transcription and translation.
Amplification vectors do not require expression control domains. All that is needed is the
ability to replicate in a host, usually conferred by an origin of replication, and a selection
tzene to facilitate rccosznition of transformants.
1. Fucosyltransferases
In some embodiments, a glycosyltransferase used in the method of the invention is a
fucosyltransferase. Fucosyltransferases are known to those of skill in the art. Exemplary
fucosyltransferases include enzymes, which transfer L-fucose from GDP-fucose to a hydroxy
position of an acceptor sugar. Fucosyltransferases that transfer from non-nuclcotide sugars to
an acceptor are also of use in the present invention.
In some embodiments, the acceptor sugar is, for example, the GlcNAc in a
Gaip(l ?3,4)GlcNAcP- group in an oligosaccharide glycoside. Suitable fucosyltransferases
for this reaction include the Gal[3(l?3.4)GlcNAcß1-a(l?3,4)fucosyltransferase (FTIII E.G.
No. 2.4.1.65), which was first characterized from human milk (see, Palcic, et al.,
Carbohydrate Res. 190: 1-11 (1989); Prieels, et al., J. Biol. Chem. 256: 10456-10463(1981):
and Nunez, et al., Can. J. Chem. 59: 2086-2095 (1981)) and the Galß(l?4)GleNAcP-
afucosyltransferases (FTIV, FTV, FTVI) which are found in human serum. FTVII (E.G. No.
2.4.1.65), a sialyl a(2?3)Gaiß((l?3)GlcNAcß fucosyltransferase, has also been
characterized. A recombinant form of the Galß(l ?3,4) GlcNAcP-
rx(l?3.4)fucosyltransferase has also been characterized (see, Dumas, et al.. Bioorg. Med.
Letters 1: 425-428 (1991) and Kukowska-Latallo, et al., Genes and Development 4: 1288-
1303 (1990)). Other exemplary fucosyltransferases include, for example, a 1,2
fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can be carried out by the
methods described in Mollicone, et al., Eur. J. Biochem. 191: 169-176 (1990) or U.S. Patent
No. 5,374.655.
2. Galactosyltransferases
In another group of embodiments, the glycosyltransferase is a galactosyltransferase.
Exemplary galactosyltransferases include a(l,3) galactosyltransferases (E.G. No. 2.4.1.151,
see. e.g.. Dabkowski et al.. Transplant Proc. 25:2921 (1993) and Yamamoto et al. Nature 345:
229-233 (1990), bovine (GenBank J04989, Joziasse et al., J. Biol. Chem. 264: 14290-14297
(1989)), murine (GenBank m26925; Larsen et al., Proc. Nat'l. Acad. Sci. USA 86: 8227-8231
(1989)). porcine (GenBank L36152; Strahan et al., Immunogenetics 41: 101-105 (1995)).
Another suitable a 1,3 galactosyltransferase is that which is involved in synthesis of the blood
group B antigen (EC 2.4.1.37. Yamamoto et al., J Biol. Chem. 265: 1146-1151 (1990)
(human)).
Also suitable for use in the methods of the invention are P(1.4) galactosyltransferases.
which include, for example, EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22 (lactose
synthetase) (bovine (D'Agostaro et al., Eur. J. Biochem. 183: 211-217(1989)), human (Masri
et al., Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nakazawa et al.. J.
Biochem. 104: 165-168 (1988)), as well as E.G. 2.4.1.38 and the ceramide
galactosyltransferase (EC 2.4.1.45. Stahl etal., J. Neurosci. Res. 38: 234-242 (1994)). Other
suitable galactosyltransferases include, for example, a1,2 galactosyltransferases (from e.g.,
Schizosaccharomyces pombe. Chapell et al., Mol. Biol. Cell 5: 519-528 (1994)). For further
suitable galactosyltransferases, see Taniguchi et al. (2002, Handbook of Glycosyltransferases
and Related Genes, Springer, Tokyo), Guo et al. (2001. Glycobiology, 11 (10):813-820), and
Breton etal. (1998,.I Biochem. 123:1000-1009).
The production of proteins such as the enzyme GalNAc TI-XIV from cloned genes by
genetic engineering is well known. Sec, e.g.. U.S. Pat. No. 4,761,371. One method involves
collection of sufficient samples, then the amino acid sequence of the enzyme is determined
by N-terminal sequencing. This information is then used to isolate a cDNA clone encoding a
full-length (membrane bound) transferase which upon expression in the insect cell line Sf9
resulted in the synthesis of a fully active enzyme. The acceptor specificity of the enzyme is
then determined using a semiquantitative analysis of the amino acids surrounding known
glycosylation sites in 16 different proteins followed by in vitro glycosylation studies of
synthetic peptides. This work has demonstrated that certain amino acid residues are
overrepresented in glycosylated peptide segments and that residues in specific positions
surrounding glycosylated serine and threonine residues may have a more marked influence on
acceptor efficiency than other amino acid moieties.
3. Sialyltransferases
Sialyltransferases arc another type of glycosyltransferase that is useful in the
recombinant cells and reaction mixtures of the invention. Examples of sialyltransferases that
are suitable for use in the present invention include ST3Gal III (e.g., a rat or human ST3Gal
III). ST3Gal IV. ST3Gal I, ST6Gal I, ST3Gal V. ST6Gal II. ST6GalNAc I. ST6GalNAc II.
and ST6GalNAc III (the sialyltransferase nomenclature used herein is as described in Tsuji et
al. Glycobiologv 6: v-\iv (1996)). An exemplary a(2,3)sialyltransfcrasc referred to as
u(2.3)sialyltransferasc (EC 2.4.99.6) transfers sialic acid to the non-reducing terminal Gal of
a Galß1?3Glc disaccharidc or glycoside. See, Van den Eijnden et al., J. Biol. Chem. 256:
3159 (1981), Weinstein et al.. J. Biol. Chem. 257: 13845 (1982) and Wen et al.. J. Biol.
Chem. 267: 21011 (1992). Another exemplary a2,3-sialyltransferase (EC 2.4.99.4) transfers
sialic acid to the non-reducing terminal Gal of the disaccharide or glycoside, see, Rearick et
al.. J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J. Biol. Chem. 267: 21004 (1992).
Further exemplary enzymes include Gal-P-l,4-GlcNAc a-2,6 sialyltransferase (See,
Kurosawa et al. Eur. J. Biochem. 219: 375-381 (1994)).
Preferably, for glycosylation of carbohydrates of glycopeptides the sialyltransferase
will be able to transfer sialic acid to the sequence Ga1ß1,4GlcNAc-, Galpl,3GlcNAc-. or
Ga1ß1.3GalNAc-. the most common penultimate sequences underlying the terminal sialic
acid on fully sialylated carbohydrate structures (see, Table 8). a2.8-Sialyltransferases
capable of transfering sialic acid to a2,3Ga1ß1,4GlcNAc are also useful in the methods of the
invention.
An example of a sialyltransferase that is useful in the claimed methods is ST3Gal III,
which is also referred to as a(2.3)sialyltransferase (IX 2.4.99.6). This enzyme catalyzes the
transfer of sialic acid to the Gal of a Galß1.3GlcNAc or Galpl,4GlcNAc glycoside (see. e.g..
Wen et al., J. Biol. Chem. 267: 21011(1992): Van den Eijnden et al., J. Biol. Chem. 256:
3159 (1991)) and is responsible for sialylation of asparagine-linked oligosaccharides in
glycopeptides. The sialic acid is linked to a Gal with the formation of an a-linkage between
the two saccharides. Bonding (linkage) between the saccharides is between the 2-position of
NeuAc and the 3-position of Gal. This particular enzyme can be isolated from rat liver
(Weinstcin et al., J. Biol. Chem. 257: 13845 (1982)); the human cDNA (Sasaki et al. (1993)
J. Biol. Chem. 268: 22782-22787; Kitagawa & Paulson (1994) J. Biol. Chem. 269: 1394-
1401) and genomic (Kitagawa et al. (1996) J. Biol. Chem. 271: 931-938) DNA sequences are
known, facilitating production of this enzyme by recombinant expression. In a preferred
embodiment, the claimed sialylation methods use a rat ST3Gal III.
An example of a sialyltransferase that is useful in the claimed methods is CST-I from
Campylobacter (see .for example, U.S. Pat. No. 6.503744, 6,096,529, and 6,210933 and
WO99/49051, and published U.S. Pat. Application 2002/2,042,369). This enzyme catalyzes
the transfer of sialic acid to the Gal of aGa1ß1.4Glc or Galpl,3GalNAc. Other exemplary
sialyltransferases of use in the present invention include those isolated from Campylobacter
jejuni, including the a(2,3) sialyltransferase. See, e.g. WO99/49051.
Other sialyltransferases, including those listed in Table 8. are also useful in an
economic and efficient large-scale process for sialylation of commercially important
glycopeptides. As a simple test to find out the utility of these other enzymes, various
amounts of each enzyme (1-100 mU/mg protein) are reacted with asialo-a1 AGP (at 1-10
mg/ml) to compare the ability of the sialyltransferase of interest to sialylate glycopeptides
relative to either bovine ST6Gal I, ST3Gal III or both sialyltransferases. Alternatively, other
glycopeptides or glycopeptides, or N-linked oligosaccharides enzymatically released from the
peptide backbone can be used in place of asialo-a1 AGP for this evaluation.
Sialyltransferases with the ability to sialylate N-linked oligosaccharides of glycopeptides
more efficiently than ST6Gal I are useful in a practical large-scale process for peptide
sialylation (as illustrated for ST3Gal III in this disclosure).
4. Other glyeosyltransferases
One of skill in the an will understand that other glyeosyltransferases can be
substituted into similar transferase cycles as have been described in detail for the
sialyltransferase. In particular, the glycosyltransferase can also be. for instance,
glucosyltranslerases, e.g., Alg8 (Stagljov et al., Proc. Natl. Acad. Sci. USA 91: 5977(1994))
or Alg5 (Heescn et al.. fiur. J. Biochem. 224: 71 (1994)).
N-acetylgalactosaminyltransferases are also of use in practicing the present invention.
Suitable N-acetylgalactosaminyltransferases include, but are not limited to, oc(I.3) N-
acetylgalactosaminyltransferase,ß(1,4) N-acetylgalactosaminyltransferases (Nagata et al.. J.
Biol. Chem. 267: 12082-12089 (1992) and Smith et al., J. Biol Chem. 269: 15162 (1994))
and peptide N-acetylgalactosaminyltransferase (Homa et al., J. Biol. Chem. 268: 12609
(1993)). Suitable N-acetylglucosaininyltransferases include GnT-I (2.4.1.101. Hull et al..
BBRC 176: 608 (1991)), GnT-11, GnT-III (Ihara et al., J. Biochem. 113: 692 (1993)), GnT-
IV, GnT-V (Shoreibah et al., J. Biol. Chem. 268: 15381 (1993)) and GnT-VI. O-Iinkcd N-
acetylglucosaminyltransferase (Bierhuizen et al., Proc. Natl. Acad. Sci. USA 89: 9326
(1992)), N-acetylglucosamine-1 -phosphate transferase (Rajput et al., Biochem J. 285: 985
(1992). and hyaluronan synthase.
Mannosyltransferases are of use to transfer modified mannose moieties. Suitable
mannosyltransferases include a(l,2) mannosyltransferase. a(1,3) mannosyltransferase. a(1,6)
mannosyltransferase.ß(1,4) mannosyltransferase, Dol-P-Man synthase, OChl, and Pmtl
(sec. Kornfeld et al.. Annu. Rev. Biochem. 54: 631-664 (1985)).
Xylosyltransferases are also useful in the present invention. See. for example,
Rodgers, et al., Biochem. J., 288:817-822 (1992); and Elbain, et al., U.S. Patent No.,
6,168,937.
Other suitable glycosyltransferase cycles are described in Ichikawa et al., JACS 114:
9283 (1992), Wong et al., J. Org. Chem. 57: 4343 (1992), and Ichikawa et al. in
Carbohydrails and Carbohydratf. Polymkrs. Yaltami, ed. (ATL Press. 1993).
Prokaryotic glyeosyltransferases are also useful in practicing the invention. Such
glyeosyltransferases include enzymes involved in synthesis of lipooligosaccharides (LOS),
which are produced by many gram negative bacteria. The I.OS typically have terminal
glycan sequences that mimic glycoconjugates found on the surface of human epithelial cells
or in host secretions (Preston et al.. Critical Reviews in Microbiology 23(3): 139-180 (1996)).
Such enzymes include, but are not limited to, the proteins of the rfa operons of species such
as E. coli and Salmonella typhimurium, which include a ß1,6 galactosyltransfcrase and a ß1.3
galactosyltransferase (see. e.g., EMBL Accession Nos. M80599 and M86935 (E. coli):
LMBL Accession No. S56361 (S. typhimurium)), a glucosyltransferase (Swiss-Prot
Accession No. P25740 (E. coli), an ß1,2-glucosyltransferase (rfaJ)(Swiss-Prot Accession No.
P27129 (E. coli) and Swiss-Prot Accession No. PI9817 (S. typhimurium)), and an B1.2-N-
acetylglucosaminyltransferase (rfaK)(EMBL Accession No. U00039 (E. coli). Other
glycosyltransferases for which amino acid sequences are known include those that arc
encoded by operons such as rfaB, which have been characterized in organisms such as
Klebsiella pneumoniae. E. coli. Salmonella typhimurium, Salmonella enterica, Yersinia
enterocolitica, Mycobacterium leprosum, and the rhl operon of Pseudomonas aeruginosa.
Also suitable for use in the present invention are glycosyltransferases that are
involved in producing structures containing lacto-N-neotetraose, D-galactosyl-p-l,4-N-
acctyl-D-glucosaminyl-ß-l,3-D-galactosyl-ß-l,4-D-glucose, and the Pk blood group
trisaccharide sequence, D-galactosyl-a-1.4-D-galactosyl-p-l,4-D-glucose, which have been
identified in the I.OS of the mucosal pathogens Neisseria gonnorhoeae and ;V. meningitidis
(Scholten et al., J. Med. Microbiol. 41: 236-243 (1994)). The genes from N. meningitidis and
N, gonorrhoeae that encode the glycosyltransferases involved in the biosynthesis of these
structures have been identified from A', meningitidis immunotypes L3 and LI (Jennings et al..
Mol. Microbiol. 18: 729-740 (1995)) and the N. gonorrhoeae mutant F62 (Gotshlich. J. Exp.
Med. 180: 2181 -2190 (1994)). In A", meningitidis, a locus consisting of three genes, IgtA,
IglB and Ig E, encodes the glycosyltransferase enzymes required for addition of the last three
of the sugars in the lacto-A'-neotetraose chain (Wakarchuk et al., J. Biol. Chem. 271: 19166-
73 (1996)). Recently the enzymatic activity of the IgtB and IgtA gene product was
demonstrated, providing the first direct evidence for their proposed glycosyltransferase
function (Wakarchuk et al.. J. Biol. Chem. 271(45): 28271-276 (1996)). In .V. gonorrhoeae.
there are two additional genes. lgtD which adds P-D-GalNAc to the 3 position of the terminal
galactose of the lacto-A'-neotetraose structure and lgtC which adds a terminal a-D-Gal to the
lactose element of a truncated LOS, thus creating the Pk blood group antigen structure
(Gotshlich (1994). supra.). In A', meningitidis, a separate immunotype LI also expresses the
P blood group antigen and has been shown to carry an IgtC gene (Jennings et al., (1995).
supra.). Neisseria glvcosyltransferases and associated genes are also described in USPN
5.545.553 (Gotschlich). Genes for a1,2-fucosyltransferase and a1,3-fucosyltransferase from
Helicobacter pylori has also been characterized (Martin et al., J. Biol. Chem. 272: 21349-
21356 (1997)). Also of use in the present invention are the glycosyltransferases of
Campylobacter jejuni {see, Taniguchi et al., 2002, Handbook of glycosyltransferases and
related genes. Springer. Tokyo).
B. Sulfotransferases
The invention also provides methods for producing peptides that include sulfated
molecules, including, for example sulfated polysaccharides such as heparin, heparan sulfate,
carragenen, and related compounds. Suitable sulfotransferases include, for example,
chondroitin-6-sulphotransferase (chicken cDNA described by Fukuta et al., J. Biol. Chem.
270: 18575-18580 (1995): GenBank Accession No. D49915), glycosaminoglycan N-
acetylglucosamine N-deacetylase/N-sulphotransferase 1 (Dixon et al.. Genomics 26: 239-241
(1995); UL18918). and glycosaminoglycan N-acetylglucosamine N-deacetylase/N-
sulphotransferase 2 (murine cDNA described in Orellana et al., J. Biol. Chem. 269: 2270-
2276 (1994) and Eriksson et al., J. Biol. Chem. 269: 10438-10443 (1994); human cDNA
described in GenBank Accession No. U2304).
C. Cell-Bound Glycosyltransferases
In another embodiment, the enzymes utilized in the method of the invention are cell-
bound glycosyltransferases. Although many soluble glycosyltransferases are known (see, for
example. U.S. Pat. No. 5.032,519), glycosyltransferases are generally in membrane-bound
form when associated with cells. Many of the membrane-bound enzymes studied thus far are
considered to be intrinsic proteins; that is, they are not released from the membranes by
sonication and require detergents for solubilization. Surface glycosyltransferases have been
identified on the surfaces of vertebrate and invertebrate cells, and it has also been recognized
that these surface transferases maintain catalytic activity under physiological conditions.
However, the more recognized function of cell surface glycosyltransferases is for intercellular
recognition (Roth, 1990, Molecular Approaches to Supracellular Phenomena,).
Methods have been developed to alter the glycosyltransferases expressed by cells.
For example. Larsen ct a!.. Proc. Natl. Acad. Sci. USA 86: 8227-823 1 (1989), report a
genetic approach to isolate cloned cDNA sequences that determine expression of cell surface
oligosaccharide structures and their cognate glycosyltransferases. A cDNA library generated
from mRNA isolated from a murine cell line known to express UDP-galactosc:.ß-D-
galactosyl-l.4-N-acetyl-D-glucosaminide a-l,3-gaIactosyltransferase was transfected into
COS-1 cells. The transfected cells were then cultured and assayed for a 1-3
galactosyltransferase activity.
Francisco etal., Proc. Natl. Acad. Sci. USA 89: 2713-2717 (1992). disclose a method
of anchoring P-lactamase to the external surface of Escherichia coli. A tripartite fusion
consisting of (i) a signal sequence of an outer membrane protein, (ii) a membrane-spanning
section of an outer membrane protein, and (iii) a complete mature P-lactamase sequence is
produced resulting in an active surface bound P-lactamase molecule. However, the Francisco
method is limited only to prokaryotic cell systems and as recognized by the authors, requires
the complete tripartite fusion for proper functioning.
D. Fusion Bnzymes
In other exemplary embodiments, the methods of the invention utilize fusion peptides
that have more than one enzymatic activity that is involved in synthesis of a desired
glycopeptide conjugate. The fusion peptides can be composed of, for example, a catalytically
active domain of a glycosyltransferase that is joined to a catalytically active domain of an
accessory enzyme. The accessory enzyme catalytic domain can, for example, catalyze a step
in the formation of a nucleotide sugar that is a donor for the glycosyltransferase, or catalyze a
reaction involved in a glycosyltransferase cycle. For example, a polynucleotide that encodes
a glycosyltransferase can be joined, in-frame, to a polynucleotide that encodes an enzyme
involved in nucleotide sugar synthesis. The resulting fusion peptide can then catalyze not
only the synthesis of the nucleotide sugar, but also the transfer of the sugar moiety to the
acceptor molecule. The fusion peptide can be two or more cycle enzymes linked into one
expressible nucleotide sequence. In other embodiments the fusion ppeptide includes the
catalytically active domains of two or more glycosyltransferases. See, for example, U.S.
Patent No. 5,641,668. The modified glycopeptides of the present invention can be readily
designed and manufactured utilizing various suitable fusion peptides (see, for example. PCT
Patent Application PCT/CA98/01180, which was published as WO 99/31224 on June 24.
1999.)
E. Immobilized Enzymes
In addition to cell-bound enzymes, the present invention also provides for the use of
enzymes that are immobilized on a solid and/or soluble support. In an exemplary
embodiment, there is provided a glycosyltransferase that is conjugated to a PEG via an intact
glycosyl linker according to the methods of the invention. The PEG-linker-enzyme conjugate
is optionally attached to solid support. The use of solid supported enzymes in the methods of
the invention simplifies the work up of the reaction mixture and purification of the reaction
product, and also enables the facile recovery of the enzyme. The glycosyltransferase
conjugate is utilized in the methods of the invention. Other combinations of enzymes and
supports will be apparent to those of skill in the art.
E. Mutagenesis of Glycosyltransferases
The novel forms of the glycosyltransferases. sialyltransferases, sulfotransferases. and
any other enzymes used in the method of the invention can be created using any of the
methods described previously, as well as others well known to those in the art. Of particular
interest are transferases with altered acceptor specificity and/or donor specificity. Also of
interest are enzymes with higher conversion rates and higher stability among others.
The techniques of rational design mutagenesis can be used when the sequence of the
peptide is known. Since the sequences as well as many of the tertiary structures of the
transferases and glucosidases used in the invention are known, these enzymes are ideal for
rational design of mutants. For example, the catalytic site of the enzyme can be mutated to
alter the donor and/or acceptor specificity of the enzyme.
The extensive tertiary structural data on the glycosyltransferases and glycosidase
hydrolases also make these enzyme idea for mutations involving domain exchanges.
Glycosyltransferases and glycosidase hydrolases are modular enzymes (see, Bourne and
Henrissat, 2001. Current Opinion in Structural Biology 11:593-600). Glycosyltransferases
are divided into two families bases on their structure: GT-A and GT-B. The
glycosyltransferases of the GT-A family comprise two dissimilar domains, one involved in
nucleotide binding and the other in acceptor binding. Thus, one could conveniently fuse the
DNA sequence encoding the domain from one gene in frame with a domain from a second
gene to create a new gene that encodes a protein with a new acceptor/donor specificity. Such
exchanges of domains could additionally include the carbohydrate modules and other
accessory domains.
The techniques of random mutation and/or directed evolution, as described above,
may also be used to create novel forms of the glycosyltransferases and glycosidases used in
the invention.
IV. In vitro and in vivo expression systems
A. Cells for the production of glycopeptides
The action of glycosyltransferases is key to the glycosylation of peptides, thus, the
difference in the expression of a set of glycosyltransferases in any given cell type affects the
pattern of glycosylation on any given peptide produced in that cell. For a review of host cell
dependent glycosylation of peptides, see Kabata and Takasaki, "Structure and Biosynthesis of
Cell Surface Carbohydrates," in Cell Surface Carbohydrates and Cell Development, 1991, pp.
1-24. Eds. Minoru Fukuda. CRC Press, Boca Raton, FL.
According to the present disclosure, the type of cell in which the peptide is produced
is relevant only with respect to the degree of remodeling required to generate a peptide
having desired glycosylation. For example, the number and sequence of enzymatic digestion
reactions and the number and sequence of enzymatic synthetic reactions that are required in
vitro to generate a peptide having desired glycosylation will vary depending on the structure
of the glycan on the peptide produced by a particular cell type. While the invention should in
no way be construed to be limited to the production of peptides from any one particular cell
type including any cell type disclosed herein, a discussion of several cell systems is now
presented which establishes the power of the present invention and its independence of the
cell type in which the peptides are generated.
In general, and to express a peptide from a nucleic acid encoding it. the nucleic acid
must be incorporated into an expression cassette, comprising a promoter element, a
terminator element, and the coding sequence of the peptide operably linked between the two.
The expression cassette is then operably linked into a vector. Toward this end, adapters or
linkers may be employed to join the nucleotide fragments or other manipulations may be
involved to provide for convenient restriction sites, removal of superfluous nucleotides,
removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair,
restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. A
shuttle vector has the genetic elements necessary for replication in a cell. Some vectors may
be replicated only in prokaryotes, or may be replicated in both prokaryotes and eukaryotes.
Such a plasmid expression vector will be maintained in one or more replication systems,
preferably two replication systems, that allow for stable maintenance within a yeast host cell
for expression purposes, and within a prokaryotic host for cloning purposes. Many vectors
with diverse characteristics are now available commercially. Vectors are usually plasmids or
phages, but may also be cosmids or mini-chromosomes. Conveniently, many commercially
available vectors will have the promoter and terminator of the expression cassette already
present, and a multi-linker site where the coding sequence for the peptide of interest can be
inserted. The shuttle vector containing the expression cassette is then transformed in E. coli
where it is replicated during cell division to generate a preparation of vector that is sufficient
to transform the host cells of the chosen expression system. The above methodology is well
know to those in the art. and protocols by which to accomplish can be found Sambrook et al.
(2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York).
The vector, once purified from the cells in which it is amplified, is then transformed
into the cells of the expression system. The protocol for transformation depended on the kind
of the cell and the nature of the vector. Transformants are grown in an appropriate nutrient
medium, and, where appropriate, maintained under selective pressure to insure retention of
endogenous DNA. Where expression is inducible, growth can be permitted of the yeast host
to yield a high density of cells, and then expression is induced. The secreted, mature
heterologous peptide can be harvested by any conventional means, and purified by
chromatography, electrophoresis, dialysis, solvent-solvent extraction, and the like.
The techniques of molecular cloning are well-known in the art. Further, techniques
for the procedures of molecular cloning can be found in Sambrook et al. (2001, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
N.Y.); Glover et al., (1985, DNA Cloning: A Practical Approach, Volumes 1 and II); Gait et
al.. (1985. Oligonucleotide Synthesis); Hames and Higgins (1985, Nucleic Acid
Hybridization ); Harries and Higgins (1984, Transcription And Translation); Frcshney et al..
(1986, Animal Cell Culture); Perbal, (1986. Immobilized Cells And Enzymes, IRL Press);
Perbal.( 1984. A Practical Guide To Molecular Cloning); Ausubel et al. (2002, Current
Protocols in Molecular Biology, John Wiley & Sons. Inc.).
B. Fungi and yeast
Peptides produced in yeast are glycosylated and the glycan structures present thereon
are primarily high mannose structures. In the case of N-glycans, the glycan structures
produced in yeast may contain as many as nine or more mannose residues which may or may
not contain additional sugars added thereto. An example of the type of glycan on peptides
produced by yeast cells is shown in Figure 4, left side. Irrespective of the number of
mannose residues and the type and complexity of additional sugars added thereto, N-glycans
as components of peptides produced in yeast cells comprise a trimannosyl core structure as
shown in Figure 4. When the glycan structure on a peptide produced by a yeast cell is a high
mannose structure, it is a simple matter for the ordinary skilled artisan to remove, in vitro
using available mannosidase enzymes, all of the mannose residues from the molecule except
for those that comprise the trimannosyl core of the glycan. thereby generating a peptide
having an elemental trimannosyl core structure attached thereto. Now, using the techniques
available in the art and armed with the present disclosure, it is a simple matter to
en/ymatically add. in vitro, additional sugar moieties to the elemental trimannosyl core
structure to generate a peptide having a desired glycan structure attached thereto. Similarly,
when the peptide produced by the yeast cell comprises a high mannose structure in addition
to other complex sugars attached thereto, it is a simple matter to enzymatically cleave off all
of the additional sugars, including extra mannose residues, to arrive at the elemental
trimannosyl core structure. Once the elemental trimannosyl core structure is produced,
generation of a peptide having desired glycosylation is possible following the directions
provided herein.
By "yeast" is intended ascosporogenous yeasts (Endomycetales). basidiosporogenous
yeasts, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous
yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceac. The later
is comprised of four subfamilies, Schizosaccharomycoideae (e.g., genus
Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., genera
Pichia. Klutyveromyces, and Saccharomyces). The basidiosporogenous yeasts include the
genera I.eucosporiJium. Rhodosporidium. Sporidiobolas. Filobasidium, and Filobasidiella.
Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae
(e.g., genera Sporobolomyces, Bullera) and Cryptococcaceae (e.g., genus Candida). Of
particular interest to the present invention are species within the genera Saccharomyces,
Pichia, Aspergillus. Trichoderma, Kluyveromyces, especially AT. lactis and K. drosophilum,
Candida, Hansenuht, Schizpsaccaromyces, Yarrowia, and Chrysoporium. Since the
classification of yeast may change in the future, for the purposes of this invention, yeast shall
be defined as described in Skinner et al., eds. 1980) Biology and Activities of Yeast (Soc.
App. Bacteriol. Symp. Series No. 9).
In addition to the foregoing, those of ordinary skill in the art are presumably familiar
with the biology of yeast and the manipulation of yeast genetics. See, for example, Bacila et
al.. eds. (1978, Biochemistry and Genetics of Yeast, Academic Press, New York); and Rose
and Harrison. (1987. The Yeasts (2nd ed.) Academic Press, London). Methods of introducing
exogenous DNA into yeast hosts are well known in the art. There are a wide variety of
methods for transformation of yeast. Spheroplast transformation is taught by Hinnen et al
(1978. Proc. Natl. Acad. Sci. USA 75:1919-1933); Beggs, (1978, Nature 275(5676): 104-
109): and Stinchcomb et al., (EPO Publication No. 45,573: herein incorporated by reference).
Electroporation is taught by Becker and Gaurante, (1991, Methods Enzymol. 194:182-187),
Lithium acetate is taught by Gietz et al. (2002, Methods Enzymol. 350:87-96) and Mount et
al. (1996, Methods Mol Biol. 53:139-145). For a review of transformation systems of non-
Saccharomyces yeasts, see Wangetal. (Crit Rev Biotechnol. 2001:21(3): 177-218). For
general procedures on yeast genetic engineering, see Barr et al., (1989, Yeast genetic
engineering, Butterworths, Boston).
In addition to wild-type yeast and fungal cells, there are also strains of yeast and fungi
that have been mutated and/or selected to enhance the level of expression of the exogenous
gene, and the purity, the post-translational processing of the resulting peptide, and the
recovery and purity of the mature peptide. Expression of an exogenous peptide may also be
direct to the cell secretory pathway, as illustrated by the expression of insulin (see (Kjeldsen,
2000, Appl. Microbiol. Biotechnol. 54:277-286, and references cited therein). In general, to
cause the exogenous peptide to be secreted from the yeast cell, secretion signals derived from
yeast genes may be used, such as those of the genes of the killer toxin (Stark and Boyd. 1986.
EM BO J. 5:1995-2002) or of the alpha phcromonc (Kurjan and Herskowitz, 1982. Cell 30:933;
Brake et al.. 1988. Yeast 4:S436).
Regarding the filamentous fungi in general, methods for genetic manipulation can be
found in Kinghorn and Turner (1992, Applied Molecular Genetics of Filamentous Fungi,
Blackie Academic and Professional, New York). Guidance on appropriate vectors can be
found in Martinelli and Kinghorn (1994, Aspergillus : 50 years, Elsevier, Amsterdam).
I. Saccharomvces
In Saccharomyces, suitable yeast vectors for use producing a peptide include YRp7
(Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEpI 3 (Broach et al.. Gene
8: 121-133, 1979). POT vectors (Kawasaki et al. U.S. Pat. No. 4,931,373, which is
incorporated by reference herein), pJDB249 and pJDB219 (Beggs, Nature 275:104-108,
1978) and derivatives thereof. Preferred promoters for use in yeast include promoters for
yeast glycolytic gene expression (Hitzeman et al., J. Biol. Chem. 255: 12073-12080, 1980;
Alberand Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No.
4.599,311) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of
Microorganisms for Chemicals. Hollaender et al., (eds.), p. 355, Plenum, New York. 1982;
Ammerer. Meth. Enzymol. 101: 192-201. 1983), and the ADH2-4c promoter (Russell et al..
Nature 304: 652-654, 1983; Irani and Kilgore, U.S. patent application Ser. No. 07/784,653,
CA 1,304,020 and EP 284 044, which are incorporated herein by reference). The expression
units may also include a transcriptional terminator. A preferred transcriptional terminator is
the TP11 terminator (Alber and Kawasaki, ibid.).
Examples of such yeast-bacteria shuttle vectors include Yep24 (Botstein et al. (1979)
Gene 8:17-24; pCl (Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642-4646), and
Yrpl7 (Stnichomb et al. (1982) J. Mol. Biol. 158:157). Additionally, aplasmid expression
vector may be a high or low copy number plasmid, the copy number generally ranging from
about 1 to about 200. In the case of high copy number yeast vectors, there will generally be
at least 10, preferably at least 20, and usually not exceeding about 150 copies of the vector in
a single host. Depending upon the heterologous peptide selected, either a high or low copy
number vector may be desirable, depending upon the effect of the vector and the recombinant
peptide on the host. See. for example. Brake et al. (1984) Proc. Natl. Acad. Sci. USA
81:4642-4646. DNA constructs of the present invention can also be integrated into the yeast
genome by an integrating vector. Examples of such vectors are known in the art. See, for
example. Botstein et al. (1979) Gene 8:17-24.
The selection of suitable yeast and other microorganism hosts for the practice of the
present invention is within the skill of the art. Of particular interest are the Saccharomyces
species S. cerevisiae. S. carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S. norbensis,
and S. oviformis. When selecting yeast host cells for expression of a desired peptide, suitable
host cells may include those shown to have, inter alia, good secretion capacity, low
proteolytic activity, and overall vigor. Yeast and other microorganisms are generally
available from a variety of sources, including the Yeast Genetic Stock Center. Department of
Biophysics and Medical Physics, University of California, Berkeley, Calif.; and the American
Type Culture Collection, Manassas VA. For a review, see Strathern et al., eds. (1981. The
Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y.)
Methods of introducing exogenous DNA into yeast hosts are well known in the art.
2. Pichia
The use of Pichia methanolica as a host cell for the production of recombinant
peptides is disclosed in PCT Applications WO 97/17450, WO 97/17451, WO 98/02536, and
WO 98/02565. DNA molecules for use in transforming P. methanolica are commonly
prepared as double-stranded, circular plasmids, which are preferably linearized prior to
transformation. For peptide production in P. methanolica, it is preferred that the promoter
and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica
alcohol utilization gene (AUG1 or AUG2). Other useful promoters include those of the
dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT)
genes, as well as those disclosed in U.S. Patent No. 5,252,726. To facilitate integration of the
DNA into the host chromosome, it is preferred to have the entire expression segment of the
plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use
in Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-
aminoimidazole carboxylase (A1RC; EC 4.1.1.21), which allows ade2 host cells to grow in
the absence of adenine. For large-scale, industrial processes where it is desirable to minimize
the use of methanol, host cells in which both methanol utilization genes (AUG1 and AUG2)
are deleted are preferred. For production of secreted peptides, host cells deficient in vacuolar
protease genes (PEP4 and PRBI) are preferred. Electroporation is used to facilitate the
introduction of a plasm id containing DNA encoding a peptide of interest into P. methanolica
cells. It is preferred to transform P. methanolica cells by electroporation using an
exponential]) decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most
preferably about 20 milliseconds. For a review of the use of Pichia pastoris for large-scale
production of antibody fragments, see Fischer et al., (1999, Biotechnol Appl Biochem. 30 ( Pt
2): 1 17-120).
3. Aspergillus
Methods to express peptides in Aspergillus spp. are well known in the art, including
but not limited to those described in Carrez et al., 1990, Gene 94:147-154; Contreras, 1991,
Bio/Technology 9:378-381; Yelton et al.., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474;
Tilburnetal., 1983, Gene 26:205-221; Kelly and. Hynes, 1985, EMBO J. 4:475-479;
Ballance et al., 1983. Biochem. Biophys. Res. Comm. 112:284-289; Buxton et al., 1985,
Gene 37:207-214. and U.S. Pat. No. 4,935,349, incorporated by reference herein in its
entirety. Examples of promoters useful in Aspergillus are found in U.S. Patent No.
5.252,726. Strains of Aspergillus useful for peptide expression are found in U.S. Patent No.
4,935.349. Commercial production of exogenous peptides is available from Novoenzymes
for Aspergillus niger and Aspergillus oryzae.
4. Trichoderma
Trichoderma has certain advantages over other species of recombinant host cells for
expression of desired peptides. This organism is easy to grow in large quantities and it has
the ability to glycosylate and efficiently secrete high yields of recombinant mammalian
peptides into the medium, making isolation of the peptide relatively easy. In addition, the
glycosylation pattern on expressed peptides is more similar to that on human peptides than
peptides expressed in many other systems. However, there are still differences in the glycan
structures on expressed peptides from these cells. For example, terminal sialic acid residues
are important to the therapeutic function of a peptide in a mammalian system, since the
presence of these moieties at the end of the glycan structure impedes peptide clearance from
the mammalian bloodstream. The mechanism behind the increased biologic half-life of
sialylated molecules is believed to lie in their decreased recognition by lectins (Drickamer,
1988. J. Biol. Chem. 263:9557-9560). However, in general fungal cells do not add terminal
sialic acid residues to glycans on peptides, and peptides synthesized in fungal cells are
therefore asialic. According to the present invention, this deficiency can be remedied using
the in vitro glycan remodeling methods of the invention described in detail elsewhere herein.
Trichoderma species useful as hosts for the production of peptides to be remodeled
include T. reesei. such as QM6a, ALK02442 or CBS383.78 (Centraalbureau voor
Schimmelcultures, Oosterstraat 1, PO Box 273, 3740 AG Baarn, The Netherlands, or.
ATCC13631 (American Type Culture Collection, Manassas VA, 10852, USA, type); T.
viride (such as CBS 189.79 (det. W. Gams); T. longibrachiatum, such as CBS816.68 (type);
T. pseudokoningii (such as MUCL19358; Mycotheque de I'Universite Catholique de
Louvain); T. saturnisporum CBS330.70 (type); T. harzianum CBS316.31 (det. W. Gams); T.
virgatwn (T. pseudokoningii) ATCC24961. Most preferably, the host is T. reesei and more
preferably, it is T. reesei strains QM94I4 (ATCC 26921), RUT-C-30 (ATCC 56765), and
highly productive mutants such as VTT-D-79125, which is derived from QM9414
(Nevalainen, Technical Research Centre of Finland Publications 26, (1985), Espoo, Finland).
The transformation of Trichoderma with DNA is performed using any technique
known in the art. including that taught in European patent No. EP0244234, Harkki (1989,
Bio/Technology 7:596-601) and Uusitalo (1991, J. Biotech. 17:35-50). Culture of
Trichoderma is supported by previous extensive experience in industrial scale fermentation
techniques; for example, see Finkelstein, 1992, Biotechnology of Filamentous Fungi:
Technology and Products, Butterworth-Heinemann, publishers, Stoneham, Mass.
5. Kluyveromyces
Yeast belonging to the genus Kluyveromyces have been used as host organisms for the
production of recombinant peptides. Peptides produced by this genus of yeast are, in
particular, chymosin (European Patent 96 430), thaumatin (European Patent 96 910),
albumin, interleukin-1ß TPA, T1MP (European Patent 361 991) and albumin derivatives
having a therapeutic function (European Patent 413 622). Species of particular interest in the
genus Kluyveromyces include K. lactis.
Methods of expressing recombinant peptides in Kluyvermyces spp. are well known in
the art. Vectors for the expression and secretion of human recombinant peptides in
Kluyvermyces are known in the art (Yeh, J. Cell. Biochem. Suppl. 14C:68, Abst. H402; Fleer.
1990. Yeast 6 (Special Issue):S449) as are procedures for transformation and expression of
recombinant peptides (Ito etal.. 1983, J. Bacterid. 153:163-168; van den Berg, 1990,
Bio/Technology 8:135-139; U.S. Patent No. 5,633,146, WO8304050A1, EP0096910.
FP0241435, EP0301670, EP0361991. all of which are incorporated by reference herein in
their entirety). For a review of genetic manipulation of Kluyveromyces lactis linear DNA
plasmids by gene targeting and plasmid shuffles, see Schaffrath et al. (1999. FFMS Microbiol
Lett. 178(2):201-210).
6. Chrysoporium
The fungal genus Chrysoporium has recently been used to expression of foreign
recombinant peptides. A description of the proceedures by which one of skill in the art can
use Chrysoporium can be used to express foreign peptides is found in WO 00/20555
(incorporated by reference herein in its entirety). Species particularly suitable for expression
system include, but are not limited to, C. botryoides, C. carmichaelii, C. crassitunicatum, C.
europae, C. evolceannui, F. fastidium, C. filiforme, C. gerogiae, C. globiferum, C. globiferum
var. articulation, C. globiferum var. niveum, C. hirundo, C. hispanicum, C. holmii, C.
indicum, C inops, C. keratinophilum, C kreiselii, C. kuzurovianum, C. lignorum. C.
lobatam, C. lucknowense, C. lucknowense Garg 27K, C. medium, C. medium var. spissescens.
C. mephiticum, C. merdarium, C. merdarium var. roseum, C. minor, C. pannicola, C.
parvum, C. parvum var. crescens, C. pilosum, C. peodomerderium, C. pyriformis, C.
queenslandicum. C. sigleri, C. sulfureum, C. synchronum, C. tropicum, C undulatum, C.
vallenarense, C. vespertilium, and C. zonatum.
7. Others
Methods for transforming Schwanniomyces are disclosed in European Patent 394
538. Methods for transforming Acremonium chrysogenum are disclosed by U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by U.S. Pat. No. 4,486,533.
Also know is an expression system specifically for Schizosaccharomyces pombe (European
Patent 385 391). General methods for expressing peptides in fission yeast.
Schizosaccharomyces pombe can be found in Giga-Hama and Kumagai (1997. Foreign gene
expression in fission yeast : Schizosaccharamyce s pombe. Springer, Berlin).
C. Mammalian systems
As discussed above, mammalian cells typically produce a heterogeneous mixture of
N-glycan structures which vary with respect to the number and arrangement of additional
sugars attached to the trimannosyl core. Typically, mammalian cells produce peptides having
a complex glycan structure, such as that shown in Figure 3, right side. Using the methods of
the present invention, a peptide produced in a mammalian cell may be remodeled in vitro to
generate a peptide having desired glycosylation by first identifying the primary glycan
structure and then determining which sugars must be removed in order to remodel the glycan
structure. As discussed herein, the sugars to be removed will determine which cleavage
enzymes will be used and thus, the precise steps of the remodeling process will vary
depending on the primary glycan structure used as the initial substrate. A sample scheme for
remodeling a glycan structure commonly produced in mammalian cells is shown in Figure 2.
The N-glycan biosynthctic pathway in mammalian cells has been well characterized
(reviewed in Moremen, 1994, Glycobiology 4:113-125). Many of the enzymes necessary for
glycan synthesis have been identified, and mutant cell lines defective in this enzymatic
pathway have been isolated including the Chinese hamster ovary (CHO) cell lines Lec23
(defective in alpha-glucosidase I) and Led 8 (novel GlcNAc-TVIIl). The glycosylation
pattern of peptides produced by these mutant cells is altered relative to normal CHO cells.
As discussed herein, the glycosylation defects in these and other mutant cells can be
exploited for the purposes of producing a peptide that lacks a complex glycan structure. For
example, peptides produced by Lec23 cells lack sialic acid residues, and thus require less
enzymatic manipulation in order to reduce the glycan structure to an elemental trimannosyl
core or to Man3GlcNAc4. Thus, peptides produced in these cells can serve as preferred
substrates for glycan remodeling. One of ordinary skill in the art could isolate or identify
other glycosylation-defective cell lines based on known methods, for example the method
described in Stanley et al., 1990, Somatic Cell Mol. Genet., 16: 211-223. Use of
glycosylation-defective cell lines, those identified and as yet unidentified, is included in the
invention for the purpose of generating preferred peptide substrates for the remodeling
processes described herein.
Expression vectors useful for expressing exogenous peptides in mammalian cells are
numerous, and are well known to those in the art. Many mammalian expression vectors are
now commercially available from companies, including Novagen. Inc (Madison. WI). Gene
Therapy Systems (San Diego, CA), Promega (Madison, WI), ClonTech Inc. (Palo Alto, CA),
and Stratagene (La Jolla, CA), among others.
There are several mammalian cell lines that are particularly adept at expressing
exogenous peptides. Typically mammalian cell lines originate from tumor cells extracted
from mammals that have become immortalized, that is to say, they can replicate in culture
essentially indeilnitely. These cell lines include, but are not limited to, CHO (Chinese
hamster ovary, e.g. CHO-K1; ATCC No. CCL 61) and variants thereof, NSO (mouse
myeloma), BNK, BHK 570 (ATCC No. CRL 10314), BHK (ATCC No. CRL 1632),
Per.C6™ (immortalized human cells, Crucell N.V., Leiden, The Netherlands), COS-1 (ATCC
No. CRL 1650), COS-7 (ATCC No. CRL 1651). HEK 293. mouse L cells. T lymphoid cell
lines, BW5147 cells and MDCK (Madin-Darby canine kidney), HeLa (human), A549 (human
lung carcinoma). 293 (ATCC No. CRL 1573; Graham et al.. 1977, Gen. Virol. 36:59-72).
BGMK (Buffalo Green Monkey kidney), Hep-2 (human epidermoid larynx carcinoma), LLC-
MK2 (African Green Monkey Kidney), McCoy, NCI-H292 (human pulmonary
inucocpidermoid carcinoma tube), RD (rhabdomyosarcoma). Vero (African Green Monkey
kidney), HEL (human embryonic lung), Human Fetal Lung-Chang, MRC5 (human
embryonic lung), MRHF (human foreskin), and WI-38 (human embryonic lung). In some
cases, the cells in which the therapeutic peptide is expressed may be cells derived from the
patient to be treated, or they may be derived from another related or unrelated mammal. For
example, fibroblast cells may be isolated from the mammal's skin tissue, and cultured and
transformed in vitro. This technology is commercially available from Transkaryotic
Therapies. Inc. (Cambridge, MA). Almost all currently used cell lines are available from the
American Type Culture Collection (ATCC. Manassas, VA) and BioWhittaker (Walkersville.
Maryland).
Mammalian cells may be transformed with DNA using any one of several techniques
that arc well known to those in the art. Such techniques include, but are not limited to.
calcium phosphate transformation (Chen and Okayama, 1988 ; Graham and van der Eb.
1973: Corsaro and Pearson, 1981, Somatic Cell Genetics 7:603), Diethylaminocthyl (DEAE)-
dextran transfection (Fujita et al., 1986; Lopata et al., 1984; Selden et al., 1986,).
electroporation (Neumann et al.. 1982. : Potter. 1988, ; Potter et al., 1984, : Wong and
Neuman, 1982 ), cationic lipid reagent transfection (Elroy-Stein and Moss. 1990; Feigner et
al.. 1987; Rose et al., 1991; Whitt et al., 1990; Hawley-Nelson et al., 1993, Focus 15:73;
Ciccarone et al., 1993, Focus 15:80), retroviral (Cepko et al., 1984; Miller and Baltimore.
1986; Pear et al.. 1993: Austin and Cepko, 1990; Bodine et al., 1991; Fckete and Cepko.
1993: LemischkaetaL 1986: Turner et al.. 1990; Williams et al., 1984: Miller and Rosman.
1989. BioTechniqucs 7:980-90; Wang and Finer, 1996, Nature Med. 2:714-6), polybrene
(Chaney et al, 1986: Kawai and Nishizawa, 1984). microinjection (Capecchi, 1980), and
protoplast fusion (Rassoulzadegan et al.. 1982; Sandri-Goldin et al., 1981: Schaffer. 1980).
among others. In general, see Sambrook et al. (2001, Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory. New York) and Ausubel et al. (2002, Current
Protocols in Molecular Biology, John Wiley & Sons, New York) for transformation
techniques.
Recently the baculovirus system, popular for transformation of insect cells, has been
adapted for stable transformation of mammalian cells (see, for review, Koat and Condrcay.
2002, Trends Biotechnol. 20:173-180, and references cited therein). The production of
recombinant peptides in cultured mammalian cells is disclosed, for example, in U.S. Pat. Nos.
4.713.339, 4,784,950; 4.579,821; and 4,656,134. Several companies offer the services of
transformation and culture of mammalian cells, including Cell Trends. Inc. (Middletown.
MD). Techniques for culturing mammalian cells are well known in the art, and further found
in l-lauscr et al. (1997. Mammalian Cell Biotechnology, Walter de Gruyer, Inc., Hawthorne,
NY), and Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor and references cited therein.
D. Insect
Insect cells and in particular, cultured insect cells, express peptides having N-linked
glycan structures that are rarely sialylated and usually comprise mannose residues which may
or may not have additional fucose residues attached thereto. Examples of the types of glycan
structures present on peptides produced in cultured insect cells are shown in Figure 6, and
mannose glycans thereof. In this situation, there may or may not be a core fucose present,
which if present, may be linked to the glycan via several different linkages.
Raculovirus-mediated expression in insect cells has become particularly well-
established for the production of recombinant peptides (Altmann et al., 1999, Glycoconjugate
1. 16:109-123). With regard to peptide folding and post-translational processing, insect cells
are second only to mammalian cell lines. I lowever, as noted above, N-glycosylation of
peptides in insect cells differs in many respects from N-glycosylation in mammalian cells
particularly in that insect cells frequently generate truncated glycan structures comprising
oligosaccharides containing just three or sometimes only two mannose residues. These
structures may be additionally substituted with fucose residues.
According to the present invention, a peptide produced in an insect cell may be
remodeled in vitro to generate a peptide with desired glycosylation by first optionally
removing any substituted fucose residues using an appropriate fucosidase enzyme. In
instances where the peptide comprises an elemental trimannosyl core structure following the
removal of fucose residues, then all that is required is the in vitro addition of the appropriate
sugars to the trimannosyl core structure to generate a peptide having desired glycosylation.
In instances when the peptide might contain only two mannose residues in the glycan
structure following removal of any fucose residues, a third mannose residue may be added
using a mannosyltransferase enzyme and a suitable donor molecule such as GDP-mannose.
and thereafter the appropriate residues are added to generate a peptide having desired
glycosylation. Optionally, monoantennary glycans can also be generated from these species.
Protocols for the use of baculovirus to transform insect cells are well known to those
in the art. Several books have been published which provide the procedures to use the
baculovirus system to express peptides in insect cells. These books include, but are not
limited to, Richardson (Baculovirus Expression Protocols, 1998, Methods in Molecular
Biology. Vol 39. Humana Pr), O'Reilly et al. (1994, Baculovirus Expression Vectors : A
Laboratory Manual, Oxford Univ Press), and King and Possee (1992, The Baculovirus
Expression System : A Laboratory Guide. Chapman & Hall). In addition, there arc also
publications such as Lucklow (1993, Curr. Opin. Biotechnol. 4:564-572) and Miller (1993.
Curr. Opin. Genet. Dev. 3:97-101).
Many patents have also been issued that related to systems for baculoviral expression
of foreign proteins. These patents include, but are not limited to. U.S. Patent No. 6.210.966
(Culture medium for insect cells lacking glutamine and containing ammonium salt), U.S.
Patent No. 6,090.584 (Use of BVACs (BaculoVirus Artificial Chromosomes) to produce
recombinant peptides). U.S. Patent No. 5,871.986 (Use of a baculovirus to express a
recombinant nucleic acid in a mammalian cell), U.S. Patent No. 5,759,809 (Methods of
expressing peptides in insect cells and methods of killing insects), U.S. Patent No. 5.753.220
(Cysteine protease gene defective baculovirus, process for its production, and process for the
production of economic peptide by using the same), U.S. Patent No. 5,750,383 (Baculovirus
cloning system), U.S. Patent No. 5,731,182 (Non-mammalian DNA virus to express a
recombinant nucleic acid in a mammalian cell), U.S. Patent No. 5,728,580 (Methods and
culture media for inducing single cell suspension in insect cell lines), U.S. Patent No.
5.583.023 (Modified baculovirus, its preparation process and its application as a gene
expression vector). U.S. Patent No. 5,571,709 (Modified baculovirus and baculovirus
expression vectors). U.S. Patent No. 5,521,299 (Oligonucleotides for detection of baculovirus
infection), U.S. Patent No. 5,516,657 (Baculovirus vectors for expression of secretory and
membrane-bound peptides), U.S. Patent No. 5,475,090 (Gene encoding a peptide which
enhances virus infection of host insects). U.S. Patent No. 5,472,858 (Production of
recombinant peptides in insect larvae), U.S. Patent No. 5.348,886 (Method of producing
recombinant eukaryotic viruses in bacteria), U.S. Patent No. 5,322,774 (Prokaryotic leader
sequence in recombinant baculovirus expression system), U.S. Patent No. 5,278,050 (Method
to improve the efficiency of processing and secretion of recombinant genes in insect
systems), U.S. Patent No. 5,244,805 (Baculovirus expression vectors), U.S. Patent No.
5,229,293 (Recombinant baculovirus). U.S. Patent No. 5,194,376 (Baculovirus expression
system capable of producing recombinant peptides at high levels), U.S. Patent No. 5.179,007
(Method and vector for the purification of recombinant peptides), U.S. Patent No. 5.169.784
(Baculovirus dual promoter expression vector), U.S. Patent No. 5,162,222 (Use of
baculovirus early promoters for expression of recombinant nucleic acids in stably
transformed insect cells or recombinant baculoviruses), U.S. Patent No. 5,155.037 (Insect
signal sequences useful to improve the efficiency of processing and secretion of recombinant
nucleic acids in insect systems), U.S. Patent No. 5,147,788 (Baculovirus vectors and methods
of use), U.S. Patent No. 5,110.729 (Method of producing peptides using baculovirus vectors
in cultured cells), U.S. Patent No. 5,077,214 (Use of baculovirus early promoters for
expression of recombinant genes in stably transformed insect cells), U.S. Patent No.
5,023.328 (Lepidopteran AKH signal sequence), and U.S. Patent Nos. 4,879,236 and
4.745,05 1 (Method for producing a recombinant baculovirus expression vector). All of the
aforementioned patents are incorporated in their entirety by reference herein.
Insect cell lines of several different species origin are currently being used for peptide
expression, and these lines are well known to those in the art. Insect cell lines of interest
include, but are not limited to, dipteran and lepidopteran insect cells in general, Sf9 and
variants thereof (fall armyworm Spodoptera frugiperda), Estigmene acrea, Trichoplusia ni,
Bombyx mori, Malacosoma disstri. drosophila lines K.cl and SL2 among others, and
mosquito.
E. Plants
Plant cells as peptide producers present a different set of issues. While N-linked
glycans produced in plants comprise a trimannosyl core structure, this pentasaccharide
backbone may comprise several different additional sugars as shown in Figure 5. For
example, in one instance, the trimannosyl core structure is substituted by a (31,2 linked xylose
residue and an ul.3 linked fucose residue. In addition, plant cells may also produce a
Man5GlcNAc2 structure. Peptides produced in plant cells are often highly antigenic as a
result of the presence of the core a1,3 fucose and xylose on the glycan structure, and are
rapidly cleared from the blood stream when introduced into a mammal due to the absence of
terminal sialic acid residues. Therefore, unless these peptides are remodeled using the
methods provided herein, they are generally considered to be unsuitable as therapeutic agents
in mammals. While some monoclonal antibodies expressed in plant cells were found to be
non-immunogenic in mouse, it is likely that the glycan chains were not immunogenic because
they were buried in the Fc region in these antibodies (Chargelegue et al., 2000, Transgenic
Res. 9(3):187-194).
Following the directions provided herein, it is now possible to generate a peptide
produced in a plant cell wherein an increased number of the glycan structures present thereon
comprise an elemental trimannosyl core structure, or a Man3GlcNAc4 structure. This is
accomplished by cleaving off any additional sugars in vitro using a combination of
appropriate glycosidases. including fucosidases. until the elemental trimannosyl core
structure or the Man3GlcNAc4 structure is arrived at. These cleavage reactions should also
include removal of any fucose or xylose residues from the structures in order to diminish the
antigenicity of the final peptide when introduced into a mammal. Plant cells having
mutations that inhibit the addition of fucose and xylose residues to the trimannosyl core
structure are known in the art (von Schaewen et al., 1993, Plant Physiology 102:1109-1 1 18).
The use of these cells to produce peptides having glycans which lack fucose and xylose is
contemplated by the invention. Upon production of the elemental trimannosyl core or
Man3GlcNAc4 structure, additional sugars may then be added thereto to arrive at a peptide
having desired glycosylation that is therefore suitable for therapeutic use in a mammal.
Transgenic plants are considered by many to be the expression system of choice for
pharmaceutical peptides. Potentially, plants can provide a cheaper source of recombinant
peptides. It has been estimated that the production costs of recombinant peptides in plants
could be between 10 to 50 times lower that that of producing the same peptide in E. culi.
While there arc slight differences in the codon usage in plants as compared to animals, these
can be compensated for by adjusting the recombinant DNA sequences (sec, Kusnadi et al.,
1997. Biotechnol. Bioeng. 56:473-484; Khoudi et al., 1999, Biotechnol. Bioeng. 135-143:
Hood et al.. 1999. Adv. Exp. Med. Biol. 464:127-147). In addition, peptide synthesis,
secretion and post-translational modification are very similar in plants and animals, with only
minor differences in plant glycosylation (see. Fischer et al.. 2000, J. Biol. Regul. Homest.
Agents 14: 83-92). Then, products from transgenic plants are also less likely to be
contaminated by animal pathogens, microbial toxins and oncogenic sequences.
The expression of recombinant peptides in plant cells is well known in the art. In
addition to transgenic plants, peptides can also produced in transgenic plant cell cultures (Lee
et al., 1997. Mol. Cell. 7:783-787), and non-transgenic plants inoculated with recombinant
plant viruses. Several books have been published that describe protocols for the genetic
transformation of plant cells: Potrykus (1995, Gene transfer to plants, Springer, New York),
Nickoloff (1995, Plant cell electroporation and electrofusion protocols, Humana Press.
Totowa. New York) and Draper (1988, Plant genetic transformation, Oxford Press, Boston).
Several methods are currently used to stably transform plant cells with recombinant
genetic material. These methods include, but are not limited to, Agrobacterium
transformation (Bechtold and Pelletier, 1998; Escudero and Hohn. 1997: Hansen and Chilton,
1999: Touraev et al.. 1997), biolistics (microprojectiles) (Finer et al.. 1999; Hansen and
Chilton. 1999; Shilito. 1999), electroporation of protoplasts (Fromm et al., 1985, Ou-Lee et
al.. 1986: Rhodes et al.. 1988; Saunders et al.. 1989; Trick et al., 1997), polyethylene glycol
treatment (Shilito. 1999: Trick et al., 1997), inplanta mircroinjection (Leduc et al., 1996;
Zhou et al., 1983), seed imbibition (Trick et al., 1997), laser beam (1996), and silicon carbide
whiskers (Thompson et al.. 1995: U.S. Patent Appln. No. 20020100077, incorporated by
reference herein in its entirety).
Many kinds of plants are amenable to transformation and expression of exogenous
peptides. Plants of particular interest to express the peptides to be used in the remodeling
method of the invention include, but are not limited to, Arabidopsis thalliana. rapeseed
(Brassica spp.; Ruiz and Blumwald, 2002. Planta 214:965-969)), soybean (Glycine max).
sunflower (Helianthus unnuus), oil palm (Elaeis guineeis), groundnut (peanut, Arachis
hypogaea: Deng et al.. 2001, Cell. Res. 11:156-160), coconut (Cocus nucifera), castor
(Ricinus communis), safflower (Carthamus tinctorius), mustard (Brassica spp. and Sinapis
alba), coriander, {Coriandrum sativum), squash (Cucurbita maxima: Spencer and Snow,
2001, Heredity 86(Pt 6):694-702), linseed/flax (Linum usitatissimum; Lamblin et al., 2001,
Physiol Plant 112:223-232), Brazil nut {Bertholletia excelsa), jojoba (Simmondsia chinensis),
maize (Zea mays; Hood et al., 1999, Adv. Exp. Med. Biol. 464:127-147; Hood et al., 1997.
Mol. Breed. 3:291-306; Petolino et al., 2000, Transgenic Research 9:1-9), alfalfa (Khoudi et
al., 1999, Biotechnol. Bioeng. 64:135-143), tobacco (Nicoliana labacum; Wright et al..
Transgenic Res. 10:177-181; Frigerio et al., 2000, Plant Physiol. 123:1483-1493; Cramer et
al., 1996, Ann. New York Acad. Sci. 792:62-8-71; Cabanes-Macheteau etal., 1999,
Glycobiology 9:365-372; Ruggiero et al., 2000, FEBS Lett. 469:132-136). canola (Bai et al.,
2001. Biotechnol. Prog. 17:168-174; Zhang etal., 2000, J. Anim. Sci. 78:2868-2878)), potato
(Tacket et al., 1998. J. Infect. Dis. 182:302-305; Richter et al., 2000, Nat. Biotechnol.
18:1167-1171; Chong et al., 2000, Transgenic Res. 9:71-78), alfalfa (Wigdorovitz et al.,
1999. Virology 255:347-353), Pea (Pisum sativum; Perrin et al., 2000, Mol. Breed. 6:345-
352), rice (Oryza sativa ; Stoger et al., 2000, Plant Mol. Biol. 42:583-590), cotton
(Gossypium hirsutism; Kornyeyev et al., 2001. Physiol Plant 113:323-331). barley (Hordeum
vulgare: Petersen et al., 2002. Plant Mol Biol 49:45-58); wheat (Triticum spp.; Pellcgrineschi
et al., 2002, Genome 45:421-430) and bean (Vicia spp.; Saalbach et al., 1994, Mol Gen Genet
242:226-236).
If expression of the recombinant nucleic acid is desired in a whole plant rather than in
cultured cells, plant cells are first transformed with DNA encoding the peptide, following
which, the plant is regenerated. This involves tissue culture procedures that are typicailv
optimized for each plant species. Protocols to regenerate plants are already well known in the
art for many species. Furthermore, protocols for other species can be developed by one of
skill in the art using routine experimentation. Numerous laboratory manuals are available
that describe procedures for plant regeneration, including but not limited to, Smith (2000.
Plant tissue culture : techniques and experiments, Academic Press, San Diego), Bhojwani and
Razdan (1996, Plant tissue culture : theory and practice, Elsevier Science Pub., Amsterdam),
Islam (1996, Plant tissue culture, Oxford & IBH Pub. Co., New Delhi, India). Dodds and.
Roberts ( 1995, Experiments in plant tissue culture. New York : Cambridge University Press.
Cambridge England). Bhojwani (Plant tissue culture : applications and limitations, Elsevier,
Amsterdam, 1990). Trigiano and Gray (2000, Plant tissue culture concepts and laboratory-
exercises,. CRC Press, Boca Raton, Fla). and Lindsey (1991, Plant tissue culture manual :
fundamentals and applications, Kluwer Academic, Boston).
While purifying recombinant peptides from plants may potentially be costly, several
systems have been developed to minimize these costs. One method directs the synthesized
peptide to the seed endosperm from where it can easily extracted (Wright et al., 2001,
Transgenic Res. 10:177-181, Guda et a... 2000. Plant Cell Res. 19:257-262; and U.S. Patent
No. 5.767.379, which is incorporated by reference herein in its entirety). An alternative
approach is the co-extraction of the recombinant peptide with conventional plant products
such as starch, meal or oil. In oil-seed rape, a fusion peptide of olcosin-hurudin when
expressed in the plant, attaches to the oil body of the seed, and can be extracted from the
plant seed along with the oil (Parmenter, 1995, Plant Mol. Biol. 29:1167-1 180; U.S. Patent
Nos. 5.650.554, 5.792.922, 5.948.682 and 6,288,304, and US application 2002/0037303. all
of which are incorporated in their entirely by reference herein). In a variation on this
approach, the oleosin is fused to a peptide having affinity for the exogenous co-expressed
peptide of interest (U.S. Patent No. 5,856,452, incorporated by reference herein in its
entirety).
Expression of recombinant peptides in plant plastids. such as the chioroplast.
generates peptides having no glycan structures attached thereto, similar to the situation in
prokaryotes. However, the yield of such peptides is vastly greater when expressed in these
plant cell organelles, and thus this type of expression system may have advantages over other
systems. For a general review on the technology for plastid expression of exogenous peptides
in higher plants, sec Hager and Beck (2000, Appl. Microbiol. Biotechnol. 54:302-310, and
references cited therein). Plastid expression has been particularly successful in tobacco (see.
for example, Staub et al.. 2000. Nat. Biotechnol. 18:333-338).
F. Transgenic animals
Introduction of a recombinant DNA into the fertilized egg of an animal (e.g., a
mammal) may be accomplished using any number of standard techniques in transgenic
animal technology. See. e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory-
Manual, Cold Spring I larbor Laboratory Press, Cold Spring Harbor, N.Y., 1986; and U.S.
Pat. No. 5,811,634. which is incorporated by reference herein in its entirety. Most
commonly, the recombinant DNA is introduced into the embryo by way of pronuclear
microinjection (Gordon et al.. 1980. PNAS 77:7380-7384: Gordon and Ruddle. 1981. Science
214:1244-1246: Brinster et al.. 1981, Cell 27:223-231; Costantini and Lacy, 1981. Nature
294:92-94). Microinjection has the advantage of being applicable to a wide variety of
species. Preimplantation embryos may also be transformed with retroviruses (Jaenisch and
Mintz, 1974. Proc. Natl. Acad. Sci. U.S.A. 71:1250-1254; Jaenisch et al., 1976. Hamatol
Bluttransfus. 19:341-356; Stuhlmann et al.. 1984, Proc. Natl. Acad. Sci. U.S.A. 81:7151-
7155). Retroviral mediated transformation has the advantage of adding single copies of the
recombinant nucleic acid to the cell, but it produces a high degree of mosaicism. Most
recently, embryonic stem cell-mediated techniques have been used (Gossler et al.. 1986.
Proc. Natl. Acad. Sci. U.S.A.. 83:9065-9069), transfer of entire chromosomal segments
(Lavitrano et al., 1989, Cell 57:717-723), and gamete transfection in conjunction with in vitro
fertilization (Lavitrano et al., 1989. Cell 57:717-723) have also been used. Several books of
laboratory procedures have been published disclosing these techniques: Cid-Arregui and
Garcia-Carranca (1998, Microinjection and Transgenesis : Strategies and Protocols, Springer.
Berlin). Clarke (2002. Transgenesis Techniques : Principles and Protocols, Humana Press.
Totowa, NJ), and Pinkert (1994. Transgenic Animal Technology : A Laboratory Handbook.
Academic Press. San Diego).
Once the combinant DNA is introduced into, the egg, the egg is incubated for a short
period of time and is then transferred into a pseudopregnant animal of the same species from
which the egg was obtained (Hogan et al., supra). In the case of mammals, typically 125
eggs are injected per experiment, approximately two-thirds of which will survive the
procedure. Twenty viable eggs are transferred into a pseudopregnant mammal, four to ten of
which will develop into live progeny. Typically, 10-30% of the progeny (in the case of mice)
carry the recombinant DNA.
While the entire animal can be used as an expression system for the peptides of the
invention, in a preferred embodiment, the exogenous peptide accumulates in products of the
animal, from which it can be harvested without injury to the animal. In preferred
embodiments, the exogenous peptide accumulates in milk, eggs, hair, blood, and urine.
If the recombinant peptide is to be accumulated in the milk of the animal, suitable
mammals are ruminants, ungulates, domesticated mammals, and dairy animals. Particularly
preferred animals are goats, sheep, camels, cows, pigs, horses, oxen, and llamas. Methods for
generating transgenic cows that accumulate a recombinant peptide in their milk are well
known: see. Newton (1999. .1. Immunol. Methods 231:159-167), Ebert et al. (1991,
Biotechnology 9: 835-838). and U.S. Patent Nos. 6,210,736, 5,849,992, 5,843,705.
5.827,690. 6.222,094. all of which arc incorporated herein by reference in their entirety. The
generation of transgenic mammals that produce a desired recombinant peptide is
commercially available from GTC Biotherapeutics, Framingham, MA.
If the recombinant peptide is to be accumulated in eggs, suitable birds include, but are
not limited to, chickens, geese, and turkeys. Other animals of interest include, but are not
limited to, other species of avians, fish, reptiles and amphibians. The introduction of
recombinant DNA to a chicken by retroviral transformation is well known in the art:
Thoraval et al. (1995, Transgenic Research 4:369-376), Bosselman et al., (1989, Science 243:
533-535), Petropoulos et al. (1992, J. Virol. 66: 3391-3397), U.S. Patent No. 5,162,215.
incorporated by reference herein in its entirety. Successful transformation of chickens with
recombinant DNA also been achieved wherein DNA is introduced into blastodermal cells
and blastodermal cells so transfected are introduced into the embryo: Brazolot et al. (1991.
Mol. Reprod. Dev. 30: 304-312), Eraser, et al. (1993. Int. J. Dev. Biol. 37: 381-385). and
Petitte et al. (1990. Development 108: 185-189). High throughput technology has been
developed to assess whether a transgenic chicken expresses the desired peptide (Harvey et al..
2002. Poult. Sci. 81:202-212. U.S. Patent No. 6,423.488, incorporated by reference herein in
its entirety). Using retroviral transformation of chicken with a recombinant DNA, exogenous
beta-lactamase was accumulated in the egg white of the chicken (Harvey et al.. 2002, Nat.
Biotechnol. 20(4):396-399). The production of chickens producing exogenous peptides in
egg is commercially available from AviGenics, Inc., Athens GA.
G. Bacteria
Recombinantly expressed peptides produced in bacteria are not generally
glycosylated. However, bacteria systems capable of glycosylating peptides are becoming
evident and therefore it is likely that glycosylated recombinant peptides may be produced in
bacteria in the future.
Numerous bacterial expression systems are known in the art. Preferred bacterial
species include, but are not limited to, E.coli. and Bacillus species.
The expression of recombinant peptides in E. coli is well known in the art. Protocols for E.
coli-based expression systems are found in U.S. Appln No. 20020064835, U.S. Patent Nos.
6.245,539, 5,606,031. 5.420,027, 5,151,511, and RE33,653, among others. Methods to
transform bacteria include, but are not limited to, calcium chloride (Cohen et al., 1972, Proc.
Natl. Acad. Sci. U.S.A.. 69:2110-2114; Hanahan, 1983, J. Mol. Biol. 166:557-580; Mandel
and Higa, 1970, J. Mol. Biol. 53:159-162) and electroporation (Shigekawa and Dower. 1988,
Biotechniques 6:742-751), and those described in Sambrook et al., 2001 (supra). For a
review of laboratory protocols on microbial transformation and expression systems, see
Saunders and Saunders (1987. Microbial Genetics Applied to Biotechnology : Principles and
Techniques of Gene Transfer and Manipulation, Croom Helm, London), Pühler (1993,
Genetic Engineering of Microorganisms, Weinheim, New York), Lee et al., (1999, Metabolic
Engineering. Marcel Dekker, New York), Adolph (1996, Microbial Genome Methods. CRC
Press. Boca Raton), and Birren and Lai (1996, Nonmammalian Genomic Analysis : A
Practical Guide, Academic Press, San Diego),
For a general review on the literature for peptide expression in E. coli see Balbas
(2001. Mol. Biotcchnol. 19:251-267). Several companies now offer bacterial strains selected
for the expression of mammalian peptides, such as the Rosetta™ strains of E. coli (Novagen,
inc.. Madison. WI; with enhanced expression of eukaryotic codons not normally used in
bacteria cells, and enhanced disulfide bond formation).
II. Cell engineering
It will be apparent from the present disclosure that the more uniform the starting
material produced by a cell, the more efficient will be the generation in vitro of large
quantities of peptides having desired glycosylation. Thus, the genetic engineering of host
cells to produce uniformly glycosylated peptides as starting material for the in vitro
enzymatic reactions disclosed herein, provides a significant advantage over using a peptide
starting material having a heterogeneous set of glycan structures attached thereto. One
preferred peptide starting material for use in the present invention is a peptide having
primarily glycan molecules which consist solely of an elemental trimannosyl core structure.
Another preferred starting material is Man3GlcNAc4. Following the remodeling process, the
preferred peptides will give rise to the greatest amount of peptides having desired
glycosylation. and thus improved clinical efficacy. However, other glycan starting material is
also suitable for use in the methods described herein, in that for example, high mannose
glycans may be easily reduced, in vitro, to elemental trimannosyl core structures using a
series of mannosidases. As described elsewhere herein, other glycan starting material may
also be used, provided it is possible to cleave off all extraneous sugar moieties so that the
elemental trimannosyl core structure or Man3GlcNAc4 is generated. Thus, the purpose of
using genetically engineered cells for the production of the peptides of the present invention
is to generate peptides having as uniform as possible a glycan structure attached thereto,
wherein the glycan structure can be remodeled in vitro to generate a peptide having desired
glycosylation. This will result in a dramatic reduction in production costs of these peptides.
Since the glycopeptides produced using this methodology will predominantly have the same
N-linked glycan structure, the post-production modification protocol can be standardized and
optimized to produce a greater batch-to-batch consistency of final product. As a result, the
final completed-chain products may be less heterogeneous than those presently available.
The products will have an improved biological half-life and bioactivity as compared to the
products of the prior art. Alternatively, if desired, the invention can be used to introduce
limited and specific heterogeneity, e.g.. by choosing reaction conditions that result in
differential addition of sugar moieties.
Preferably, though not as a rigid requirement, the genetically engineered cell is one
which produces peptides having glycan structures comprised primarily of an elemental
trimannosyl core structure or Man3GlcNAc4. At a minimum, the proportion of these
preferred structures produced by the genetically engineered cell must be enough to yield a
peptide having desired glycosyiation following the remodeling protocol.
In general, any eukaryotic cell type can be modified to become a host cell of the
present invention. First, the glycosyiation pattern of both endogenous and recombinant
glycopeptides produced by the organism are determined in order to identify suitable
additions/deletions of enzymatic activities that result in the production of elemental
trimannosyl core glycopeptides or Man3GlcNAc4 glycopeptides. This will typically entail
deleting activities that use trimannosyl glycopeptides as substrates for a glycosyltransferasc
reaction and inserting enzymatic activities that degrade more complex N-linked glycans to
produce shorter chains. In addition, genetically engineered cells may produce high mannosc
glycans, which may be cleaved by mannosidase to produce desired starting glycan structures.
The mannosidase may be active in vivo in the cell (i.e., the cell may be genetically engineered
to produce them), or they may be used in in vitro post production reactions.
Techniques for genetically modifying host cells to alter the glycosyiation profile of
expressed peptides are well-known. See, e.g., Altmann et al. (1999, Glycoconjugate J. 16:
109-123), Ailoretal. (2000, Glycobiology 10(8): 837-847), Jarvis et al., (In vitrogen
Conference, March. 1999, abstract), Hollisterand Jarvis, (2001. Glycobiology 11(1): 1-9).
and Palacpac et al.. (1999, PNAS USA 96: 4697), Jarvis et al., (1998. Curr. Opin. Biotechnol.
9:528-533), Gerngross (U.S. Patent Publication No. 20020137134), all of which disclose
techniques to "mammalianize" insect or plant cell expression systems by transfecting insect
or plant cells with glycosyltransferase genes.
Techniques also exist to genetically alter the glycosyiation profile of peptides
expressed in E. coli. E. coli has been engineered with various glycosyltransferases from the
bacteria Neisseria meningitidis and Azorhizobium to produce oligosaccharides in vivo (Bettler
et al. 1999. Glycoconj. J. 16:205-212). E. coli which has been genetically engineered to
over-express Neisseria meningitidis ß1,3 N acetyl glucosaminyltransferase IgtA gene will
efficiently glycosylate exogenous lactose (Priem et al., 2002. Giycobiology 12:235-240).
Fungal cells have also been gefletically modified to produce exogenous
glycosyltransferases (Yoshida et al., 1999, Giycobiology, 9(l):53-58; Kalsner et al.. 1995.
Glycoconj. J. 12:360-370; Schwientek and Ernst, 1994. Gene 145(2):299-303: Chiba et al,
1995, Biochem J. 308:405-409).
Thus, in one aspect, the present invention provides a cell that glycosylates a
glycopeptide population such that a proportion of glycopeptides produced thereby have an
elemental trimannosyl core or a Man3GlcNAc4 structure. Preferably, the cell produces a
peptide having a glycan structure comprised solely of an elemental trimannosyl core. At a
minimum, the proportion of peptides having an elemental trimannosyl core or a
Man3GlcNAc4 structure is enough to yield peptides having desired glycosylation following
the remodeling process. The cell has introduced into it one or more heterologous nucleic acid
expression units, each of which may comprise one or more nucleic acid sequences encoding
one or more peptides of interest. The natural form of the glycopeptide of interest may
comprise one or more complex N-linked glycans or may simply be a high man nose glycan.
The cell may be any type of cell and is preferably a eukaryotic cell. The cell may be a
mammalian cell such as human, mouse, rat, rabbit, hamster or other type of mammalian cell.
When the cell is a mammalian cell, the mammalian cell may be derived from or contained
within a non-human transgenic mammal where the cell in the mammal encodes the desired
glycopeptide and a variety of glycosylating and glycosidase enzymes as necessary for the
production of desired glycopeptide molecules. In addition, the cell may be a fungal cell,
preferably, a yeast cell, or the cell may be an insect or a plant cell. Similarly, when the cell is
a plant cell, the plant cell may be derived from or contained within a transgenic plant,
wherein the plant encodes the desired glycopeptide and a variety of glycosylating and
glycosidase enzymes as are necessary for the production of desired glycopeptide molecules.
In some embodiments the host cell may be a eukaryotic cell expressing one or more
heterologous glycosyltransferase enzymes and/or one or more heterologous glycosidase
enzymes, wherein expression of a recombinant glycopeptide in the host cell results in the
production of a recombinant glycopeptide having an elemental trimannosyl core as the
primary glycan structure attached thereto.
In some embodiments the heterologous glycosyltransferase enzyme useful in the cell
may be selected from a group consisting of any known glycosyltransferase enzyme included
for example, in the list of Glycosyltransferase Families available in 1aniguchi et al. (2002.
Handbook of Glycosyltransferases and Related Genes. Springer, New York).
In other embodiments, the heterologous glycosylasc enzyme may be selected from a
group consisting of mannosidase 1, mannosidase 2, mannosidase 3. and other mannosidases,
including, but not limited to, microbial mannosidases. Additional disclosure regarding
enzymes useful in the present invention is provided elsewhere herein.
In yet other embodiments, the host cell may be a eukaryotic cell wherein one or more
endogenous glycosyltransferase enzymes and/or one or more endogenous glycosidase
enzymes have been inactivated such that expression of a recombinant glycopeptide in the
host cell results in the production of a recombinant glycopeptide having an elemental
trimannosyl core as the primary glycan structure attached thereto.
In additional embodiments, the host cell may express heterologous
glycosyltransferase enzymes and/or glycosidase enzymes while at the same time one or more
endogenous glycosyltransferase enzymes and/or glycosidase enzymes are inactivated.
Endogenous glycosyltransferase enzymes and/or glycosidase enzymes may be inactivated
using any technique known to those skilled in the art including, but not limited to, antisensc
techniques and techniques involving insertion of nucleic acids into the genome of the host
cell. In some embodiments, the endogenous enzymes may be selected from a group
consisting of GnT-1. a selection of mannosidases, xylosyltransferase, core al,3
fucosyltransferase, serine/threonine O-mannosyltransferases, and the like.
Alternatively, an expression system that naturally glycosylates peptides such that the
N-linked glycans are predominantly the trimannosyl core type, or the Man3GlcMAc4 type,
can be exploited. An example of a cell type that produces the trimannosyl core is Sf9 cells.
Other such expression systems can be identified by analyzing glycopeptides that are naturally
or recombinantly expressed in cells and selecting those which exhibit the desired
glycosylation characteristics. The invention should be construed to include any and all such
cells for the production of the peptides of the present invention.
V. Purification of glycan remodeled and/or glycoconjugated peptides
If the modified glycoprotein is produced intracellular^ or secreted, as a first step, the
particulate debris, either host cells, lysed fragments, is removed, for example, by
centrifugation or ultrafiltration; optionally, the protein may be concentrated with a
commercially available protein concentration filter, followed by separating the peptide
variant from other impurities by one or more steps selected from immunoaffinity
chromatography, ion-exchange column fractionation (e.g.. on diethylaminoethyl (DFAE) or
matrices containing carboxymethyl or sulfopropyl groups), chromatography on Blue-
Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharosc, WGA-
Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl. or
protein A Sepharose, SDS-PAGE chromatography, silica chromatography,
chromatofocusing. reverse phase HPLC (RP-HPLC), gel filtration using, e.g., Sephadex
molecular sieve or size-exclusion chromatography, chromatography on columns that
selectively bind the peptide, and ethanol, pH or ammonium sulfate precipitation, membrane
filtration and various techniques.
Modified peptides produced in culture are usually isolated by initial extraction from
cells, enzymes, etc.. followed by one or more concentration, salting-out. aqueous ion-
exchange, or size-exclusion chromatography steps. Additionally, the modified glycoprotein
may be purified by affinity chromatography. Then, HPLC may be employed for final
purification steps.
A protease inhibitor, e.g., phenylmethylsulfonyifluoride (PMSF) may be included in
any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent
the growth of adventitious contaminants.
Within another embodiment, supernatants from systems which produce the modified
peptide of the invention are first concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
Following the concentration step, the concentrate may be applied to a suitable purification
matrix. For example, a suitable affinity matrix may comprise a ligand for the peptide, a lectin
or antibody molecule bound to a suitable support. Alternatively, an anion-exchange resin
may be employed, for example, a matrix or substrate having pendant DEAF groups. Suitable
matrices include acrylamide. agarose, dextran. cellulose, or other types commonly employed
in protein purification. Alternatively, a cation-exchange step may be employed. Suitable
cation exchangers include various insoluble matrices comprising sulfopropyl or
carboxymethyl groups. Sulfopropyl groups are particularly preferred.
Then, one or more RP-HPLC steps employing hydrophobic RP-HPLC media, e.g..
silica gel having pendant methyl or other aliphatic groups, may be employed to further purify
a peptide variant composition. Some or all of the foregoing purification steps, in various
combinations, can also be employed to provide a homogeneous modified glycoprotein.
The modified peptide of the invention resulting from a large-scale fermentation may
be purified by methods analogous to those disclosed by Urdal et al, J. Chromatog. 296: 171
(1984). This reference describes two sequential, RP-HPLC steps for purification of
recombinant human 1L-2 on a preparative HPLC column. Alternatively, techniques such as
affinity chromatography may be utilized to purify the modified glycoprotein.
VI. Preferred Peptides and Nucleic Acids Encoding Preferred Peptides
The present invention includes isolated nucleic acids encoding various peptides and
proteins, and similar molecules or fragments thereof. The invention should not be construed
to be limited in any way solely to the use of these peptides in the methods of the invention,
but rather should be construed to include any and all peptides presently available or which
become available to those in the art. In addition, the invention should not be construed to
include only one particular nucleic acid or amino acid sequence for the peptides listed herein,
but rather should be construed to include any and all variants, homologs. mutants, etc. of each
of the peptides. It should be noted that when a particular peptide is identified as having a
mutation or other alteration in the sequence for that peptide, the numbering of the amino
acids which identify the alteration or mutation is set so that the first amino acid in the mature
peptide sequence is amino acid no. 1, unless otherwise stated herein.
Preferred peptides include, but are not limited to human granulocyte colony
stimulating factor (G-CSF), human interferon alpha (IFN-alpha), human interferon beta (1FN-
beta), human Factor VII (Factor VII), human Factor IX (Factor IX), human follicle
stimulating hormone (FSH), human erythropoietin (EPO), human granulocyte/macrophage
colony stimulating factor (GM-CSF), human interferon gamma (IFN-gamma), human alpha-
1-protease inhibitor (also known as alpha-1-antitrypsin or alpha-]-trypsin inhibitor: A-l-PI).
glucocerebrosidase, human tissue-type activator (TPA), human interleukin-2 (IL-2), human
Factor VIII (Factor VIII). a 75 kDa tumor necrosis factor receptor fused to a human IgG
immunoglobulin Fc portion, commercially known as ENBREL™ or ETANERCEPT™
(chimeric TNFR), human urokinase (urokinase), a Fab fragment of the human/mouse
chimeric monoclonal antibody that specifically binds glycoprotein IIb/ IIIa and the
vitronectin alphav beta3 receptor, known commercially as REOPRO™ or ABCIXIMAB
(chimeric anti-glycoprotein Ilb/IIIa). a mouse/human chimeric monoclonal antibody that
specifically binds human HER2, known commercially as HERCEPTIN™ (chimeric anti-
11ER2), a human/mouse chimeric antibody that specifically binds the A antigenic site or the F
protein of respiratory syncytial virus commercially known as SYNAGIS™ or
PAL1VIZUMAB (chimeric anti-RSV), a chimeric human/mouse monoclonal antibody that
specifically binds CD20 on human B-cells. known commercially as RITUXAN™ or
RITUXAMAB (chimeric anti-CD20), human recombinant DNase (DNase), a chimeric
human/mouse monoclonal antibody that specifically binds human tumor necrosis factor,
known commercially as REMICADE™ or INFLIXIMAB (chimeric anti-TNF), human
insulin, the surface antigen of a hepatitis B virus (adw subtype; HBsAg), and human growth
hormone (HGH), alpha-galactosidase A (Fabryzyme™), a-Iduronidase (Aldurazyme™),
anlithrombin (antithrombin III, AT-III), human chorionic gonadotropin (hCG), interferon
omega, and the like.
The isolated nucleic acid of the invention should be construed to include an RNA or a
DNA sequence encoding any of the above-identified peptides of the invention, and any
modified forms thereof, including chemical modifications of the DNA or RNA which render
the nucleotide sequence more stable when it is cell free or when it is associated with a cell.
As a non-limiting example, oligonucleotides which contain at least one phosphorothioate
modification are known to confer upon the oligonucleotide enhanced resistance to nucleases.
Specific examples of modified oligonucleotides include those which contain
phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl orcycloalkyl
intersugar linkages, or short chain heteroatomic or heterocyclic intersugar ("backbone")
linkages. In addition, oligonucleotides having morpholino backbone structures (U.S. Patent
No: 5.034,506) or polyamide backbone structures (Nielsen et al., 1991, Science 254: 1497)
may also be used.
Chemical modifications of nucleotides may also be used to enhance the efficiency
with which a nucleotide sequence is taken up by a cell or the efficiency with which it is
expressed in a cell. Any and all combinations of modifications of the nucleotide sequences
are contemplated in the present invention.
The present invention should not be construed as being limited solely to the nucleic
and amino acid sequences disclosed herein. As described in more detail elsewhere herein,
once armed with the present invention, it is readily apparent to one skilled in the art that other
nucleic acids encoding the peptides of the present invention can be obtained by following the
procedures described herein (e.g., site-directed mutagenesis, frame shift mutations, and the
like), and procedures that are well-known in the art.
Also included are isolated nucleic acids encoding fragments of peptides, wherein the
peptide fragments retain the desired biological activity of the peptide. In addition, although
exemplary nucleic acids encoding preferred peptides are disclosed herein in relation to
specific SEQ ID NOS. the invention should in no way be construed to be limited to any
specific nucleic acid disclosed herein. Rather, the invention should be construed to include
any and all nucleic acid molecules having a sufficient percent identity with the sequences
disclosed herein such that these nucleic acids also encode a peptide having the desired
biological activity disclosed herein. Also contemplated are isolated nucleic acids that are
shorter than full length nucleic acids, wherein the biological activity of the peptide encoded
thereby is retained. Methods to determine the percent identity between one nucleic acid and
another are disclosed elsewhere herein as are assays for the determination of the biological
activity of any specific preferred peptide.
Also as disclosed elsewhere herein, any other number of procedures may be used for
the generation of derivative, mutant, or variant forms of the peptides of the present invention
using recombinant DNA methodology well known in the art such as, for example, that
described in Sambrook et al. (1989. Molecular Cloning: A Laboratory Manual. Cold Spring
Harbor Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in
Molecular Biology, Green & Wiley, New York). Procedures for the introduction of amino
acid changes in a peptide or polypeptide by altering the DNA sequence encoding the peptide
are well known in the art and are also described in Sambrook et al. (1989, supra); Ausubel et al.
(1997, supra).
The invention includes a nucleic acid encoding a G-CSF, IFN-alpha. IFN-beta, Factor
VII. Factor IX. FSM. L.PO. GM-CSF. IFN-gamma, A-l-Pl. glucocerebrosidase, TPA, IL-2,
Factor VIII, chimeric TNFR. urokinase, chimeric anti-glycoprotein IIb/IIa, chimeric anti-
11ER2, chimeric anti-RSV, chimeric anti-CD20, DNase, chimeric anti-TNF . human insulin.
HBsAg, and HGH. wherein a nucleic acid encoding a tag peptide is covalently linked thereto.
That is, the invention encompasses a chimeric nucleic acid wherein the nucleic acid sequence
encoding a tag peptide is covalently linked to the nucleic acid encoding a peptide of the
present invention. Such tag peptides are well known in the art and include, for instance,
green fluorescent protein (GFP), myc, myc-pyruvate kinase (myc-PK), Hisf,. maltose binding
protein (MBP). an influenza virus hemagglutinin tag polypeptide, a flag tag polypeptide
(FLAG), and a glutathione-S-transferase (GST) tag polypeptide. However, the invention
should in no way be construed to be limited to the nucleic acids encoding the above-listed tag
peptides. Rather, any nucleic acid sequence encoding a peptide which may function in a
manner substantially similar to these tag peptides should be construed to be included in the
present invention.
The nucleic acid comprising a nucleic acid encoding a tag peptide can be used to
localize a peptide of the present invention within a cell, a tissue, and/or a whole organism
(e.g., a mammalian embryo), detect a peptide of the present invention secreted from a cell,
and to study the role(s) of the peptide in a cell. Further, addition of a tag peptide facilitates
isolation and purification of the "tagged" peptide such that the peptides of the invention can
be produced and purified readily.
The invention includes the following preferred isolated peptides: G-CSF, IFN-alpha,
IFN-beta, Factor VII. Factor IX, FSH, EPO, GM-CSF, IFN-gamma, A-l-PI,
glucocerebrosidase, TPA, IL-2, Factor VIII, chimeric TNFR, urokinase, chimeric anti-
glycoprotein llb/IUa. chimeric anti-HER2, chimeric anti-RSV, chimeric anti-CD20, DNase,
chimeric anti-TNF, human insulin. HBsAg, HGH, alpha-galactosidase A,, a-Iduronidase.
antithrombin III, hCG. and interferon omega, and the like.
The present invention should also be construed to encompass "derivatives,"
"mutants", and "variants" of the peptides of the invention (or of the DNA encoding the same)
which derivatives, mutants, and variants are peptides which are altered in one or more amino
acids (or, when referring to the nucleotide sequence encoding the same, are altered in one or
more base pairs) such lhal the resulting peptide (or DNA) is not identical to the sequences
recited herein, but has the same biological property as the peptides disclosed herein, in that
the peptide has biological/biochemical properties of G-CSF, IFN-alpha. IFN-beta. Factor VII.
Factor IX, FSH, EPO. GM-CSF, IFN-gamma. A-l-Pl, glucocerebrosidase, TPA. IL-2, Factor
VIII, chimeric TNFR. urokinase, chimeric anti-glycoprotein Ub/IIla, chimeric anti-HER2,
chimeric anti-RSV. chimeric anti-CD20, DNase, chimeric anti-TNF. human insulin. IIBsAg.
and HGH.
Further included are fragments of peptides that retain the desired biological activity of
the peptide irrespective of the length of the peptide. It is well within the skill of the artisan to
isolate smaller than full length forms of any of the peptides useful in the invention, and to
determine, using the assays provided herein, which isolated fragments retain a desired
biological activity and are therefore useful peptides in the invention.
A biological property of a protein of the present invention should be construed to
include, but not be limited to include the ability of the peptide to function in the biological
assay and environments described herein, such as reduction of inflammation, elicitation of an
immune response, blood-clotting, increased hematopoietic output, protease inhibition,
immune system modulation, binding an antigen, growth, alleviation of treatment of a disease,
DNA cleavage, and the like.
A. G-CSF
The present invention encompasses a method for the modification of the glycan
structure on G-CSF. G-CSF is well known in the art as a cytokine produced by activated T-
cells, macrophages, endothelial cells, and stromal fibroblasts. G-CSF primarily acts on the
bone marrow to increase the production of inflammatory leukocytes, and further functions as
an endocrine hormone to initiate the replenishment of neutrophils consumed during
inflammatory functions. G-CSF also has clinical applications in bone marrow replacement
following chemotherapy.
A remodeled G-CSF peptide may be administered to a patient selected from the group
consisting of a non-myeloid cancer patient receiving myelosuppressive chemotherapy, a
patient having Acute Myeloid Leukemia (AML) receiving induction or consolidation
chemotherapy, a non-myeloid cancer patient receiving a bone marrow transplant, a patient
undergoing peripheral blood progenitor cell collection, a patient having severe chronic
neutropenia, and a patient having persistent neutropenia and also having advanced HIV
infection. Preferably, the patient is a human patient.
While G-CSF has been shown to be an important and useful compound for
therapeutic applications in mammals especially humans, present methods for the production
of G-CSF from recombinant cells results in a product having a relatively short biological lite,
an inaccurate glycosylation pattern that could potentially lead to immunogenicity, loss of
function, and an increased need for both larger and more frequent doses in order to achieve
the same effect, and the like.
G-CSF has been isolated and cloned, the nucleic acid and amino acid sequences of
which are presented as SEQ ID NO:1 and SEQ ID NO:2, respectively (Figure 58A and 58B,
respectively). The present invention encompasses a method for modifying G-CSF.
particularly as it relates to the ability of G-CSF to function as a potent and functional
biological molecule. The skilled artisan, when equipped with the present disclosure and the
teachings herein, will readily understand that the present invention provides compositions and
methods for the modification of G-CSF.
The present invention further encompasses G-CSF variants, as well known in the art.
As an example, but in no way meant to be limiting to the present invention, a G-CSF variant
has been described in U.S. Patent No. 6,166, 183. in which a G-CSF comprising the natural
complement of lysine residues and further linked to one or two polyethylene glycol
molecules is described. Additionally, U.S. Patent Nos. 6,004,548, 5,580,755, 5,582,823, and
5.676,941 describe a G-CSF variant in which one or more of the cysteine residues at position
I 7. 36, 42, 64, and 74 arc replaced by alanine or alternatively serine. U.S. Patent No.
5.416,195 describes a G-CSF molecule in which the cysteine at position 17, the aspartic acid
at position 27, and the serines at positions 65 and 66 are substituted with serine, serine,
proline, and proline, respectively. Other variants are well known in the art. and are described
in, for example, U.S. Patent No. 5,399,345.
The expression and activity of a modified G-CSF molecule of the present invention
can be assayed using methods well known in the art, and as described in, for example, U.S.
Patent No. 4,810.643. As an example, activity can be measured using radio-labeled
thymidine uptake assays. Briefly, human bone marrow from healthy donors is subjected to a
density cut with Ficoll-Hypaque (1.077 g/ml. Pharmacia, Piscataway, NJ) and low densih
cells are suspended in lscove's medium (GIBCO, La Jolla, CA) containing 10% fetal bovine
scrum, glutamine and antibiotics. About 2 X 104 human bone marrow cells are incubated
with either control medium or the G-CSF or the present invention in 96-well flat bottom
plates at about 37º C in 5% CO2 in air for about 2 days. Cultures are then pulsed for about 4
hours with 0.5 µCi/well of 3H-thymidine (New England Nuclear, Boston, Mass.) and uptake
is measured as described in, for example, Ventua, et al.(1983. Blood 61:781). An increase in
3H-thymidine incorporation into human bone marrow cells as compared to bone marrow cells
treated with a control compound is an indication of a active and viable G-CSF compound.
B. IFN alpha., 1FN beta and 1FN omega
The present invention further encompasses a method for the remodeling and
modification of IFN alpha. IFN beta and IFN omega. IFN alpha is part of a family of
approximately twenty peptides of approximately 18kDa in weight. IFN omega is very similar
in structure and function to IFN alpha. IFN omega is useful for treatment of hepatitis C virus
infection when an immune response to IFN alpha is mounted in the host rendering that
treatment ineffective. Antibodies raised against IFN alpha do not cross-react with IFN
omega. Thus, treatment of hepatitis C may continue using IFN omega when IFN alpha
therapy is no longer possible.
IFN alpha, omega, and IFN beta, collectively known as the Type I interferons, bind to
the same cellular receptor and elicit similar responses. Type I IFNs inhibit viral replication,
increase the lytic potential of NK cells, modulate MIIC molecule expression, and inhibit
cellular proliferation, among other things. Type I IFN has been used as a therapy for viral
infections, particularly hepatitis viruses, and as a therapy for multiple sclerosis.
Current compositions of Type I IFN are, as described above, useful compounds for
both the modulation of aberrant immunological responses and as a therapy for a variety of
diseases. However, they are hampered by decreased potency and function, and a limited half-
life in the body as compared to natural cytokines comprising the natural complement of
glycosylation.
A remodeled interferon-alpha peptide may be administered to a patient selected from
the group consisting of a patient having hairy cell leukemia, a patient having malignant
melanoma, a patient having follicular lymphoma, a patient having condylomata acuminata, a
patient having AIDS-related Kaposi's sarcoma, a patient having Hepatitis C. a patient having
Hepatitis B, a patient having a human papilloma virus infection, a patient having Chronic
Mycloid Leukemia ((ML), a patient having chronic phase Philadelphia chromosome (III)
positive Chronic Myelogenous Leukemia, a patient having non-Hodgkin's lymphoma (NHL),
a patient having lymphoma, a patient having bladder cancer, and a patient having renal
cancer. Preferably, the patient is a human patient.
A remodeled interferon-beta peptide may be administered to a patient selected from
the group consisting of a patient having multiple sclerosis (MS), a patient having Hepatitis B.
a patient having Hepatitis C, a patient having human papilloma virus infection, a patient
having breast cancer., a patient having brain cancer, a patient having colorectal cancer, a
patient having pulmonary fibrosis, and a patient having rheumatoid arthritis. Preferably, the
patient is a human patient.
A remodeled interferon-omega peptide may be administered to a patient selected from
the group consisting of a patient having hairy cell leukemia, a patient having malignant
melanoma, a patient having follicular lymphoma, a patient having condylomata acuminata, a
patient having AIDS-related Kaposi's sarcoma, a patient having Hepatitis C, a patient having
Hepatitis B, a patient having a human papilloma virus infection, a patient having Chronic
Myeloid Leukemia (CML), a patient having chronic phase Philadelphia chromosome (Ph)
positive Chronic Myelogenous Leukemia, a patient having non-Hodgkin's lymphoma (NHL),
a patient having lymphoma, a patient having bladder cancer, and a patient having renal
cancer. Preferably, the patient is a human patient.
The prototype nucleotide and amino acid sequence for IFN alpha is set forth herein as
SEQ ID NO:3 and SEQ ID NO:4, respectively (Figure 59A and 59B, respectively). The
prototype nucleotide and amino acid sequence for IFN omega is set forth herein as SEQ ID
NO:74 and SEQ ID NO:75, respectively (Figures 59C and 59D, respectively). IFN beta
comprises a single gene product of approximately 20 kDa, the nucleic acid and amino acid
sequence of which are presented herein as SEQ ID NO:5 and SEQ ID NO:6 (Figure 60A and
60B, respectively). The present invention is not limited to the nucleotide and amino acid
sequences herein. One of skill in the art will readily appreciate that many variants of IFN
alpha exist both naturally and as engineered derivatives. Similarly. IFN beta has been
modified in attempts to achieve a more beneficial therapeutic profile, Eamples of modified
Type I IFNs are well known in the art (see Table 9), and are described in. for example U.S.
Patent No. 6,323.006, in which cysteine-60 is substituted for tyrosine, U. S. Patent Nos.
4.737.462, 4.588.585. 5.545,723, and 6.127.332 where an IFN beta with a substitution of a
variety of amino acids is described. Additionally, U.S. Patent Nos. 4,966,843, 5,376,567,
5.795.779 describe IFN alpha-61 and IFN-alpha-76. U.S. Patent Nos .4.748,233 and
4,695,543 describe IFN alpha gx-1, whereas U.S. Patent No. 4,975,276 describes IFN alpha-
54. In addition, U.S. Patent Nos. 4,695,623, 4,897,471, 5,661,009, and 5,541,293 all describe
a consensus IFN alpha sequence to represent all variants known at the date of filing. While
this list of Type I IFNs and variants thereof is in no way meant to be exhaustive, one of skill
in the art will readily understand that the present invention encompasses IFN beta and IFN
alpha molecules, derivatives, and variants known or to be discovered in the future.
Methods of expressing IFN in recombinant cells are well known in the art, and is
easily accomplished using techniques described in, for example U.S. Patent No. 4,966.843,
and in Sambrook et al. (2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in Molecular
Biology. Green & Wiley, New York). Assays to determine the biological activity of a Type I
IFN modified by the present invention will be well known to the skilled artisan. For
example, the assay described in Rubinstein et al., (1981, Journal of Virology 37:755-758) is
commonly used to determine the effect of an Type I IFN by measuring the cytopathic effects
of viral infection, on a population of cells. This method is only one of many known in the art
for assaying the biological function of a Type IFN.
C. Factor VIIa
The present invention further encompasses a method for the remodeling and
modification of Factor VII. The blood coagulation pathway is a complex reaction comprising
many events. An intermediate event in this pathway is Factor VII, a proenzyme that
participates in the extrinsic pathway of blood coagulation by converting (upon its activation
to Factor VIIa) Factor X to Xa in the presence of tissue factor and calcium ions. Factor Xa in
turn then converts prothrombin to thrombin in the presence of Factor Va, calcium ions and
phospholipid. The activation of Factor X to Factor Xa is an event shared by both the intrinsic
and extrinsic blood coagulation pathways, and therefore, Factor VIIa can be used for the
treatment of patients with deficiencies or inhibitors of Factor VIII. There is also evidence to
suggest that Factor VIIa may participate in the intrinsic pathway as well therefore increasing
the prominence and importance of the role of Factor VII in blood coagulation.
Factor VII is a single-chain glycoprotein with a molecular weight of approximately 50
kDa. In this form, the factor circulates in the blood as an inactive zymogen. Activation of
Factor VII to VIIa may be catalyzed by several different plasma proteases, such as Factor
XIIa. Activation of Factor VII results in the formation of a heavy chain and a light chain held
together by at least one disulfide bond. Further, modified Factor VII molecules that cannot
be converted to Factor VIIa have been described, and are useful as anti-coagulation remedies,
such as in the case of blood clots, thrombosis, and the like. Given the importance of Factor
VII in the blood coagulation pathway, and its use as a treatment for both increased and
decreased levels of coagulation, it follows that a molecule that has a longer biological half-
life, increased potency, and in general, a therapeutic profile more similar to wild-type Factor
VII as it is synthesized and secreted in the healthy human would be beneficial and useful as a
treatment for blood coagulation disorders.
A remodeled Factor VII peptide may be administered to a patient selected from the
group consisting of a hemophiliac patient having a bleeding episode, a patient having
Hemophilia A. a patient with Hemophilia B. a patient having Hemophilia A, wherein the
patient also has antibodies to Factor VIII, a patient having Hemophilia B, wherein the patient
also has antibodies to Factor IX, a patient having liver cirrhosis, a cirrhotic patient having an
orthotopic liver transplant, a cirrhotic patient having upper gastrointestinal bleeding, a patient
having a bone marrow transplant, and a patient having a liver resection. Preferably, the
patient is a human patient.
Factor VII has been cloned and sequenced, and the nucleic acid and amino acid
sequences are presented herein as SEQ ID NO:7 and SEQ ID NO:8 (Figure 61A and 61B,
respectively). The present invention should in no way be construed as limited to the Factor
VII nucleic acid and amino acid sequences set forth herein. Variants of Factor VII are
described in. for example, U.S. Patent Nos. 4,784,950 and 5.580,560. in which lysine-38.
lysine-32, arginine—290. arginine-341, isoleucine-42, tyrosine-278, and tyrosine-332 is
replaced by a variety of amino acids. Further, U.S. Patent Nos. 5,861,374. 6,039,944,
5,833.982, 5,788.965, 6.183,743, 5,997,864, and 5,817,788 describe Factor VII variants that
are not cleaved to form Factor VIIa. The skilled artisan will recognize that the blood
coagulation pathway and the role of Factor VII therein are well known, and therefore many
variants, both naturally occurring and engineered, as described above, are included in the
present invention.
Methods for the expression and to determine the activity of Factor VII arc well known
in the art, and are described in, for example, U.S. Patent No. 4,784,950. Briefly, expression
of Factor VII. or variants thereof, can be accomplished in a variety of both prokaryotic and
eukaryotic systems, including E. coli. CHO cells, BHK cells, insect cells using a baculovirus
expression system, all of which are well known in the art.
Assays for ihc activity of a modified Factor VII prepared according to the methods of
the present invention can be accomplished using methods well known in the art. As a non-
limiting example. Quick et al. (Hemorragic Disease and Thrombosis, 2nd ed.. Leat Febiger.
Philadelphia. 1966). describes a one-stage clotting assay useful for determining the biological
activity of a Factor VII molecule prepared according to the methods of the present invention.
P. Factor IX
The present invention further encompasses a method for remodeling and/or modifying
Factor IX. As described above, Factor IX is vital in the blood coagulation cascade. A
deficiency of Factor IX in the body characterizes a type of hemophilia (type B). Treatment of
this disease is usualh limited to intravenous tranfusion of human plasma protein concentrates
of Factor IX. However, in addition to the practical disadvantages of time and expense,
transfusion of blood concentrates involves the risk of transmission of viral hepatitis, acquired
immune deficiency syndrome or thromboembolic diseases to the recipient.
While Factor IX has demonstrated itself as an important and useful compound for
therapeutic applications, present methods for the production of Factor IX from recombinant
cells (U.S. Patent No. 4,770,999) results in a product with a rather short biological life, an
inaccurate glycosylation pattern that could potentially lead to immunogenicity, loss of
function, an increased need for both larger and more frequent doses in order to achieve the
same effect, and the like.
A remodeled Factor IX peptide may be administered to a patient selected from the
group consisting of a hemophiliac patient having a bleeding episode and also having
Hemophilia B, a patient having Hemophilia B, a patient having Hemophilia B and having
antibodies to Factor IX. a patient having liver cirrhosis, a cirrhotic patient having an
orthotopic liver transplant, a cirrhotic patient having upper gastrointestinal bleeding, a patient
having a bone marrow transplant, and a patient having a liver resection. A remodeled Factor
IX peptide may also be administered to control and/or prevent hemorrhagic episodes in a
patient having Hemophilia B, congenital Factor IX deficiency, or Christmas disease. A
remodeled Factor IX peptide may also be administered to a patient to control and/or prevent
hemorrhagic episodes in the patient during surgery. Preferably, the patient is a human
patient.
The nucleic and amino acid sequences of Factor IX is set forth herein as SEQ ID
NO:9 and SEQ ID NO: 10 (Figure 62A and 62B, respectively), flic present invention is in no
way limited to the sequences set forth herein. Factor IX variants are well known in the art, as
described in. for example, U.S. Patent Nos. 4,770.999, 5,521,070 in which a tyrosine is
replaced by an alanine in the first position, U.S. Patent No. 6,037,452, in which Factor XI is
linked to an alkylenc oxide group, and U.S. Patent No. 6,046,380, in which the DNA
encoding Factor IX is modified in at least one splice site. As demonstrated herein, variants of
Factor IX are well known in the art. and the present disclosure encompasses those variants
known or to be developed or discovered in the future.
Methods for determining the activity of a modified Factor IX prepared according to
the methods of the present invention can be carried out using the methods described above, or
additionally, using methods well known in the art, such as a one stage activated partial
thromboplastin time assay as described in. for example. Biggs (1972. Human Blood
Coagulation Haemoslasis and Thrombosis (Ed. 1). Oxford, Blackwell, Scientific, pg. 614).
Briefly, to assay the biological activity of a Factor IX molecule developed according to the
methods of the present invention, the assay can be performed with equal volumes of
activated partial thromboplastin reagent, Factor IX deficient plasma isolated from a patient
with hemophilia B using sterile phlebotomy techniques well known in the art, and normal
pooled plasma as standard, or the sample. In this assay, one unit of activity is defined as that
amount present in one milliliter of normal pooled plasma. Further, an assay for biological
activity based on the ability of Factor IX to reduce the clotting time of plasma from Factor
IX-deficient patients to normal can be performed as described in, for example, Proctor and
Rapaport (1961, Amer. J. Clin. Path. 36: 212).
E. FSH
The present invention further includes a method for remodeling and/or modifying
FSH. Human reproductive function is controlled in part by a family of hetcrodimeric human
glycoprotein hormones which have a common 92 amino acid glycoprotein alpha subunit, but
differ in their hormone-specific beta subunits. The family includes follicle-stimulating
hormone (FSH), luteinizing hormone (LH), thyrotropin or thyroid-stimulating hormone
(TSH), and human chorionic gonadotropin (hCG). Human FSH and LH are used
therapeutically to regulate various aspects of metabolism pertinent to reproduction in the
human female. For example, FSH partially purified from urine is used clinically to stimulate
follicular maturation in anovulatory women with anovulatory syndrome or luteal phase
deficiency. Luteinizing hormone (LH) and FSH are used in combination to stimulate the
development of ovarian follicles for in vitro fertilization. The role of FSH in the reproductive
cycle is sufficiently well-known to permit therapeutic use, but difficulties have been
encountered due. in part, to the heterogeneity and impurity of the preparation from native
sources. This heterogeneity is due to variations in glycosylation pattern.
FSH is a valuable tool in both in vitro fertilization and stimulation of fertilization in
vivo, but as stated above, its clinical efficacy has been hampered by inconsistency in
glycosylation of the protein. It therefore seems apparent that a method for remodeling FSH
will be of great benefit to the reproductive sciences.
A remodeled FSH peptide may be administered to a patient selected from the group
consisting of a patient undergoing intrauterine insemination (IUI), a patient undergoing in
vitro fertilization (IVF). and an infertile patient. A remodeled FSH peptide may also be
administered to induce or increase ovulation in a patient, to stimulate development of an
ovarian follicle in a patient, to induce gametogenic follicle growth in a patient, to stimulate,
induce or increase follicle development and subsequent ovulation in a patient, or to treat
infertility in a patient. Preferably, the patient is a human female patient. A remodeled FSH
peptide may also be administered to a patient having a pituitary deficiency or to a patient
during puberty. Preferably this patient is a human male patient.
FSH has been cloned and sequenced, the nucleic and amino acid sequences of which
are presented herein as SEQ ID NOT 1, SEQ ID NO: 12, respectively (alpha subunit) and
SEQ ID NO: 13 and SEQ ID NO: 14, respectively (beta subunit) (Figure 63A. 63B, 63C and
63D, respectively). The skilled artisan will readily appreciate that the present invention is not
limited to the sequences depicted herein, as variants of FSH are well known in the art. As a
non-limiting example. U.S. Patent No. 5,639,640 describes the beta subunit comprising two
different amino acid sequences and U.S. Patent No. 5,338,835 describes a beta subunit
comprising an additional amino acid sequence of approximately twenty-seven amino acids
derived from the beta subunit of human chorionic gonadotropin. Therefore, the present
invention comprises FSH variants, both natural and engineered by the human hand, all well
known in the art.
Methods to express FSH in cells, both prokaryotic and cukaryotic, are well known in
the art and abundantly described in the literature (U.S. Patent Nos. 4,840,896, 4.923.805.
5.156.957). Further, methods for evaluating the biological activity of a remodeled FSH
molecule of the present invention are well known in the art, and arc described in, for
example, U.S. Patent No. 4,589, 402, in which methods for determining the effect of FSH on
fertility, egg production, and pregnancy rates is described in both non-human primates and
human subjects.
F. EPO
The present invention further comprises a method of remodeling and/or modifying
EPO. EPO is an acidic glycoprotein of approximately 34 kDa and may occur in three natural
forms: alpha, beta, and asialo. The alpha and beta forms differ slightly in carbohydrate
components but have the same potency, biological activity and molecular weight. The asialo
form is an alpha or beta form with the terminal sialic acid removed. EPO is present in very
low concentrations in plasma when the body is in a healthy state wherein tissues receive
sufficient oxygenation from the existing number of erythrocytes. This normal concentration
is enough to stimulate replacement of red blood cells which are lost normally through aging.
The amount of erythropoietin in the circulation is increased under conditions of hypoxia
when oxygen transport by blood cells in the circulation is reduced. Hypoxia may be caused
by loss of large amounts of blood through hemorrhage, destruction of red blood cells by over-
exposure to radiation, reduction in oxygen intake due to high altitudes or prolonged
unconsciousness, or various forms of anemia. Therefore EPO is a useful compound for
replenishing red blood cells after radiation therapy, anemia, and other life-threatening
conditions.
A remodeled EPQ. peptide may be administered to a patient selected from the group
consisting of a patient having anemia, an anemic patient having chronic renal insufficiency,
an anemic patient having end stage renal disease, an anemic patient undergoing dialysis, an
anemic patient having chronic renal failure, an anemic Zidovudine-treated HIV infected
patient, an anemic patient having non-myeloid cancer and undergoing chemotherapy, and an
anemic patient scheduled to undergo non-cardiac, non-vascular surgery. A remodeled EPO
peptide may also be administered to a patient undergoing surgery to reduce the need for an
allogenic blood transfusion. A remodeled EPO peptide may also be administered to a patient
at increased risk for a perioperative blood transfusion with significant anticipated blood loss.
Preferably, the patient is a human patient.
In light of the importance of EPO in aiding in the recovery from a variety of diseases
and disorders, the present invention is useful for the production of EPO with a natural, and
therefore more effective saccharide component. EPO, as it is currently synthesized, lacks the
full glycosylation complement, and must therefore be administered more frequently and in
higher doses due to its short life in the body. The invention also provides for the production
of PEGylated EPO molecules with greatly improved half-life compared with what might be
achieved by maximizing desirable glycoforms.
EPO has been cloned and sequenced, and the nucleotide and amino acid sequences arc
present herein as SEQ ID NO: 15 and SEQ ID NO: 16, respectively (Figure 64A and 64B,
respectively). It will be readily understood by one of skill in the art that the sequences set
forth herein are only an example of the sequences encoding and comprising EPO. As an
example, U.S. Patent No. 6,187,564 describes a fusion protein comprising the amino acid
sequence of two or more EPO peptides, U.S. Patent Nos. 6,048,971 and 5,614,184 describe
mutant EPO molecules having amino acid substitutions at positions 101, 103, 104, and 108.
U.S. Patent No. 5.106.954 describes a truncated EPO molecule, and U.S. Patent No.
5.888.772 describes an EPO analog with substitutions at position 33, 139, and 166.
Therefore, the skilled artisan will realize that the present invention encompasses EPO and
EPO derivatives and variants as are well documented in the literature and art as a whole.
Additionally, methods of expressing EPO in a cell are well known in the art. As
exemplified in U.S. Patent Nos. 4,703.008, 5,688,679, and 6,376.218, among others, EPO can
be expressed in prokaryotic and eukaryotic expression systems. Methods for assaying the
biological activity of EPO are equally well known in the art. As an example, the Krystal
assay (Krystal, 1983. Exp. Hematol. 11:649-660) can be employed to determine the activity
of EPO prepared according to the methods of the present invention. Briefly, the assay
measures the effect of erythropoietin on intact mouse spleen cells. Mice are treated with
phenylhydrazine to stimulate production of erythropoietin-responsive red blood cell
progenitor cells. Alter treatment, the spleens are removed, intact spleen cells are isolated and
incubated with various amounts of wild-type erythropoietin or the erythropoietin proteins
described herein. After an overnight incubation. 3H-thymidinc is added and its incorporation
into cellular DNA is measured. The amount of 3H-thymidine incorporation is indicative of
erythropoietin-stimulated production of red blood cells via interaction of erythropoietin with
its cellular receptor. The concentration of the erythropoietin protein of the present invention,
as well as the concentration of wild-type erythropoietin, is quantified by competitive
radioimmunoassay methods well known in the art. Specific activities are calculated as
international units measured in the Krystal assay divided by micrograms as measured as
immunoprecipitable protein by radioimmunoassay.
Several different mutated EPO's with different glycosylation patterns have been
reported. Many have improved stimulation of reticulocytosis activity without effecting the
half-life of the peptide in the blood stream of the animal. It is contemplated that mutated
FPO peptides can be used in place of the native F.PO peptides in any of the glycan
remodeling, glycoPEGylation and/or glycoconjugation embodiments described herein.
Preferred mutations of FPO are listed in the following table, but not limited to those listed in
the table (see, for example, Chern et al., 1991, Eur. J. Biochem. 202:225-229; Grodberget al.,
1993. Fur. J. Biochem. 218:597-601; Burns et al., 2002, Blood 99:4400-4405; U.S. Patent
No. 5,614.184; GenBank Accession No. AAN76993; O'Connel) et al.. 1992. J. Biol. Chem.
267:25010-25018: Elliott et al., 1984. Proc. Natl. Acad. Sci. U.S.A. 81:2708-2712; Biosscl et al..
1993, J. Biol. Chem. 268:15983-15993). The most preferred mutations of EPO are Arg139
to Ala139. Arg143 to Ala143 and Lys154 to .Ala154. The preferred native FPO from which to
make these mutants is the 165 aa form, which is depicted in Fig. 65; however other native
forms of EPO may also be used. Finally, the mutations described in Table 10 may be
combined with each other and with other mutations to make EPO peptides that are useful in
the present invention.
G. GM-CSF
The present invenlion encompasses a method for the modification of GM-CSF. GM-
CSF is well known in the art as a cytokine produced by activated T-cells. macrophages,
endothelial cells, and stromal fibroblasts. GM-CSF primarily acts on the bone marrow to
increase the production of inflammatory leukocytes, and further functions as an endocrine
hormone to initiate the replenishment of neutrophils consumed during inflammatory
functions. Further GM-CSF is a macrophage-activating factor and promotes the
differentiation of Lagerhans cells into dendritic cells. Like G-CSF, GM-CSF also has clinical
applications in bone marrow replacement following chemotherapy.
While G-CSF has demonstrated itself as an important and useful compound for
therapeutic applications, present methods for the production of G-CSF from recombinant
cells results in a product with a rather short biological life, an inaccurate glycosylation pattern
that could potentially lead to immunogenicity. loss of function, an increased need for both
larger and more frequent doses in order to achieve the same effect, and the like.
A remodeled GM-CSF peptide may be administered to a patient selected from the
group consisting of a patient having Acute Myelogenous Leukemia (AML) or acute non-
lymphocytic leukemia (ANLL), a patient undergoing leukapheresis to collect hematopoietic
progenitor cells from the peripheral blood, a patient undergoing transplantation of autologous
peripheral blood progenitor cells, a non-1 lodgkin's lymphoma (NHL) patient undergoing an
autologous bone marrow transplant, a Hodgkin's disease patient undergoing an autologous
bone marrow transplant, and an acute lymphoblastic leukemia (ALL) patient undergoing an
autologous bone marrow transplant. A remodeled GM-CSF peptide may also be
administered to a patient to accelerate myeloid engraftment. to shorten time to neutrophil
recovery following chemotherapy, to mobilize hematopoietic progenitor cells into the
peripheral blood for collection by leukapheresis, or to promote myeloid reconstitution after
autologous or allogeneic bone marrow transplantation (BMT). A remodeled GM-CSF
peptide may also be administered to a patient in which bone marrow transplantation has
failed or in which myeloid engraftment is delayed. Preferably, the patient is a human patient.
GM-CSF has been isolated and cloned, the nucleic acid and amino acid sequences of
which are presented as SEQ ID NO: 17 and SEQ ID NO: 18, respectively (Figure 66A and
66B. respectively). The present invention encompasses a method for modifying GM-CSF.
particularly as it relates to the ability of GM-CSF to function as a potent and functional
biological molecule. The skilled artisan, when equipped with the present disclosure and the
teachings herein, will readily understand that the present invention provides compositions and
methods for the modification of GM-CSF.
The present invention further encompasses GM-CSF variants, as well known in the
art. As an example, but in no way meant to be limiting to the present invention, a GM-CSF
variant has been described in WO 86/06358, where the protein is modified for an alternative
quaternary structure. Further, U.S. Patent No. 6,287,557 describes a GM-CSF nucleic acid
sequence ligated into the genome of a herpesvirus for gene therapy applications.
Additionally, European Patent Publication No. 0288809 (corresponding to PCT Patent
Publication No. WO 87/02060) reports a fusion protein comprising IL-2 and GM-CSF. The
IL-2 sequence can be at either the N- or C-terminal end of the GM-CSF' such that after acid
cleavage of the fusion protein, GM-CSF having either N- or C-terminal sequence
modifications can be generated. Therefore. GM-CSF derivatives, mutants, and variants are
well known in the ad, and are encompassed within the methods of the present invention.
The expression and activity of a modified GM-CSF molecule of the present invention
can be assayed using methods well known in the art, and as described in, for example, U.S.
Patent No. 4,810,643. As an example, activity can be measured using radio-labeled
thymidine uptake assays. Briefly, human bone marrow from healthy donors is subjected to a
density cut with Ficoll-Hypaque (1.077 g/ml. Pharmacia, Piscataway. NJ) and low density
cells are suspended in Iscove's medium (GIBCO, La Jolla, CA) containing 10% fetal bovine
serum, glutamine and antibiotics. About 2 X 104 human bone marrow cells are incubated
with either control medium or the GM-CSF or the present invention in 96-well flat bottom
plates at about 37° C in 5% CO2 in air for about 2 days. Cultures are then pulsed for about 4
hours with 0.5 u.Ci/\vell of 3H-thymidine (New Fngland Nuclear. Boston. Mass.) and uptake
is measured as described in. for example. Ventua, et al.(1983, Blood 61:781). An increase in
JH-thymidine incorporation into human bone marrow cells as compared to bone marrow cells
treated with a control compound is an indication of a active and viable GM-CSF compound.
H. IFN-gamma
It is an object of the present invention to encompass a method of modifying and/or
remodeling IFN-gamma. IFN-gamma, otherwise known as Type II interferon, in contrast to
IFN alpha and IFN beta, is a homodimeric glycoprotein comprising two subunits of about 21 -
24 kDa. The size variation is due to variable glycosylation patterns, usually not replicated
when reproduced recombinantly in various expression systems known in the art. IFN-gamma
is a potent activator of macrophages, increases MHC class I molecule expression, and to a
lesser extent, a MHC class II molecule stimulatory agent. Further, IFN-gamma promotes T-
cell differentiation and isotype switching in B-cells. IFN-gamma is also well documented as
a stimulator of neutrophils, NK cells, and antibody responses leading to phagocyte-mediated
clearance. IFN-gamma has been proposed as a treatment to be used in conjunction with
infection by intracellular pathogens, such as tuberculosis and leishmania, and also as an anti-
proliferative therapeutic, useful in conditions with abnormal cell proliferation as a hallmark,
such as various cancers and other neoplasias.
IFN-gamma has demonstrated potent immunological activity, but due to variations in
glycosylation from systems currently used to express IFN-gamma. the potency, efficacy,
biological half-life, and other important factors of a therapeutic have been variable at best.
The present invention encompasses methods to correct this crucial defect.
A remodeled interferon-gamma peptide may be administered to a patient selected
from the group consisting of a patient having chronic granulomatous disease, a patient having
malignant osteopetrosis, a patient having pulmonary fibrosis, a patient having tuberculosis, a
patient having Cryptococcal meningitis, and a patient having pulmonary Mycobacterium
avium complex (MAC) infection. Preferably, the patient is a human patient.
The nucleotide and amino acid sequences of IFN-gamma are presented herein as SEQ
ID NO: 19 and SEQ ID NO:20, respectively (Figure 67A and 67B, respectively). It will be
readily understood that the sequences set forth herein are in no way limiting to the present
invention. In contrast, variants, derivatives, and mutants of IFN-gamma are well known to
the skilled artisan. As an example. U.S. Patent No. 6,083,724 describes a recombinant avian
IFN-gamma and U.S. Patent No. 5.770,191 describes C-terminus variants of human IFN-
gamma. In addition, U.S. Patent No. 4,758,656 describes novel IFN-gamma derivatives, and
methods of synthesizing them in various expression systems. Therefore, the present
invention is not limited to the sequences of IFN-gamma disclosed elsewhere herein, but
encompasses all derivatives, variants, muteins, and the like well known in the art.
Expression systems for IFN-gamma are equally well known in the art, and include
prokaryotic and eukaryotic systems, as well as plant and insect cell preparations, methods of
which are known to the skilled artisan. As an example, U.S. Patent No. 4,758,656 describes a
system for expressing IFN-gamma derivatives in E. coli, whereas U.S. Patent No. 4.889,803
describes an expression system employing Chinese hamster ovary cells and an SV40
promoter.
Assays for the biological activity of a remodeled IFN-gamma prepared according to
the methods disclosed herein will be well known to one of skill in the art. Biological assays
for IFN-gamma expression can be found in. for example, U.S. Patent No. 5,807,744. Briefly,
IFN-gamma is added to cultures of CD34++CD38-cells (100 cells per well) stimulated by
cytokine combinations to induce proliferation of CD34++CD38- cells, such as IL-3, c-kit
ligand and either IL-1. IL-6 or G-CSF. Cell proliferation, and generation of secondary
colony forming cells will be profoundly inhibited in a dose dependent way, with near
complete inhibition occurring at 5000 U/milliliter of IFN-gamma. As a confirmatory test to
the inhibitory effect of IFN-gamma, addition of IFN-gamma antibodies can be performed as a
control.
I. alpha-Protease inhibitor (q-antitrypsin)
The present invention further includes a method for the remodeling of alpha-protease
inhibitor (A-1 -PI. a-1-antitrypsin or a-1-trypsin inhibitor), also known as alpha-antitrypsin.
A-l-PI is a glycoprotein having molecular weight of 53 kDa. A-l-PI plays a role in
controlling tissue destruction by endogenous serine proteases, and is the most pronounced
serine protease inhibitor in blood plasma. In particular, A-l-PI inhibits various elastases
including neutrophil elastase. Elastase is a protease which breaks down tissues, and can be
particularly problematic when its activity is unregulated in lung tissue. This protease
functions by breaking down foreign proteins. However, when API is not present in sufficient
quantities to regulate elastase activity, the elastase breaks down lung tissue. In time, this
imbalance results in chronic lung tissue damage and emphysema. In fact, a genetic
deficiency of A-l-PI has been shown to be associated with premature development of
pulmonary emphysema. A-l-PI replenishment has been successfully used for treatment of
this form of emphysema. Further, a deficiency of A-1-PI may also contribute to the
aggravation of other diseases such as cystic fibrosis and arthritis, where leukocytes move in
to the lungs or joints to fight infection.
Therefore, A-l-PI could conceivably be used to treat diseases where an imbalance
between inhibitor and protease(s), especially neutrophil elastase, is causing progression of a
disease state. Antiviral activity has also been attributed to A-l-PI. In light of this, it logically
follows that the present invention is useful for the production of A-l-PI that is safe, effective,
and potent in the ever changing atmosphere of the lungs.
A remodeled A-1-PI peptide may be administered to a patient selected from the group
consisting of a patient having congenital alpha-1-antitrypsin deficiency and emphysema, a
patient having cystic fibrosis, and a patient having pulmonary fibrosis. Preferably, the patient
is a human patient.
A-1-PI has been cloned and sequenced, and is set forth in SEQ ID NO:21 and SEQ ID
NO:22 (Figure 68A and 68B, respectively). As is understood by one of skill in the art,
natural and engineered variants of A-1-PI exist, and are encompassed in the present
invention. As an example, U.S. Patent No. 5,723,316 describes A-l-PI derivatives having
amino acid substitutions at positions 356-361 and further comprises an N-terminal extension
of approximately three amino acids. U. S. Patent No. 5,674,708 describes A-l-PI analogs
with amino acid substitutions at position 358 in the primary amino acid sequence. The
skilled artisan will readily realize that the present invention encompasses A-l-PI variants,
derivatives, and mutants known or to be discovered.
Methods for the expression and determination of activity of a remodeled A-l-PI
produced according to the methods of the present invention are well known in the art. and are
described in, for example. U.S. Patent No. 5.674,708 and U.S. Patent No. 5.723,316. Briefly,
biological activity can be determined using assays for antichymotrypsin activity by
measuring the inhibition of the chymotrypsin-catalyzed hydrolysis of substrate N-suc-Ala--
Ala-Pro-Phe-p-nitroanilide (0.1 ml of a 10 mM solution in 90% DMSO), as described in,
for example. DelMar et al. (1979, Anal. Biochem. 99: 316). A typical chymotrypsin assay
contains, in 1.0 milliliters: 100 mM Tris-CI buffer, pH 8.3, 0.005% (v/v) Triton X-100,
bovine pancreatic chymotrypsin (18 kmmol) and A-l-PI of the present invention. The assay
mixture is pre-incubated at room temperature for 5 minutes, substrate (0.01 ml of a 10 mM
solution in 90% DMSO) is added and remaining chymotrypsin activity is determined by the
rate of change in absorbance at 410nm caused by the release of p-nitroanilinc. Measurements
of optical absorbance are conducted at 25° C using a spectrophotometer fitted with a
temperature controlled sample compartment.
J. Glucocerebrosidase
The invention described herein further includes a method for the modification of
glucocerebrosidase. Glucocerebrosidase is a lysosomal glycoprotein enzyme which catalyzes
the hydrolysis of the glycolipid glucocerebroside to glucose and ceramide. Variants of
glucocerebrosidase are sold commercially as Cerezyme™ and Ceredase™, and is an
approved therapeutic for the treatment of Gaucher disease. Ceredase™ is a placental derived
form of glucocercbrosidase with complete N-linked structures. Cerezyme™ is a recombinant
variant of glucocerebrosidase which is 497 amino acids in length and is expressed in CI JO
cells. The 4 N-linked glycans of Cerezyme have been modified to terminate in the
trimannose core.
Glucocerebrosidase is presently produced in recombinant mammalian cell cultures,
and therefore reflects the glycosylation patterns of those cells, usually rodent cells such as
Chinese hamster ovary cells or baby hamster kidney cells, which differ drastically from those
of human glycosylation patterns, leading to, among other things, immunogenicity and lack of
potency.
A remodeled glucocerebrosidase peptide may be administered to a patient selected
from the group consisting of a patient having a lysosomal storage disease, a patient having a
glucocerebrosidase deficiency, and a patient having Gaucher disease. Preferably, the patient
is a human patient.
The nucleic acid and amino acid sequences of glucocerebrosidase are set forth herein
as SBQ ID NO:23 and 24 (Figure 69A and 69B, respectively). However, as will be
appreciated by the skilled artisan, the sequences represented herein are prototypical
sequences, and do not limit the invention. In fact, variants of glucocerebrosidase are well
known, and are described in, for example. U.S. Patent 6,015,703 describes enhanced
production of glucocerebrosidase analogs and variants thereof. Further, U.S. Patent No.
6.087.131 describes the cloning and sequencing of yet another glucocerebrosidase variant. It
is the intention of the present invention to encompass these and other derivatives, variants,
and mutants known or to be discovered in the future.
Methods for the expression of glucocerebrosidase are well known in the art using
standard techniques, and are described in detail in, for example, U.S. Patent No. 6.015,703.
Assays for the biological efficacy of a glucocerebrosidase molecule prepared according to the
methods of the present invention are similarly well known in the art. and a mouse Gaucher
disease model for evaluation and use of a glucocerebrosidase therapeutic is described in. for
example. Marshall et al. (2002, Mol. Ther. 6:179).
K. TPA
The present invention further encompasses a method for the remodeling of tissue-type
activator (TPA). TPA activates plasminogen to form plasmin which dissolves fibrin, the
main component of the protein substrate of the thrombus. TPA preparations were developed
as a thrombolytic agents having a very high selectivity toward the thrombus in the
thrombolytic treatment for thrombosis which causes myocardial infarction and cerebral
infarction.
Further, various modified TPA's have been produced by genetic engineering for the
purpose of obtaining higher affinity to fibrin and longer half-life in blood than that of natural
TPA. TPA's are proteins that are generally extremely difficult to solubilize in water. In
particular, the modified TPA's are more difficult to solubilize in water than natural TPA,
making very difficult the preparation of modified TPA's. Modified TPA's are thus difficult to
dissolve in water at the time of the administration to a patient. However, the modified TPA's
have various advantages, such as increased affinity for fibrin and longer half-life in blood. It
is the object of the present invention to increase the solubility of modified TPA's.
A remodeled TPA peptide may be administered to a patient selected from the group
consisting of a patient suffering from an acute myocardial infarction and a patient suffering
from an acute ischemic stroke. A remodeled TPA peptide may also be administered to a
patient to improve ventricular function following an acute myocardial infarction, to reduce
the incidence of congestive heart failure following an acute myocardial infarction, or to
reduce mortality associated with acute myocardial infarction. A remodeled TPA peptide may
also be administered to a patient to improve neurological recovery following an acute
ischemic stroke or to reduce the incidence of disability or paralysis following an acute
ischemic stroke. Preferably, the patient is a human patient.
The nucleic and amino acid sequences of TPA are set forth herein as SEQ ID NO:25
and SEQ ID NO:26, respectively (Figure 70A and 70B, respectively). As described above,
variants of TPA have been constructed and used in therapeutic applications. For example,
U.S. Patent 5.770,425 described TPA variants in which some of all of the fibrin domain has
been deleted. Further, U.S. Patent 5,736,134 describes TPA in which modifications to the
amino acid at position 276 are disclosed. The skilled artisan, when equipped with the present
disclosure and the teachings herein, will readily realize that the present invention comprises
the TPA sequences set forth herein, as well as those variants well known to one versed in the
literature.
The expression of TPA from a nucleic acid sequence encoding the same is well
known in the art. and is described, in detail, in, for example, U.S. Patent No. 5,753.486.
Assays for determining the biological properties of a TPA molecule prepared according to the
methods of the present invention are similarly well known in the art. Briefly, a TPA
molecule synthesized as disclosed elsewhere herein can be assayed for their ability to lysc
fibrin in the presence of saturating concentrations of plasminogen, according to the method of
Carlsen et al. (1988, Anal. Biochem. 168: 428 ). The in vitro clot lysis assay measures the
activity of tissue-type activators by turbidimetry using a microcentrifugal analyzer. A
mixture of thrombin and TPA is centrifuged into a mixture of fibrinogen and plasminogen to
initiate clot formation and subsequent clot dissolution. The resultant profile of absorbance
versus time is analyzed to determine the assay endpoint. Activities of the TPA variants are
compared to a standard curve of TPA. The buffer used throughout the assay is 0.06M sodium
phosphate. pH 7.4 containing 0.01% (v/v) TWEEN 80 and 0.01% (w/v) sodium azide.
1 luman thrombin is at a concentration of about 33 units/ml. Fibrinogen (at 2.0 mg/ml
clottable protein) is chilled on wet ice to precipitate fibronectin and then gravity filtered.
Glu-plasminogen is at a concentration of 1 mg/ml. The analyzer chamber temperature is set
at 37° C. The loader is set to dispense 20 microliters of TPA (about 500 nanograms/milliliter
to about 1.5 micrograms per milliliter) as the sample for the standard curve, or 20 microliters
of variant TPAs at a concentration to cause lysis within the range of the standard curve.
Twenty microliters of thrombin as the secondary reagent, and 200 microliters of a 50:1 (v/v)
fibrinogen: plasminogen mixture as the primary reagent. The absorbance/time program is
used with a 5 min incubation time, 340-nanometer-filter and 90 second interval readings.
L. IL-2
The present invention further encompasses a method for the remodeling and
modification of IL-2. IL-2 is the main growth factor of T lymphocytes and increases the
humoral and cellular immune responses1 by stimulating cytotoxic CDS T ceils and NK cells.
IL-2 is therefore crucial in the defense mechanisms against tumors and viral infections. IL-2
is also used in therapy against metastatic melanoma and renal adenocarcinoma, and has been
used in clinical trials in many forms of cancer. Further, IL-2 has also been used in HIV
infected patients where it leads to a significant increase in CD4 counts.
Given the success IL-2 has demonstrated in the management and treatment of life-
threatening diseases such as various cancers and AIDS, it follows that the methods of the
present invention would be useful for developing an IL-2 molecule that has a longer
biological half-life, increased potency, and in general, a therapeutic profile more similar to
wild-type IL-2 as it is synthesized secreted in the healthy human.
A remodeled IL-2 peptide may be administered to a patient selected from the group
consisting of a patient having metastatic renal cell carcinoma, a patient having metastatic
melanoma, a patient having ovarian cancer, a patient having Acute Myelogenous Leukemia
(AML), a patient having non-Hodgkin"s lymphoma (NHL), a patient infected with HIV, and
a patient infected with Hepatitis C. A remodeled IL-2 peptide may also be useful for
administeration to a patient as a cancer vaccine adjuvant. Preferably, the patient is a human
patient.
IL-2 has been cloned and sequenced, and the nucleic acid and amino acid sequences
are presented herein as SEQ ID NO:27 and SEQ ID NO:28 (Figure 71A and 71B,
respectively). The present invention should in no way be construed as limited to the IL-2
nucleic acid and amino acid sequences set forth herein. Variants of IL-2 are described in, for
example, U.S. Patent No. 6,348,193, in which the asparagine at position 88 is substituted for
arginine, and in U.S. Patent No. 5,206,344, in which a polymer comprising IL-2 variants with
various amino acid substitutions is described. The present invention encompasses these IL-2
variants and others well known in the art.
Methods for the expression and to determine the activity of IL-2 are well known in
the art, and are described in. for example, U.S. Patent No. 5,417,970. Briefly, expression of
IL-2. or variants thereof, can be accomplished in a variety of both prokaryotic and eukaryotic
systems, including E. coli, CHO cells, BHK cells, insect cells using a baculovirus expression
system, all of which are well known in the art.
Assays for the activity of a modified IL-2 prepared according to the methods of the
present invention can proceed as follows. Peripheral blood lymphocytes can be separated
from the erythrocytes and granulocytes by centrifuging on a Ficoll-Hypaque (Pharmacia,
Piscataway. NJ) gradient by the method described in, for example, A. Boyum et al. (Methods
in Enzymology, 1984, Vol. 108, page 88, Academic Press, Inc.). Lymphocytes are
subsequently washed about three times in culture medium consisted of RPMI 1640 (Gibco-
BRL, La Jolla, CA) plus 10% AB human serum (CTS Purpan, Toulouse, France) inactivated
by heat (1 hour at 56" C). 2 mM sodium pyruvate, 5 mM HEPHS, 4 mM L-glutamine. 100
U/ml penicillin. 100 µg/ml streptomycin and 0.25 u,g/ml amphotericin B (complete medium).
Adhesive cells (monocytes and macrophages) arc eliminated by adhesion to plastic and the
remainder of the cells are suspended in complete medium at a concentration of about 5 to 10
X105 cells per milliliter and seeded in culture flasks at a density of about 1-2 X 105 cells per
square centimeter. Flasks are then incubated at 37° C in a 5% C02 atmosphere for about I
hour, after which the non-adhesive lymphocytes are recovered by aspiration after gentle
agitation of the culture flasks.
Non-adhesive lymphocytes are washed once and cultivated at a concentration of about
105 cells per milliliter in complete medium in the presence of the IL-2 of the present
invention for about 48 hours in an incubator as described above. The cells are then washed
once.
The cytotoxic activity of the cells is evaluated after about 4 hours of contact with
target cells of the human T lymphoid line C8166-45/C63 (MT1 cells) resistant to NK cell
cytotoxicity, as described by Salahuddin et al. (1983, Virology 129: 51-64; 1984. Science:
223, 703-707). 6 X 105 HT1 cells are radio-tagged with about 200 ^Ci of 5,Cr (sodium
chromate. Amersham, Arlington Heights. IL) at 37° C for about 1 hour in complete medium
without serum, and then washed several times. The target cells and effective cells are
distributed in round-bottomed microtitration plates with varying ratios of effective cells to
target cells (50:1, 10:1, 1:1). The microtitration plates are centrifuged and, after incubation as
described above, the supernatant from each well is recovered and the radioactivity is
measured using a gamma counter. Cytotoxicity is determined from the quantity of 3 Cr
released by dead target cells. Non-specific cytotoxicity is determined from the amount of
radioactivity spontaneously released from the target cells in the absence of effective cells.
The present method is just one of many well known in the art for measuring the
cytotoxicity of effector cells, and is should not be construed as limiting to the present
invention.
M. Factor VIII
The invention further encompasses a method for the remodeling and modification of Factor
VIII. As described earlier for Factor VII and Factor IX, Factor VIII is a critical component of
the blood coagulation pathway. Human Factor VIII, (antihemophilic factor; FVIII:C) is a
human plasma protein consisting of 2 peptides (light chain molecular weight of 80 kDa and
heavy chain molecular weight variable from 90 to 220 kDa, depending on glycosylation
state). It is an essential cofactor in the coagulation pathway and is required for the
conversion of Factor X into its active form (Factor Xa). Factor VIII circulates in plasma as a
non-covalent complex with von Willibrand Factor (aka FVIII:RP), a dimer of a 2050 aa
peptide (See, U.S. Patent No. 6,307,032). Blood concentrations of Factor VIII below 20% of
normal cause a bleeding disorder designated hemophilia A. Factor VIII blood levels less than
1% result in a severe bleeding disorder, with spontaneous joint bleeding being the most
common symptom.
Similar to other blood coagulation factors, Factor VIII is a therapeutic with a great
deal of potential for the treatment of various bleeding disorders, such as hemophilia A and
hemophilia B. Due to the glycosylation of the heavy chain, current methods for the
preparation of Factor VIII from recombinant cells results in a product that is not as effective
as natural Factor VIII. Purification methods from human plasma result in a crude
composition that is less effective and more difficult to prepare than recombinant Factor VIII.
The current invention seeks to improve this situation.
A remodeled Factor VIII peptide may be administered to a patient selected from the
group consisting of a patient having von Willebrand's disease, a patient having Hemophilia
A. a patient having Factor VIII:C deficiency, a patient having fibrinogen deficiency, a patient
having Factor XIII deficiency, and a patient having acquired Factor VIII inhibitors (acquired
hemophilia). A remodeled Factor VIII peptide may also be administered to a patient to
prevent, treat or control bleeding or hemorrhagic episodes. Preferably, the patient is a human
patient.
The nucleic acid and amino acid sequences of Factor VIII are presented herein as
SEQ ID NO:29 and SEQ ID NO:30, respectively (Figure 72A and 72B, respectively). The
art is rife with variants of Factor VIII, as described in. for example, U.S. Patent No.
5,668,108, in which the aspartic acid at position 1241 is replaced by a glutamic acid with the
accompanying nucleic acid changes as well. U.S. Patent No. 5,149,637 describes a Factor
VIII variants comprising the C-terminal fraction, either glycosylated or unglycosylated. and
U.S. Patent No. 5,661.008 describes a Factor VIII variant comprising amino acids 1 -740
linked to amino acids 1649 to 2332 by at least 3 amino acid residues. Therefore, variants,
derivatives, modifications and complexes of Factor VIII are well known in the art, and are
encompassed in the present invention.
Expression systems for the production of Factor VIII are well known in the art, and
include prokaryotic and eukaryotic cells, as exemplified in U.S. Patent Nos. 5.633,150.
5.804,420. and 5.422.250.
To determine the biological activity of a Factor VIII molecule synthesized according
the methods of the present invention, the skilled artisan will recognize that the assays
described herein for the evaluation of Factor VII and Factor IX are applicable to Factor VIII.
N. Urokinase
The present invention also includes a method for the remodeling and/or modification
of urokinase. Urokinase is a serine protease which activates plasminogen to plasmin. The
protein is synthesized in a variety of tissues including endothelium and kidney, and is
excreted in trace amounts into urine. Purified urokinase exists in two active forms, a high
molecular weight form (HUK; approximately 50 kDa) and a low molecular weight form
(LUK; approximately 30 kDa). LUK has been shown to be derived from IIUK by a
proteolysis after lysine 135, releasing the first 135 amino acids from HUK. Conventional
wisdom has held that 1 IUK or LUK must be converted to proteolylically active forms by the
proteolytic hydrolysis of a single chain precursor, also termed prourokinase. between lysine
158 and isoleucine 159 to generate a two-chain activated form (which continues to
correspond to either HUK or LUK). The proteolytically active urokinase species resulting
from this hydrolytic clip contains two amino acid chains held together by a single disulfide
bond. The two chains formed by the activation clip are termed the A or A1 chains (HUK or
LUK. respectively), and the B chain comprising the protease domain of the molecule.
Urokinase has been shown to be an effective thrombolytic agent. However, since it is
produced naturally in trace quantities the cost of the enzyme is high for an effective dosage.
Urokinase has been produced in recombinant cell culture, and DNA encoding urokinase is
known together with suitable vectors and host microorganisms. Present compositions
comprising urokinase and methods for producing urokinase rccombinantly arc hampered b\ a
product that has deficient glycosylation patterns, and given the complex proteolytic cleavage
events surrounding the activation of urokinase, this aberrant glycosylation leads to a less
effective and less potent product.
A remodeled urokinase peptide may be administered to a patient selected from the
group consisting of a patient having an embolism, a patient having an acute massive
pulmonary embolism, and a patient having coronary artery thrombosis. Preferably, the
patient is a human patient. A remodeled urokinase peptide may also be used to restore
patency to an intravenous catheter, including a central venous catheter obstructed by clotted
blood or fibrin.
The sequence of the nucleotides encoding the primary amino acid chain of urokinase
are depicted in SHQ ID NO:33 and SFQ ID NO:34 (Figure 73A and 73B. respectively).
Variants of urokinase are well known in the art, and therefore the present invention is not
limited to the sequences set forth herein. In fact, the skilled artisan will readily realize that
urokinase variants described in, for example U.S. Patent Nos. 5,219,569, 5,648,253. and
4,892,826, exist as functional moieties, and are therefore encompassed in the present
invention.
The expression and evaluation of a urokinase molecule prepared according to the
methods of the present invention are similarly well known in the art. As a non-limiting
example, the expression of urokinase in various systems is detailed in U.S. Patent No.
5,219,569. An assay for determining the activity and functionality of a urokinase prepared in
accordance to the methods set forth herein are described throughout the literature, and are
similar to assays for other plasminogen and fibrin related assays described elsewhere
throughout. One example of an assay to determine the activity of an urokinase molecule
synthesized as described herein can be as described in, for example, Ploug. et al. (1957.
Biochim. Biophys. Acta 24: 278-282), using fibrin plates comprising 1.25% agarose, 4.1
mg/ml human fibrinogen, 0.3 units/ml of thrombin and 0.5 jug/ml of soybean trypsin
inhibitor.
O. Human DNasc
The present invention further encompasses a method for the remodeling and/or
modification of recombinant human DNase. Human DNase I has been tested as a therapeutic
agent and was shown to diminish the viscosity of cystic fibrosis mucus in vitro. It has been
determined that purulent mucus contains about 10-13 mg/ml of DNA. an ionic polymer
predicted to affect the rheologic properties of airway fluids. Accordingly, bovine pancreatic
DNase I, an enzyme that degrades DNA, was tested as a mucolytic agent many years ago but
did not enter clinical practice, because of side effects induced by antigenicity and/or
contaminating proteases. Recombinant human DNase is currently used as a therapeutic agent
to alleviate the symptoms of diseases such as cystic fibrosis.
A remodeled rDNase peptide may be administered to a patient having cystic fibrosis.
A remodeled rDNase peptide may also be administered to a cystic fibrosis patient to improve
pulmonary function. Preferably, the patient is a human patient.
Similar to DNase derived from bovine sources, recombinant human DNase poses
some problems, mostly due to lowered efficacy due to improper glycosylation imparted by
mammalian expression systems currently in use. The present invention describes a method
for remodeling DNasc, leading to increased efficacy and better therapeutic results.
The nucleotide and amino acid sequences of human DNAse are presented herein as
SEQ ID NO:39 and SEQ ID NO:40 (Figure 74A and 74B, respectively). Variants of the
peptide comprising DNase are well known in the art. As an example, U.S. Patent No.
6,348,343 describes a human DNase with multiple amino acid substitutions throughout the
primary structure. Additionally, U.S. Patent No. 6,391,607 describes a hyperactive variant of
DNase with multiple amino acid substitutions at positions 9, 14, 74, 75, and 205. The present
examples, and others well known in the art or to be discovered in the future are encompassed
in the present invention.
Expression systems for producing a DNase peptide are well known to the skilled
artisan, and have been described in prokaryotic and eukaryotic systems. For example, PCT
Patent Publication No. WO 90/07572 describes these methods in considerable detail.
Assays to determine the biological activity of a DNase molecule developed according
to the methods of the present invention are well known in the art. As an example, but in no
way meant to be limiting to the present invention, an assay to determine the DNA-hydrolytic
activity of"human DNase I is presented herein. Briefly, two different plasmid digestion
assays are used. The first assay ("supcrcoiled DNA digestion assay") measures the
conversion of supcrcoiled double-stranded plasmid DNA to relaxed (nicked), linear, and
degraded forms. The second assay ("linear DNA digestion assay") measured the conversion
of linear double-stranded plasmid DNA to degraded forms. Specifically. DNase prepared
according to the methods of the present invention is added to 160 microliters of a solution
comprising 25 micrograms per milliliter of either supercoiled plasmid DNA or EcoRI-
digested linearized plasmid DNA in 25 mM HEPES. pH 7.1. 100 µg/ml bovine serum
albumin. 1 mM MgCl2. 2.5 mM CaCl2, 150 mM NaCl, and the samples are incubated at room
temperature. At various times, aliquots of the reaction mixtures are removed and quenched
by the addition of 25 mM EDTA, together with xylene cyanol, bromophenol blue, and
glycerol. The integrity of the plasmid DNA in the quenched samples is analyzed by
electrophoresis of the samples on agarose gels. After electrophoresis, the gels are stained
with a solution of ethidium bromide and the DNA in the gels is visualized by ultraviolet light.
The relative amounts of supercoiled, relaxed, and linear forms of plasmid DNA are
determined by scanning the gels with a fluorescent imager (such as the Molecular Dynamics
Model 575 Fluorlmager) and quantitating the amount of DNA in the bands of the gel that
correspond to the different forms.
P. Insulin
The invention further includes a method for remodeling insulin. Insulin is well
known as the most effective treatment for type I diabetes, in which the beta islet cells of the
pancreas do not produce insulin for the regulation of blood glucose levels. The ramifications
of diabetes and uncontrolled blood glucose include circulatory and foot problems, and
blindness, not to mention a variety of other complications that either result from or arc
exacerbated by diabetes.
Prior to the cloning and sequencing of human insulin, porcine insulin was used as a
treatment for diabetes. Insulin is now produced recombinantly, but the short, 51 amino acid
sequence of the mature molecule is a complex structure comprising multiple sulfide bonds.
Current methods to recombinantly produce insulin result in a product that lacks similarity to
the native protein as produced in healthy non-diabetic subjects. The present invention seeks
to repair this flaw.
A remodeled insulin._peptide may be administered to a patient selected from the group
consisting of a patient having Type I Diabetes (diabetes mellitus) and a patient having Type 2
diabetes mellitus who requires basal (long-acting) insulin for the control of hyperglycemia.
A remodeled insulin peptide may also be administered to a diabetic patient to control
hyperglycemia. Preferably, the patient is a human patient.
The nucleotide and amino acid sequence of human insulin is portrayed in SEQ ID
NO:43 and SEQ ID NO:44, respectively (Figure 75A and 75B, respectively). Variants of
insulin are abundant throughout the art. U.S. Patent No. 6,337,194 describes insulin fusion
protein analogs. U.S. Patent No. 6.323.311 describes insulin derivatives comprising a cyclic
anhydride of a dicarboxylic acid, and U.S. Patent No. 6,251,856 describes an insulin
derivative comprising multiple amino acid substitutions and a lipophilic group. The skilled
artisan will recognize that the following examples of insulin derivatives are in no way
exhaustive, but simply represent a small sample of those well known in the art. Therefore,
the present invention comprises insulin derivatives known or to be discovered.
Expression systems for the production of insulin are well known in the art, and can be
accomplished using molecular biology techniques as described in, for example, Sambrook et
al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
New York).
Assays to determine the functionality of an insulin molecule prepared according to the
methods of the present invention are similarly well known in the art. For example, an in vivo
model of glucose depression can be used to evaluate the biological activity of insulin
synthesized using the methods of the present invention. Useful for this purpose is a rat
model. The animals are fasted overnight (16 hours) prior to the experiment, and then
anesthetized with intraperitoneal!}' administered sodium pentobarbital or another suitable
anesthetic such as ketamine. Each animal receives an i.v. injection (tail vein) of the particular
insulin derivative (20 u.g/ml/kg). Blood, samples are taken from the jugular vein 15 and 5
minutes before injection and 15, 30, 60, 90, 120, 180, and 240 minutes after injection. Blood
glucose levels are measured with a blood glucose monitor, available from a variety of
commercial suppliers.
Q. Hepatitis B Vaccines (HBsAg)
The present invention further comprises a method for the remodeling the antigen used
in hepatitis B vaccines (HbsAg or Hepatitis B sAg). HBsAg is a recombinantly produced
surface antigen of the hepatitis B S-protein, and is used to illicit an immune response to the
hepatitis B virus, an increasing dangerous virus that results in, among other things, liver
disease including cirrhosis and carcinoma, and results in over 1 million deaths worldwide
annually. Currently the HBsAg vaccine is administered three times over a six month interval
to illicit a protective and neutralizing immune response.
HBsAg is currently produced in yeast strains, and therefore reflects the glycosylation
patterns native to a fungus. The present invention provides a method to remodel HBsAg,
resulting in among other things, improved immunogenicity. antibodies with improved affinity
for the virus, and the like.
A remodeled HBsAg peptide may be administered to a patient to immunize the
patient against disease caused by a Hepatitis B virus. A remodeled HBsAg peptide may also
be administered to a predialysis patient or a dialysis patient to immunize the patient against
disease caused by a Hepatitis B virus. Preferably, the patient is a human patient.
The sequences of the S-protein from a Hepatitis B virus (HBsAg) nucleic acid and
primary amino acid chain are set forth herein as SEQ ID NO:45 and SEQ ID NO:46 (Figure
76A and 76B, respectively). The nucleotide is 1203 bases in length. The amino acid is 400
residues long. The last 226 amino acid residues are the small S-antigen, which is used in the
GlaxoSmithKline vaccine and the Merck vaccine. Fifty-five amino acids upstream from the
small S-antigen is the Pre-S start codon. The Pre-S + S regions are the middle S antigen,
which is used in the Aventis Pasteur vaccine. From the first start codon to the Pre-S start
codon comprises the rest of the S-protein, and is called the large S-protein. This is but one
example of the HBsAg used in vaccines, and other subtypes are well known, as exemplified
in GenBank Ace Nos.: AF415222, AF415221, AF415220, and AF415219. The sequences
presented herein are simply examples of HBsAg known in the art. Similar antigens have
been isolated from other strains of hepatitis B virus, and may or may not have been evaluated
for antigenicity and potential as vaccine candidates. The present invention therefore
encompasses hepatitis B vaccine S-protein surface antigens known or to be discovered.
Kxpression of an HBsAg in an expression system is a routine procedure for one of
skill in the art, and is described in, for example, U.S. Patent No. 5,851,823. Assays for the
immunogenicity of a vaccine are well known in the art, and comprise various tests for the
production of neutralizing antibodies, and employ techniques such as FLISA, neutralization
assays. Western blots, immunoprecipitation, and the like. Briefly, a sandwich ELISA for the
detection of effective anti-HBsAg antibodies is described. The Enzygnost HBsAg assay
(Aventis Behring, King of Prussia, PA) is used for such methods. Wells are coated with anti-
HBs. Serum plasma or purified protein and appropriate controls are added to the wells and
incubated. After washing, peroxidase-labeled antibodies to HBsAg are reacted with the
remaining antigenic determinants. The unbound enzyme-linked antibodies are removed by
washing and the enzyme activity on the solid phase is determined by methods well known in
the art. The enzymatically catalyzed reaction of hydrogen peroxide and chromogen is
stopped by adding diluted sulfuric acid. The color intensity is proportional to the HBsAg
concentration of the sample and is obtained by photometric comparison of the color intensity
of the unknown samples with the color intensities of the accompanying negative and positive
control sera.
R. Human Growth Hormone
The present invention further encompasses a method for the remodeling of human
growth hormone (HGH). The isoform of HGH which-is secreted in the human pituitary,
consists of 191 amino acids and has a molecular weight of about 21,500. The isoform of
HGH which is made in the placenta is a glycosylated form. HGH participates in much of the
regulation of normal human growth and; development, including linear growth
(somatogenesis), lactation, activation of macrophages, and insulin-like and diabetogenic
effects, among others.
HGH is a complex hormone, and its effects are varied as a result of interactions with
various cellular receptors. While compositions comprising HGH have been used in the
clinical setting, especially to treat dwarfism, the efficacy is limited by the absence of
glycosylation of the HGH produced recombinantly.
A remodeled HGH peptide may be administered to a patient selected from the group
consisting of a patient having a growth hormone deficiency, a patient having Turner
syndrome, a patient having growth failure due to a iack of adequate endogenouse growth
hormone secretion, a patient having growth failure due to Prader-Willi syndrome (PWS), a
patient having growth failure associated with chronic renal insufficiency, and a patient having
AIDS associated wasting or cachexia. A remodeled HGH peptide may also be administered
to a patient having short stature. Preferably, the patient is a human patient.
The nucleic and amino acid sequence of HGH are set forth elsewhere herein as SFQ
ID NO:47 and SFQ ID NO:48 (Figure 77A and 77B, respectively). The skilled artisan will
recognize that variants, derivatives, and mutants of HGH are well known. Examples can be
found in U.S. Patent No. 6.143,523 where amino acid residues at positions 10, 14, 18,21,
167. 171. 174. 176 and 179 are substituted, and in U.S. Patent No. 5,962,411 describes splice
variants of HGH. The present invention encompasses these HGH variants known in the art of
to be discovered.
Methods for the expression of HGH in recombinant cells is described in, for example,
U.S. Patent No. 5,795,745. Methods for expression of HGH in, inter alia, prokaryotes,
eukaryotes, insect cell systems, plants, and in vitro translation systems are well known in the
art.
An HGH molecule produced using the methods of the current invention can be
assayed for activity using a variety of methods known to the skilled artisan. For example.
U.S. Patent 5,734.024 describes a method to determine the biological functionality of an
expressed HGH.
S. Anti-Thrombin 111
Antithrombin (antithrombin III. AT-I1H is a Dotent inhibitor of the coagulation
cascade in blood. It is a non-vitamin K-dependent protease that inhibits the action of
thrombin as well as other procoagulant factors (e.g., Factor Xa). Congenital antithrombin III
deficiency is an autosomal dominant disorder in which an individual inherits one copy of a
defective gene. This condition leads to increased risk of venous and arterial thrombosis, with
onset of clinical manifestations typically presenting in young adulthood. Severe congenital
antithrombin III deficiency, in which the individual inherits two defective genes, is an
autosomal recessive condition associated with increased thrombogenesis, typically noted in
infancy. Acquired antithrombin III deficiency most commonly is seen in situations where
there is inappropriate activation of the coagulation system. Common conditions that result in
acquired antithrombin III deficiency include disseminated intravascular coagulation,
microangiopathic hemolytic anemias due to endothelial damage (i.e.. Hemolytic-uremic
syndrome), and vcno-occlusive disease (VOD) seen in patients undergoing bone marrow
transplant. AT-III deficiency may be corrected acutely by infusions of AT-III concentrates.
A remodeled AT-III peptide may be administered to a patient selected from the group
consisting of a patient having a hereditary AT-III deficiency in connection with a surgical or
obstetrical procedure and a hereditary AT-III deficient patient having a thromboembolism.
Preferably, the patient is a human patient.
Antithrombin III (AT-III) is an a2 -glycoprotein of molecular weight 58,000. It is
sold commercially as Thrombate III™ (Bayer Corp., West Haven, CT). The nucleic acid
and amino acid sequences of human antithrombin III are displayed in Figure 78A (SEQ ID
NO:63) and 78B (SEQ ID NO:64), respectively.
Methods to make anti-thrombin III are well know to those in the art. For example,
published nucleic acid and amino acid sequences are available for human antithrombin III
(see, U.S. Patent No. 4,517,294) and mutants of human antithrombin III (see, U.S. Patent
Nos. 5,420.252, 5.618,713, 5.700,663). The methods of the invention may be used with any
of these amino acid sequences and any nucleic acid sequences that encode them, but are not
limited to these sequences. Exemplary methods to produce recombinant antithrombin III are
well known in the art, and several are described in U.S. Patent Nos. 5,420,252, 5.843,705,
6.441,145 and 5.994,628. Exemplary methods to purify recombinant antithrombin III are
described in U.S. Patent Nos. 5.989,593, 6,268,487, 6,395.888, 6.395,881, 6,451,978 and
6.518,406.
There are many known uses for recombinant antithrombin III. Antithrombin III can
be used as a anticoagulant during surgery (U.S. Patent Nos. 5,252,557, 5,182,259), as part of
a pharmaceutical preparation or method to inhibit thrombosis.. (U.S. Patent Nos. 5,565,471.
6,001,820), and to reduce the adverse side effects of cellular transplantation (U.S. Patent No.
6,387,366). Additionally, antithrombin III preparations can be used to increase placental
blood flow (U.S. Patent No. 5,888,964).' inhibit fertilization (U.S. Patent No. 5.545,615), treat
asthma (U.S. Patent No. 6,355,626) and treat arthritis (U.S. Patent No. 5,252,557) and other
inflammatory processes (U.S. Patent No. 6,399,572). Antithrombin III can also be used to
manufacture replacement blood plasma (U.S. Patent Nos. 4,900,720) or prepare a stabilized
cellular blood product (U.S. Patent No. 6,139,878) for transfusions. Antithrombin 111 may be
administered as a pharmaceutical preparation (U.S. Patent Nos. 5,084,273, 5,866,122,
6,399.572, 6.156.731 and 6.514,940) or using gene therapy methodology (U.S. Patent No.
6.410,015). Compositions comprising antithrombin III can be used as tissue adhesives (U.S.
Patent No. 6,500,427) or lubricants for medical devices that are introduced to the patient
(U.S. Patent No. 6,391,832). Antithrombin III can also be used to coat endovascular stents
(U.S. Patent Nos. 6,355.055, 6,240,616, 5.985,307, 5,685,847 and 5.222,971), ocular
implants (U.S. Patent No. 5,944,753) and prostheses in general (U.S. Patent Nos. 6,503.556.
6,491,965 and 6,451.373). Antithrombin III can also be used in methods to locate an internal
bleeding site in a patient (U.S. Patent No. 6,314,314) and to determine hemostatic
dysfunction in a patient (U.S. Patent No. 6,429,017).
T. Human Chorionic Gonadotropin
Human Chorionic Gonadotropin (hCG) is a glycoprotein composed of an alpha
subunit and a beta subunit. HCG is closely related to two other gonadotropins, luteinizing
hormone (LI I) and follicle stimulating hormone (FSH), as well as thyroid stimulating
hormone (TSH), all three of which are glycoprotein hormones. The alpha subunits of these
various glycoprotein hormones are structurally very similar, but the beta subunits differ in
amino acid sequence.
The nucleic acid and amino acid sequences of the human chorionic gonadotropin oc-
subunit are displayed in Figures 79A (SLQ ID NO:69) and 79B (SEQ ID NO:70),
respectively. The nucleic acid and amino acid sequences of the human chorionic
gonadotropin P-subunit are displayed in Figures 79C (SEQ ID NO:71) and 79D (SEQ ID
NO:72), respectively.
Human chorionic gonadotropin is used in an infertility treatment to promote ovulation
or release of an egg from the ovary in women who do not ovulate on their own. Human
chorionic gonadotropin is also given to young males to treat undescended or underdeveloped
testicles. It is used in men to stimulate the production of testosterone. Some physicians also
prescribe human chorionic gonadotropin for men having erictile dysfunctionor lack of sexual
desire, and for treatment of male "menopause.'"
A remodeled hCG peptide may be administered to a patient selected from the group
consisting of a patient undergoing assisted reproductive technology (ART), a patient
undergoing in vitro fertilization (IVF), a patient undergoing embryo transfer, an infertile
patient, a male patient having prepubertal cryptoorchidism not due to anatomical obstruction,
and a male patient having hypogonadotropic hypogonadism. A remodeled hCG peptide may
also be administered to induce final follicular maturation and early luteinization in an infertile
female patient, wherein the infertile female patient has undergone pituitary desensitization
and pretreatment with follicle stimulating hormones. A remodeled hCG peptide may also be
administered to induce ovulation and pregnancy in an anovulatory infertile patient.
Preferably, the patient is a human patient.
Methods to make human chorionic gonadotropin are well known in the art. The
hetcrodimeric hCG can be recombinantly made in any one of many expression systems
currently used for industrial manufacture of recombinant proteins. One method of making
recombinant hCG is described in U.S. Patent No. 5,639,639. Methods for making
recombinant hetcrodimeric proteins by expressing both subunits in the same cell are, in
general, well known in the art. and several methods are described in the U.S. Patent Nos.
5.643,745 (expression in a filamentous fungus), 5,985,611 and 6,087,129 (expression in
secretory cells). Alternatively, each subunit can be expressed individually in cells, and the
two subunits later brought together in vitro for assembly into the heterdimer.
Methods for using human chorionic gonadotropin are numerous and well known in
the art. Commonly, hCG is used to induce or synchronize ovulation in mammals (see, U.S.
Patent Nos. 6,489,288, 5,589,457, 5,532,155, 4,196,123, 4,062,942 and 4,845,077).
Additionally, hCG can be used in pregnancy tests, and in particular agglutination-based tests
(see, U.S. Patent Nos. 3,991,175, 4,003,988, 4,071,314 and 4,088,749). hCG can also be
used in a contraceptive vaccine (see, U.S. Patent Nos. 4,161,519 and 4,966,888). In addition,
hCG can be used to treat conditions related to aging and altered hormonal balance such as
benign prostatic hypertrophy (see, U.S. Patent No. 5,610,136) and central nervous system
diseases common in the elderly (see, U.S. Patent No. 4,791,099).
Alternatively. hCG can be used to detect and treat cancers that express hCG or one of
its subunits. hCG-expressing tumors include, but are not limited to, breast, prostate, ovary
and stomach carcinomas, and neuroblastomas such as Karposi's sarcoma. Antibodies can be
raised to hCG which has been glycoremodeled so as to have glycan structures similar to those
found on the tumor-expressed hCG, and these antibodies may be used to detect hCG-
expressing tumors in patients according to methods well known in the art (see, U.S. Patent
Nos. 4.31 1,688, 4.478,815 and 4.323,546). Additionally, remodeled hCG can be used to
raise an immune response to a tumor that is expressing hCG (see, U.S. Patent Nos. 5.677,275.
5,762,931,5,877,148,4,970,071 and 4,966,753).
hCG can also be used in methods to generally immunomodulate an animal, such as
described in U.S. Patent Nos. 5,554,595, 5,851,997 and 5,700,781. In addition, hCG can be
used as an inhibitor of the matrix metalloprotease in conditions benefiting from such
treatment, such as chronic inflammatory diseases, multiple sclerosis and angiogenesis-
dependent diseases (see, U.S. Patent No. 6,444,639).
U. a-lduronidase
a-Iduronidase is sold commercially as Aldurazyme™ (BioMarin and Genzyme). It is
useful for replacement therapy for the treatment of MPS I, a lysosomal storage disease. MPS
I (also known as Hurler disease) is a genetic disease caused by the deficiency of alpha-L-
iduronidase, an enzyme normally required for the breakdown of certain complex
carbohydrates known as glycosaminoglycans (GAGs). The normal breakdown of GAGs is
incomplete or blocked if the enzyme is not present in sufficient quantity. The cell is then
unable to excrete the carbohydrate residues and they accumulate in the lysosomes of the cell
and cause MPS I.
A remodeled alpha-iduronidase peptide may be administered to a patient selected
from the group consisting of a patient having a lysosomal storage disease, a patient having an
alpha-L-iduronidase deficiency, a patient having mucopolysaccaridosis I (MPS I), and a
patient having Hurler disease. Preferably, the patient is a human patient.
Methods to produce and purify a-iduronidase, as well as methods to treat certain
genetic disorders including a-L-iduronidase deficiency and mucopolysaccharidosis I (MPS 1)
are described in U.S. Patent No. 6,426.208. The nucleic acid and amino acid sequences of
human a-iduronidase are found in Figures 80A (SEQ ID NO:65) and 80B (SEQ ID NO:66),
respectively.
V. a-Galactosidase A
a-Galactosidase A (also known as agalsidase beta) is sold commercially as
Fabrazyme™ (Genzyme). a-Galactosidase A is useful for the treatment of Fabry disease.
Fabry disease is a rare, inherited disorder caused by the deficiency of the essential enzyme oc-
galactosidase. Without this enzyme. Fabry patients are unable to breakdown a fatty acid
substance in their body called globotriasylceramide (GL-3), which accumulates in cells in the
blood vessels of the heart, kidney, brain and other vital organs. The progressive buildup of
this substance puts patients a risk for stroke, heart attack, kidney damage and debilitating
pain. Most patients develop kidney failure during adulthood, and severe organ complications
lead to death around age forty.
A remodeled alpha-galactosidase A peptide may be administered to a patient selected
from the group consisting of a patient having a lysosomal storage disease, a patient having an
alpha-galactosidase A deficiency, and a patient having Fabry disease. Preferably, the patient
is a human patient.
The a-galactosidase A enzyme is a lysosomal enzyme which hydrolyzes
globotriaosylceramide and related glycolipids which have terminal a-galactosidase linkages.
It is a 45 kDa N-glycosylated protein encoded on the long arm of the X chromosome. The
initial glycosylated forms (Mr=55,000 to 58,000) synthesized in human fibroblasts or Chang
liver cells are processed to a mature glycosylated form (Mr=50,000). The mature active
enzyme as purified from human tissues and plasma is a homodimer (Bishop et al.. 1986.
Proc. Natl. Acad. Sci. USA 83: 4859-4863). The nucleic acid and amino acid sequences of
a-galactosidase A are found in Figures 81A (SEQ ID NO:67) and 81B (SEQ ID NO:68).
Other useful nucleic acid and amino acid sequences of alpha-galactosidase A are found in
U.S. Patent No. 6,329,191.
References teaching how to make alpha-galactosidase A are found in U.S. Patent Nos.
5.179.023 and 5.658,567 (expression in insect cells), U.S. Patent No. 5,356,804 (expression
and secretion from mammalian cells, including CHO cells), U.S. Patent No. 5.401.451
(expression in mammalian cells). U.S. Patent No. 5.580,757 (expression in mammalian cells
as a fusion protein) and U.S. Patent No. 5,929.304 (expression in plant cells). Methods for
purifying recombinant alpha-galactosidase A are found in U.S. Patent No. 6,395,884.
References teaching how to use alpha-galactosidase A to treat patients include, but are
not limited to, U.S. Patent No. 6,066,626 (gene therapy) and U.S. Patent No. 6,461,609
(treatment with the protein). Mutant forms of alpha-galactosidase A that are useful in the
methods of the invention include, but are not limited to, those described in U.S. Patent No.
6,210,666.
W. Antibodies
The present invention further comprises a method for the remodeling of various
antibody preparations including chimeric antibody preparations, including, chimeric TNFR,
chimeric anti-glycoprotein lib/Ilia, chimeric anti-HER2, chimeric anti-RSV, chimeric anti-
CD20, and chimeric anti-TNF. Chimeric antibody preparations comprise a human Fc portion
from an IgG antibody and the variable regions from a monoclonal antibody specific for an
antigen. Other preparations comprise a receptor, for example the 75 kDa TNF receptor, fused
to a human IgG Fc portion. These molecules further include Fab fragments comprising light
and heavy chains from human and mice. A chimeric TNFR is useful in the treatment of
inflammatory diseases, such as rheumatoid arthritis. Chimeric anti-glycoprotein lib/Ilia is
useful in the treatment of cardiac abnormalities, blood clotting, and platelet function
disturbances. A chimeric anti-HER2 is useful as a treatment for breast cancer, chimeric anti-
RSV is useful for the treatment of respiratory syncytial virus, chimeric anti-CD20 is useful
for the treatment of Non-Hodgkin's lymphoma, and chimeric anti-TNF is used for treatment
of Crohn's disease.
While these chimeric antibodies have proved useful in the management of varied
diseases, administration has to be fairly frequent and at fairly high doses due to the relatively
short half-life of a recombinant protein produced in rodent cells. While a majority of the
chimeric antibody is human, and therefore regarded as "self by the immune system, they are
degraded and destroyed due to non-native glycosylation patterns. The present invention
proposes to repair this problem, greatly increasing the efficacy of these novel medicines.
Antibodies and Methods.of their Generation
The term "antibody." as used herein, refers to an immunoglobulin molecule which is
able to specifically bind to a specific epitope on an antigen. Antibodies can be intact
immunoglobulins derived from natural sources or from recombinant sources and can be
immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of
immunoglobulin molecules. The antibodies in the present invention may exist in a variety of
forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv. Fab and
F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999.
Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow
et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
By the term "synthetic antibody" as used herein, is meant an antibody which is
generated using recombinant DNA technology, such as, for example, an antibody expressed
by a bacteriophage as described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA molecule encoding the
antibody and which DNA molecule expresses an antibody peptide, or an amino acid sequence
specifying the antibody, wherein the DNA or amino acid sequence has been obtained using
synthetic DNA or amino acid sequence technology which is available and well known in the
art.
Monoclonal antibodies directed against full length or peptide fragments of a peptide
or peptide may be prepared using any well known monoclonal antibody preparation
procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A
Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynski et al. (1988, Blood. 72:109-
115). Quantities of the desired peptide may also be synthesized using chemical synthesis
technology. Alternatively. DNA encoding the desired peptide may be cloned and expressed
from an appropriate promoter sequence in cells suitable for the generation of large quantities
of peptide. Monoclonal antibodies directed against the peptide are generated from mice
immunized with the peptide using standard procedures as referenced herein.
Nucleic acid encoding the monoclonal antibody obtained using the procedures
described herein may be cloned and sequenced using technology which is available in the art.
and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4): 125-
168) and the references cited therein. Further, the antibody of the invention may be
"humanized" using the technology described in Wright et al., (supra) and in the references
cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77(4):755-759).
To generate a phage antibody library, a cDNA library is first obtained from mRNA
which is isolated from cells, e.g., the hybridoma, which express the desired peptide to be
expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are
produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are
obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to
generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes.
The procedures for making a bacteriophage library comprising heterologous DNA are well
known in the art and are described, for example, in Sambrook and Russell (2001, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, NY).
Bacteriophage which encode the desired antibody, may be engineered such that the
peptide is displayed on the surface thereof in such a manner that it is available for binding to
its corresponding binding peptide, e.g., the antigen against which the antibody is directed.
Thus, when bacteriophage which express a specific antibody are incubated in the presence of
a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell.
Bacteriophage which do not express the antibody will not bind to the cell. Such panning
techniques are well known in the art and are described for example, in Wright et al., (supra).
Processes such as those described above, have been developed for the production of
human antibodies using Ml3 bacteriophage display (Burton et al., 1994, Adv. Immunol.
57:191-280). Essentially, a cDNA library is generated from mRNA obtained from a
population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin
genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into Ml3
expression vectors creating a library of phage which express human antibody fragments on
their surface. Phage which display the antibody of interest are selected by antigen binding
and are propagated in bacteria to produce soluble human immunoglobulin. Thus, in contrast
to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding
human immunoglobulin rather than cells which express human immunoglobulin.
Remodeling glycans of antibody molecules
The specific glycosylation ofone class of peptides, namely immunoglobulins, has a
particularly important effect on the biological activity of these peptides. The invention should
not be construed to be limited solely to immunoglobulins of the IgG class, but should also be
construed to include immunoglobulins of the IgA, IgE and IgM classes of antibodies.
Further, the invention should not be construed to be limited solely to any type of
traditional antibody structure. Rather, the invention should be construed to include all types
of antibody molecules, including, for example, fragments of antibodies, chimeric antibodies,
human antibodies, humanized antibodies, etc.
A typical immunoglobulin molecule comprises an effector portion and an antigen
binding portion. For a review of immunoglobulins, see Harlow et al., 1988. Antibodies: A
Laboratory Manual. Cold Spring Harbor, New York, and Harlow et al., 1999, Using
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY. The effector
portion of the immunoglobulin molecule resides in the Fc portion of the molecule and is
responsible in part for efficient binding of the immunoglobulin to its cognate cellular
receptor. Improper glycosylation of immunoglobulin molecules particularly in the CH2
domain of the Fc portion of the molecule, affects the biological activity of the
immunoglobulin.
More specifically with respect to the immunoglobulin IgG, IgG effector function is
governed in large part by whether or not the IgG contains an N-acetylglucosamine (GlcNAc)
residue attached at the 4-0 position of the branched mannose of the trimannosyl core of the
N-glycan at Asparagine (Asn) 297 in the CH2 domain of the IgG molecule. This residue is
known as a "bisecting GlcNAc." The purpose of adding bisecting GlcNAc to the N-glycan
chains of a natural or recombinant IgG molecule or a IgG-Fc-containing chimeric construct is
to optimize Fc immune effector function of the Fc portion of the molecule. Such effector
functions may include antibody-dependent cellular cytotoxicity (ADCC) and any other
biological effects that require efficient binding to FcyR receptors, and binding to the CI
component of complement. The importance of bisecting GlcNAc for achieving maximum
immune effector function of IgG molecules has been described (Lifely et al., 1995.
Glycobiology 5 (8): 813-822: Jeffris et al.. 1990. Biochem. J. 268 (3): 529-537).
The glycans found at the N-glycosylation site at Asn 297 in the CH2 domain of IgG
molecules have been structurally characterized for IgG molecules found circulating in human
and animal blood plasma, IgG produced by myeloma cells, hybridoma cells, and a variety of
transfected immortalized mammalian and insect cell lines. In all cases the N-glycan is cither
a high mannose chain or a complete ( Man3, GlcNAc4, GaI2, NeuAc2, Fuel) or variably
incomplete biantennary chain with or without bisecting GlcNAc (Raju et al., 2000.
Glycobiology 10 (5): 477-486: Jeffris et al., 1998, Immunological. Rev. 163L59-76: Lerouge
et al., 1998, Plant Mol. Biol. 38: 31 -48; James et al.. 1995, Biotechnology 13: 592-596).
The present invention provides an in vitro customized glycosylated immunoglobulin
molecule. The immunoglobulin molecule may be any immunoglobulin molecule, including,
but not limited to, a monoclonal antibody, a synthetic antibody, a chimeric antibody, a
humanized antibody, and the like. Specific methods of generating antibody molecules and
their characterization are disclosed elsewhere herein. Preferably, the immunoglobulin is
IgG. and more preferably, the IgG is a humanized or human IgG, most preferably, IgG I.
The present invention specifically contemplates using |31,4-mannosyl-glycopeptide
(31.4-N-acetylglucosaminyltransferase. GnT-III: EC2.4.I.I44 as an in vitro reagent to
glycosidically link N-acetylglucosamine (GlcNAc) onto the 4-0 position of the branched
mannose of the trimannosyl core of the N-glycan at Asn 297 in the CH2 domain of an IgG
molecule. However, as will be appreciated from the disclosure provided herein, the invention
should not be construed to solely include the use of this enzyme to provide a bisecting
GlcNAc to an immunoglobulin molecule. Rather, it has been discovered that it is possible to
modulate the glycosylation pattern of an antibody molecule such that the antibody molecule
has enhanced biological activity, i.e., effector function, in addition to potential enhancement
of other properties, e.g., stability, and the like.
There is provided in the present invention a general method for removing fucose
molecules from the Asn(297) N-linked glycan for the purpose of enhancing binding to Fc-
gammaRIHA, and enhanced antibody-dependent cellular cytotoxicity (see, Shields et al.,
2002, J. Biol. Chem. 277:26733-26740). The method entails contacting the antibody
molecule with a fucosidase appropriate for the linkage of the fucose molecule(s) on the
antibody glycan(s). Alternately, the recombinant antibody can be expressed in cells that do
express fucosytransferases, such as the Lee ] 3 varient of CHO cells. The removal of fucose
from the glycan(s) of the antibody can be done alone, or in conjunction with other methods to
remodel the glycans. such as adding a bisecting GlcNAc. Expression of antibodies in cells
lacking GnT-1 may also result in Fc glycans lacking core fucose, which can be further
modified by the present invention.
There is provided in the present invention a general method for introducing a
bisecting GlcNAc for the purpose of enhancing Fc immune effector function in any
preparation of IgG molecules containing N-linked oligosaccharides in the CH2 domain,
typically at Asn 297. The method requires that the population of IgG molecules is brought to
a state of glycosylation such that the glycan chain is an acceptor for GnT-IIl. This is
accomplished in any one of three ways: 1) by selection or genetic manipulation of a host
expression system that secretes IgG with N-glycan chains that are substrates for GnT-III; 2)
by treatment of a population of IgG glycoforms with exoglycosidases such that the glycan
structure(s) remaining after exoglycosidase treatment is an acceptor for GnT-III; 3) some
combination of host selection and exoglycosidase treatment as in 1) and 2) above plus
successive additions of GlcNAc by GnT-1 and GnT-II to create an acceptor for GnT-III.
For example, IgG obtained from chicken plasma contains primarily high mannose
chains and would require digestion with one or more a-mannosidases to create a substrate for
addition of GlcNAc to the al,3 mannose branch of the trimannosyl core by GnT-I. This
substrate could be the elemental trimannosyl core, Man3GlcNAc2. Treatment of this core
structure with a combination of GnT-I, GnT-II, and GnT-III using UDP-GlcNAc as a sugar
donor creates Man3GlcNAc5 as shown in Figure 1. The order of action of these
glycosyltransferases may be varied to optimize the production of the desired product.
Optionally, this structure can then be extended by treatment with ß1,4 galactosyltransfcrase.
If required, the galactosylated oligosaccharide can be further extended using a2,3- or a2,6-
sialyltransferase to achieve a completed biantennary structure. Using this method
biantennary glycan chains can be remodeled as required for the optimal Fc immune effector
function of any therapeutic IgG under development (Figure 3).
Alternatively. IgG molecules found in the plasma of most animals or IgG which is
secreted as a recombinant product by most animal cells or by transgenic animals typically
include a spectrum of biantennary glycoforms including complete (NeuAc2, Gal2, GlcNAc4.
Man3, ±Fucl) (Figure 3) and variably incomplete forms, with or without bisecting GlcNAe
(Raju et al., 2000, Glycobiology 10 (5): 477-486; Jeffris et al.. 1998, Immunological Rev.
163: 59-76). To ensure that bisecting GlcNAe is present in the entire population of
immunoglobulin molecules so produced, the mixture of molecules can be treated with the
following exoglycosidases, successively or in a mixture: neuraminidase, P-galactosidase, p-
hexosaminidase, a-fucosidase. The resulting trimannosyl core can then be remodeled using
glycosyltransferases as noted above.
In some cases it may be desired to abolish effector function from existing antibody
molecules. The present invention also includes modifying the Fc glycans with appropriate
glycosidases and glycosyltransferases to eliminate effector function. Also anticipated is the
addition of sugars modified with PEG or other polymers that serve to hinder or abolish
binding of Fc receptors or complement to the antibody.
In addition, IgG secreted by transgenic animals or stored as "plantibodies" by
transgenic plants have been characterized. An IgG molecule produced in a transgenic plant
having N-glycans that containß1,2 linked xylose and/or with exoglycosidases to remove those residues, in addition to the above described
exoglycosidases in order to create the trimannosyl core or a Man3GlcNAc4 structure, and are
then treated with glycosyltransferases to remodel the N-glycan as described above.
The primary novel aspect of the current invention is the application of appropriate
glycosyltransferases, with or without prior exoglycosidasc treatment, applied in the correct
sequence to optimize the effector function of the antibody. In one exemplary embodiment, a
bisecting GlcNAe is introduced into the glycans of IgG molecules or or other JgG-Fc-
chimeric constructs where bisecting GlcNAe is required. In another exemplary embodiment,
the core fucose is removed from the glycans of IgG molecules or other IgG-Fc-chimeric
constructs.
X. TNF receptor-IgG Fc fusion protein
The nucleotide and amino acid sequences of the 75 kDa human TNI7 receptor are set
forth herein as SEQ ID NO:31 and SEQ ID NO:32, respectively (Figure 82A and 82B,
respectively). The amino acid sequences of the light and heavy variable regions of chimeric
anti-HER2 are set forth as SEQ ID NO:35 and SEQ ID NO:36, respectively (Figure 83A and
83B. respectively). The amino acid sequences of the heavy and light variable regions of
chimeric anti-RSV are set forth as SEQ ID NO:38 and SEQ ID NO:37, respectively (Figure
84A and 84B, respectively). The amino acid sequences of the non-human variable regions of
anti-TNF are set forth herein as SEQ ID NO:41 and SEQ ID NO:42, respectively (Figure 85A
and 85B, respectively). The nucleotide and amino acid sequence of the Fc portion of human
IgG is set forth as SEQ ID NO:49 and SEQ ID NO:50 (Figure 86A and 86B, respectively).
A remodeled chimeric ENBREL™ may be administered to a patient selected from the
group consisting of a patient having rheumatoid arthritis and a patient having polyarticular-
coursc juvenile arthritis. A remodeled chimeric ENBREL™ may also be administered to an
arthritis patient to reduce signs, symptoms, or structural damage in the patient. Preferably,
the patient is a human patient.
A remodeled Synagis™ antibody may be administered to a patient to immunize the
patient against infection by respiratory syncytial virus (RSV). A remodeled Synagis™
antibody may also be administered to a patient to prevent or reduce the severity of a lower
respiratory tract disease caused by RSV. Preferably, the patient is a human patient.
Y. MAb anti-glycoprotein Hb/llla
The amino acid sequences of a murine anti-glycoprotein Ilb/IIIa antibody variable
regions are set forth in SEQ ID NO:52 (murine mature variable light chain. Figure 87) and
SEQ ID NO: 54 (murine mature variable heavy chain, Figure 88). These murine sequences
can be combined with human IgG amino acid sequences SEQ ID NO:51 (human mature
variable light chain, Figure 89), SEQ ID NO: 53 (human mature variable heavy chain, Figure
90), SEQ ID NO: 55 (human light chain. Figure 91) and SEQ ID NO: 56 (human heavy
chain. Figure 92) according to the proceedures found in U.S. Patent No. 5,777.085 to create a
chimeric humanized murine anti-glycoprotein llb/llla antibody. Other anti-glycoprotein
Ilb/IIIa humanized antibodies are found in U.S. Patent No. 5,877,006. A cell line expressing
the anti-glycoprotein Ilb/IIIa MAb 7E3 can be commercially obtained from the ATCC
(Manassas, VA) as accession no. HB-8832.
Indications for selected antibodies
A remodeled Reopro™ may be administered to a patient selected from the group
consisting of a patient undergoing percutaneous coronary intervention and a patient having
unstable angina, wherein the patient is scheduled for percutaneous coronary intervention
within 24 hours. A remodeled Reopro™ may also be administered to a patient undergoing
percutaneous coronary intervention to reduce or prevent a cardiac ischemic complication in
the patient. Preferably, the patient is a human patient.
A remodeled Herceptin™ may be administered to a patient having metastatic breast
cancer that overexpresses the HER2 protein. Preferably, the patient is a human patient.
A remodeled Remicade™ antibody may be administered to a patient selected from the
group consisting of a patient having rheumatoid arthritis, a patient having Crohn's disease,
and a patient having fistulizing Crohn's disease. A remodeled Remicade™ antibody may
also be administered to a rheumatoid arthritis patient to reduce signs and symptoms of
rheumatoid arthritis in the patient. A remodeled Remicade™ antibody may also be
administered to a Crohn's disease patient to reduce signs and symptoms of Crohn's disease in
the patient. Preferably, the patient is a human patient.
Z. MAb anti-CD20
The nucleic acid and amino acid sequences of a chimeric anti-CD20 antibody are set
forth in SEQ ID NO: 59 (nucleic acid sequence of murine variable region light chain, Figure
93A), SEQ ID NO:60 (amino acid sequence of murine variable region light chain, Figure
93B), SEQ ID NO.61 (nucleic acid sequence of murine variable region heavy chain. Figure
94A) and SEQ IDNO.62 (amino acid sequence of murine variable region heavy chain.
Figure 94B). In order to humanize a murine antibody, the TCAE 8 (SEQ ID NO:57, Figure
95A - 95E), which contains the human IgG heavy and light constant domains, may be
conveniently used. By cloning the above murine variable region encoding DNA into the
TCAE 8 vector according to instructions given in U.S. Patent No. 5,736,137, a vector is
created (SEQ ID-NO: 58, Figure 96A - 96E) which when transformed into a mammaliam cell
line, expresses a chimeric anti-CD20 antibody. Other humanized anti-CD20 antibodies are
found in U.S. Patent No. 6,120,767. A ceil line expressing the anti-CD20 MAb C273 can be
commercially obtained from the ATCC (Manassas, VA) as accession no. FIB-9303.
The skilled artisan will readily appreciate that the sequences set forth herein are not
exhaustive, but are rather examples of the variable regions, receptors, and other binding
moieties of chimeric antibodies. Further, methods to construct chimeric or "humanized"
antibodies are well known in the art, and are described in, for example, U.S. Patent No.
6.329.511 and U.S. Patent No. 6,210.671. Coupled with the present disclosure and methods
well known throughout the art, the skilled artisan will recognize that the present invention is
not limited to the sequences disclosed herein.
The expression of a chimeric antibody is well known in the art, and is described in
detail in, for example, U.S. Patent No. 6,329,511. Expression systems can be prokaryotic,
eukaryotic, and the like. Further, the expression of chimeric antibodies in insect cells using a
baculovirus expression system is described in Putlitz et al. (1990, Bio/Technology 8:651-
654). Additionally, methods of expressing a nucleic acid encoding a fusion or chimeric
protein are well known in the art, and are described in, for example. Sambrook et al. (2001,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York)
and Ausubel et al. (1997, Current Protocols in Molecular Biology, Green & Wiley, New-
York).
Determining the function and biological activity of a chimeric antibody produced
according to the methods of the present invention is a similarly basic operation for one of
skill in the art. Methods for determining the affinity of an antibody by competition assays are
detailed in Berzofsky (J. A. Berzofsky and I. J. Berkower, 1984, in Fundamental
Immunology (ed. W. F. Paul). Raven Press (New York). 595). Briefly, the affinity of the
chimeric antibody is compared to that of the monoclonal antibody from which it was derived
using a radio-iodinated monoclonal antibody.
A remodeled anti-CD20 antibody may be administered to a patient having relapsed or
refractory low grade or follicular, CD20-positive, B-cell non-Hodgkin's lymphoma.
Preferably, the patient is a human patient.
VII. Pharmaceutical Compositions
In another aspect, the invention provides a pharmaceutical composition. The
pharmaceutical composition includes a pharmaceutically acceptable diluent and a covalent
conjugate between a non-naturally-occurring, water-soluble polymer, therapeutic moiety or
biomolecule and a glycosylated or non-glycosylated peptide. The polymer, therapeutic
moiety or biomoleculc is conjugated to the peptide via an intact glycosyl linking group
interposed between and covalently linked to both the peptide and the polymer, therapeutic
moiety or biomoleculc.
Pharmaceutical compositions of the invention are suitable for use in a variety of drug
delivery systems. Suitable formulations for use in the present invention are found in
Remington's Pharmaceutical Sciences* Mace Publishing Company, Philadelphia, PA, 17th
ed. (1985). For a brief review of methods for drug delivery, see, Langer, Science 249:1527-
1533(1990).
The pharmaceutical compositions may be formulated for any appropriate manner of
administration, including for example, topical, oral, nasal, intravenous, intracranial,
intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration,
such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a
wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as
mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose,
sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres {e.g.,
polylactate polyglycolate) may also be employed as carriers for the pharmaceutical
compositions of this invention. Suitable biodegradable microspheres are disclosed, for
example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
Commonly, the pharmaceutical compositions are administered parenterally, e.g.,
intravenously. Thus, the invention provides compositions for parenteral administration which
comprise the compound dissolved or suspended in an acceptable carrier, preferably an
aqueous carrier, e.g., water, buffered water, saline, PBS and the like. The compositions may
contain pharmaceutically acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents,
wetting agents, detergents and the like.
These compositions may be sterilized by conventional sterilization techniques, or may
be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or
lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to
administration. The pH of the preparations typically will be between 3 and 11, more
preferably from 5 to 9 and most preferably from 7 and 8.
In some embodiments the peptides of the invention can be incorporated into
liposomes formed from standard vesicle-forming lipids. A variety of methods are available
for preparing liposomes, as described in, e.g., Szoka et al.,Ann. Rev. Biophys. Bioeng. 9: 467
(1980). U.S. Pat. Nos. 4.235,871, 4,501,728 and 4,837,028. The targeting of liposomes using
a variety of targeting agents (e.g., the sialyl galactosides of the invention) is well known in
the art (see. e.g.. U.S. Patent Nos. 4,957.773 and 4.603.044).
Standard methods for coupling targeting agents to liposomes can be used. These
methods generally involve incorporation into liposomes of lipid components, such as
phosphatidylethanolamine, which can be activated for attachment of targeting agents, or
derivatized lipophilic compounds, such as lipid-derivatized peptides of the invention.
Targeting mechanisms generally require that the targeting agents be positioned on the
surface of the liposome in such a manner that the target moieties are available for interaction
with the target, for example, a cell surface receptor. The carbohydrates of the invention may
be attached to a lipid molecule before the liposome is formed using methods known to those
of skill in the art (e.g.. alkylation or acylation of a hydroxyl group present on the
carbohydrate with a long chain alkyl halide or with a fatty acid, respectively). Alternatively,
the liposome may be fashioned in such a way that a connector portion is first incorporated
into the membrane at the time of forming the membrane. The connector portion must have a
lipophilic portion, which is firmly embedded and anchored in the membrane. It must also
have a reactive portion, which is chemically available on the aqueous surface of the liposome.
The reactive portion is selected so that it will be chemically suitable to form a stable chemical
bond with the targeting agent or carbohydrate, which is added later. In some cases it is
possible to attach the target agent to the connector molecule directly, but in most instances it
is more suitable to use a third molecule to act as a chemical bridge, thus linking the connector
molecule which is in the membrane with the target agent or carbohydrate which is extended,
three dimensionally. off of the vesicle surface. The dosage ranges for the administration of
the peptides of the invention are those large enough to produce the desired effect in which the
symptoms of the immune response show some degree of suppression. The dosage should not
be so large as to cause adverse side effects. Generally, the dosage will vary with the age.
condition, sex and extent of the disease in the animal and can be determined by one of skill in
the art. The dosage can be adjusted by the individual physician in the event of any
counterindications.
Additional pharmaceutical methods may be employed to control the duration of
action. Controlled release preparations may be achieved by the use of polymers to conjugate,
complex or adsorb the peptide. The controlled delivery may be exercised by selecting
appropriate macromolecules (for example, polyesters, polyamino carboxymethylcellulose,
and protamine sulfate) and the concentration of macromolecules as well as the methods of
incorporation in order to control release. Another possible method to control the duration of
action by controlled release preparations is to incorporate the peptide into particles of a
polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or
ethylene vinylacetate copolymers.
In order to protect peptides from binding with plasma proteins, it is preferred that the
peptides be entrapped in microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules
and poly (methymethacrylate) microcapsules, respectively, or in colloidal drug delivery
systems, for example, liposomes, albumin microspheres, microemulsions. nanoparticlcs. and
nanocapsules or in macroemulsions. Such teachings are disclosed in Remington's
Pharmaceutical Sciences (16th Ed., A. Oslo, ed., Mack, Easton. Pa.. 1980).
The peptides of the invention are well suited for use in targetable drug delivery
systems such as synthetic or natural polymers in the form of macromolecular complexes,
nanocapsules, microspheres, or beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, liposomes, and resealed erythrocytes. These systems
are known collectively as colloidal drug delivery systems. Typically, such colloidal particles
containing the dispersed peptides are about 50 nm-2 urn in diameter. The size of the colloidal
particles allows them to be administered intravenously such as by injection, or as an aerosol.
Materials used in the preparation of colloidal systems are typically sterilizable via filter
sterilization, nontoxic, and biodegradable, for example albumin, ethylcellulose, casein,
gelatin, lecithin, phospholipids, and soybean oil. Polymeric colloidal systems are prepared by
a process similar to the coacervation of microencapsulation.
In an exemplary embodiment, the peptides are components of a liposome, used as a
targeted delivery system. When phospholipids are gently dispersed in aqueous media, they
swell, hydrate, and spontaneously form multilamellar concentric bilayer vesicles with layers
of aqueous media separating the lipid bilayer. Such systems are usually referred to as
multilamellar liposomes or multilamellar vesicles (MLVs) and have diameters ranging from
about 100 nm to about 4 um. When MLVs are sonicated, small unilamellar vesicles (SUVS)
with diameters in the range of from about 20 to about 50 nm are formed, which contain an
aqueous solution in the core of the SUV.
Examples of lipids useful in liposome production include phosphatidyl compounds,
such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, and
phosphatidylethanolamine. Particularly useful are diacylphosphatidylglycerols. where the
lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
are saturated. Illustrative phospholipids.include egg phosphatidylcholine,
dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
In preparing liposomes containing the peptides of the invention, such variables as the
efficiency of peptide encapsulation, lability of the peptide, homogeneity and size of the
resulting population of liposomes, peptide-to-lipid ratio, permeability instability of the
preparation, and pharmaceutical acceptability of the formulation should be considered.
Szoka, et al. Annual Review of Biophysics and Bioengineering, 9: 467 (1980); Deamer, et al.,
in Liposomes, Marcel Dekker, New York, 1983, 27: Hope, et al., Chem. Phys. Lipids, 40: 89
(1986)).
The targeted delivery system containing the peptides of the invention may be
administered in a variety of ways to a host, particularly a mammalian host, such as
intravenously, intramuscularly, subcutaneously, intra-peritoneally, intravascularly. topically,
intracavitarily. transdermally. intranasally, and by inhalation. The concentration of the
peptides will vary upon the particular application, the nature of the disease, the frequency of
administration, or the like. The targeted delivery system-encapsulated peptide may be
provided in a formulation comprising other compounds as appropriate and an aqueous
physiologically acceptable medium, for example, saline, phosphate buffered saline, or the
like.
The compounds prepared by the methods of the invention may also find use as
diagnostic reagents. For example, labeled compounds can be used to locate areas of
inflammation or tumor metastasis in a patient suspected of having an inflammation. For this
use. the compounds can be labeled with 125I, 14C, or tritium.
EXPERIMENTAL EXAMPLES
The invention is now described with reference to the following Examples. These
Examples are provided for the purpose of illustration only and the invention should in no way
be construed as being limited to these Examples, but rather should be construed to encompass
any and all variations which become evident as a result of the teaching provided herein.
The materials and methods used in the experiments presented in this Example are now
described.
A. General Procedures
1. Preparation of CMP-SA-PEG
This example sets forth the preparation of CMP-SA-PEG.
Preparation of 2-(benzyloxycarboxamido)-glycylamido-2-deoxy-D-
mannopyranose. N-benzyloxycarbonyl-glycyl-N-hydroxysuccinimide ester (3.125 g, 10.2
mmol) was added to a solution containing D-mannosamine-HCl (2 g, 9.3 mmol) and
triethylamine (1.42 mL,10.2 mmol) dissolved in MeOH (10 mL) and H2C) (6 mL). The
reaction was stirred at room temperature for 16 hours and concentrated using
rotoevaporation. Chromatography (silica, 10% MeOH/CH2Cl2) yielded 1.71 g (50% yield) of
product as a white solid: Rf= 0.62 (silica; CHCl3:MeOH:H20, 6/4/1); 1H NMR (CD3OD,
500 MHz) 8 3.24-3.27 (m, 2H), 3.44 (t. 1H), 3.55 (t, 1H), 3.63-3.66 (m, 1H), 3.76-3.90 (m.
6H), 3.91 (s, 2H), 4.0 (dd, 2 H), 4.28 (d, 1H, J = 4.4), 4.41 (d, III, J - 3.2), 5.03 (s, 1H), 5.10
(m. 3H), 7.29-7.38(m, I OH).
Preparation of 5-(N-benzyloxycarboxamido)glycylamido-3,5-dideoxy-D-glycero-
D-galacto-2-nonulopyranosuronate. 2-(N-Benzyloxycarboxamido) glycylamide-2-deoxy-
D-mannopyranose (1.59 g, 4.3 mmol) was dissolved in a solution of 0.1 M HEPES (12 mL.
pH 7.5) and sodium pyruvate (4.73 g, 43 mmol). Neuraminic acid aldolase (540 U of enzyme
in 45 mL of a 10 mM phosphate buffered solution containing 0.1 M NaCl at pH 6.9) and the
reaction mixture was heated to 37°C for 24 hr. The reaction mixture was then centrifuged
and the supernatant was chromatographed (C18 silica, gradient from H20 (100%) to 30%
MeOl 1/watcr). Appropriate fractions were pooled, concentrated and the residue
chromatographed (silica, gradient from 10% MeOH/ CH2Cl2 to CH2Cl2/MeOH/ H20 6/4/1).
Appropriate fractions were collected, concentrated and the residue resuspended in water.
After freeze-drying, the product (1.67 g, 87% yield) was obtained as a white solid: R1 = 0.26
(silica. CHCl3/MeOH/H20 6/4/1); 'H NMR (D20. 500 MHz) 5 1.82 (t, lH),2.20(m, 1H),
3.49 (d. 1H). 3.59(dd, 1H). 3.67-3.86 (m, 2H), 3.87(s, 2H), 8.89-4.05 (m, 3H), 5.16 (s, 2H).
7.45 (m, 5H).
Preparation of 5-glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-
nonulopyranosuronate. 5-(N-Benzyloxycarboxamido)glycylamido-3,5-dideoxy-l)-glyccro-
F)-galacto-2-nonulopyranosuronate (1.66 g .3.6 mmol) was dissolved in 20 mL of 50%
water/methanol. The flask was repeatedly evacuated and placed under argon and then 10%
Pd/C (0.225 g) was added. After repeated evacuation, hydrogen (about 1 atm) was then
added to the flask and the reaction mixture stirred for 18 hr. The reaction mixture was
filtered through celite, concentrated by rotary evaporation and freeze-dried to yield 1.24 g
(100% yield) of product asawhite solid: Rf=0.25 (silica, IPA/H20/NH4OH 7/2/1); 'H
NMR (D20. 500 MHz) 5 1.83 (t, 1H. J = 9.9), 2.23 (dd, 1H, J = 12.9, 4.69), 3.51-3.70 (m.
211), 3.61(s, 2H). 3.75-3.84 (m, 2H), 3.95-4.06(m, 3H).
Preparation of cytidine-5'-monophosphoryl-[5-(N-fluorenylmethoxy-
earboxamido)glycylamido-3,5-dideoxy-ß-D-glycero-D-galacto-2-nonulopyranosuronate].
A solution containing 5-glycylamido-3,5-dideoxy-D-glycero-D-galacto-2-
nonulopyranosuronatc (0.55 g, 1.70 mmol) dissolved in 20 mL H20 was added to a solution
of Iris (1.38 g, 1 1.4 mmol). 1 M MgCl2 (1.1 mL) and BSA (55 mg). The pH of the solution
was adjusted to 8.8 with 1M NaOH (2 mL) and CTP-2Na+ (2.23 g, 4.2 mmol) was added.
The reaction mixture pi 1 was controlled with a pH controller which delivered 1 M NaOH as
needed to maintain pH 8.8. The fusion protein (sialyltransferase/CMP-neuraminic acid
synthetase) was added to the solution and the reaction mixture was stirred at room
temperature. After 2 days, an additional amount of fusion protein was added and the reaction
stirred an additional 40 hours. The reaction mixture was precipitated in EtOH and the
precipitate was washed 5 times with cold EtOH to yield 2.3 grams of a white solid. About
1.0 g of the crude product was dissolved in 1.4 dioxane (4 mL), H20 (4 mL) and saturated
NaHCCh(3 ml.) and a solution of FMOC-C1 (308 mg, 1.2 mmol) dissolved in 2 ml dioxane
was added dropwisc. After stirring for 16 hr at room temperature, the reaction mixture was
concentrated to about 6 ml. by rotary evaporation and purified using chromatography (C18
silica, gradient 100% H20 to 30% MeOH/ H20). Appropriate fractions were combined and
concentrated. The residue was dissolved in water and freeze-dried to yield 253 mg of a white
solid: R, - 0.50 (silica. IPA/H20/NH4OH 7/2/1); 'H NMR (D20, 500 MHz) 5 1.64 (dt. Ill, J
- 12.0, 6.0). 2.50 (dd. 1H, J= 13.2, 4.9), 3.38 (d. J = 9.67, 1H), 3.60 (dd. J=l 1.65. 6.64, IH).
3.79 (d. J=4.11, 1H), 3.87 (dd, J= 12.24, 1.0, 1H), 3.97 (m, 2H), 4.07 (td, J = 10.75, 4.84,
1H), 4.17 (dd, J = 10.68, 1.0, 1 H), 4.25 (s, 2H), 4.32 (t, J =4.4, 1H), 4.37 (t, J=5.8 1H), 4.6-
4.7 (m, obscured by solvent peak), 5.95 (d, J = 4. 1 H), 6.03 (d, J = 7.4, 1H), 7.43-7.53 (m,
3H), 7.74 (m, 2H), 7.94 (q, J = 7, 3H) . MS (ES); calc. for C35H42N5O18P ([M-H]+), 851.7:
found 850.0.
Preparation of cytidine-5,-monophosphoryl-(5-glycylamido-3,5-dideoxy-ß-D-
glycero-D-galacto-2-nonulopyranosuronate). Diisopropylamine (83 µL. 0.587 µmol) was
added to a solution of cytidine-5'-monophosphoryl-[5-(N-fluorenyl-
methoxycarboxamido)glycylamido-3,5-dideoxy-ß-D-glyceJro-D-galacto-2-
nonulopyranosuronate] (100 mg, 0.117 mmol) dissolved in water (3 mL) and methanol (I
mL). The reaction mixture was stirred 16 hr at room temperature and the reaction methanol
removed from the reaction mixture by rotary evaporation. The crude reaction mixture was
llltered through a C18 silica gel column using water and the efluant was collected and freeze-
dried to yield (87 mg, 100%) of product as a white solid: Rf = 0.21 (silica, IPA/H20/NH4OH
7/2/1): 'H NMR (D20. 500 MHz) 5 1.66 (td, IH, J =5.3), 2.50 (dd, IH, J = 13.2. 4.6), 3.43 (d.
J =' 9.58. 1H). 3.63 (dd. .1-11.9, 6.44. 1H), 3.88 (dd. J = 11.8, 1.0. 1H). 3.95 (td. J= 9.0. 2.3.
IH), 4.10 (t, J = 10.42. 1H), 4.12 (td, J = 10.34. 4.66, 1 H), 4.18 (d, J = 10.36, IH), 4.24 (m,
211), 4.31 (t. .1-4.64. Ill), 4.35 (t, IH). 6.00 (d, J = 4.37. 1 H), 6.13 (d. J = 7.71, IH). 7.98 (d.
.1=7.64, IH). MS (KS); calc. for C21H32M5O11P ([M-H]"), 629.47; found 627.9.
Preparation of cytidine-5'-monophosphoryl-[5-(N-methoxy-polyoxyethylene-(l
kDa)-3-oxypropionamido)-glycylamido-3,5-dideoxy-ß-D-glycero-D-galacto-2-
nonulopyranosuronate]. Benzyltriazol-1 -yloxy-tris(dimethylamino)-phosphonium
hexafluorophosphate (BOP, 21 mg, 48 µmol) was added to a solution of
methoxypolyoxyethylene-(l kDa average molecular weight)-3-oxypropionic acid (48 mg, 48
µmol) dissolved in anhydrous DMF (700 uL) and triethylamine (13 µL, 95 µmol). After 30
min, a solution containing cytidine-5'-monophosphoryl-(5-glycylamido-3.5-dideoxy-ß-D-
glycero-D-galacto-2-nonulopyranosuronate) (30 mg, 48 µmol), water (400 µl) and
triethylamine (13 uL, 95 µmol) was added. This solution was stirred 20 min at room
temperature and then chromatographed (C18 silica, gradient of methanol/water). Appropriate
fractions were collected, concentrated, the residue dissolved in water and freeze-dried to
afford 40 mg (50% yield) of a white solid: Rr = 0.36 (silica, IPA/H20/NH4OH 7/2/1); 'H
NMR (D20. 500 MHz) 8 1.66 (td, 1H, J =5.3), 2.50 (dd, 1H, J = 13.2, 4.6), 2.64 (t, J=5.99.
311) 3.43 (d. .1 - 9.58. 1H), 3.63 (m, 1H), 3.71 (s. 7011), 3.79 (m, obscured by 3.71 peak). 3.82
(t.J=6.19, 1H) 3.88 (dd. J = 11.8. 1.0, 1H), 3.95 (td, J= 9.0, 2.3, 1H), 3.98 (t, .1= 5.06, 1H),
4.12 (td. J = 10.34, 4.66, 1 H), 4.18 (d, J = 10.36, 1H), 4.23 (d, J=4.85, 2H), 4.31 (t, J=4.64.
1H). 4.35 (t, 1H). 6.00 (d. J = 4.55, 1 H), 6.13 (d, J = 7.56, 1H), 7.98 (d, J=7.54. 1H). MS
(MALDI), observe [M-HJ; 1594.5, 1638.5, 1682.4, 1726.4, 1770.3, 1814.4, 1858.2, 1881.5,
1903.5, 1947.3.
Preparation of cytidine-5'-monophosphoryl-[5-(N-methoxy-poIyoxyethylene-(10
kDa)-oxycarboxamido)-glycylamido-3,5-dideoxy-ß-D-glycero-D-galacto-2-
nonulopyranosuronate]. Cytidine-5'-monophosphory-(5-glycylamido-3,5-dideoxy-ß-D-
glycera-D-galacto-2-nonulopyranosuronate) (2.5 mg, 4 µmol) and water (180 µL) was added
to a solution of (Methoxypolyoxyethylene-(10 kDa, average molecular weight)-oxycarbonyl-
(N-oxybenzotriazole) ester (40 mg. 4 µmol) in anhydrous DMF (800 uL) containing
triethylamine (1.1 µL, 8 µmol) and the reaction mixture stirred for 1 hr at room temperature.
The reaction mixture was then diluted with water (8 mL) and was purified by reversed phase
flash chromatography (C1 8 silica, gradient of methanol/water). Appropriate fractions were
combined, concentrated, the residue dissolved in water and freeze-dried yielding 20 mg (46%
yield) of product as a white solid: Rt = 0.35 (silica, IPA/H2O/NH4OH 7/2/1); 1H NMR (D20,
500 MHz) d 1.66 (td. 1H). 2.50 (dd. 1H), 2.64 (t, 3H) 3.55-3.7 (m. obscured by 3.71 peak).
3.71 (s. 48811), 3.72-4.0 (m, obscured by 3.71 peak), 4.23 (m, 3H), 4.31 (t, 1H), 4.35(1, 1 H).
6.00 (d, J -4.77, 1 H), 6.12 (d. J = 7.52, 1H), 7.98 (d, J=7.89, 1H). MS (MALDI). observe
[M-CMP+Na]; 10780.
2. Preparation of CMP-SA-PEG II
This example sets forth the general procedure for making CMP-SA-PEG, and specific-
procedures for making CMP-SA-PEG (1 kDa) and CMP-SA-PEG (20 kDa).
General procedures Preparing Cytidine-5'-monophosphoryl-(5-glycylamido-3,5-
dideoxy-ß-D-glycero;-D-galacto-2-nonulopyranosuronate). Cytidine-5'-monophosphoryl-
(5-glycylamido-3.5-dideoxy-ß-D-glycero-D-galacto-2-nonulopyranosuronate (870 mg, 1.02
mmol) was dissolved in 25 mL of water and 5.5 mL of 40 wt% dimethylamine solution in
H20 was added. The reaction was stirred for 1 hr and the excess dimethyl amine was then
removed by rotary evaporation. The aqueous solution was filtered through a C-l 8 silica gel
column and the column was washed with water. The eluants were combined and lyophilized
to afford 638 mg (93%) of a white solid. R, = 0.10 (silica, IPA/H20/NH4OH; 7/2/1). 1H
NMR (D20, 500 MHz) 8 1.66 (td. IH, J ==5.3), 2.50 (dd, 1H, J = 13.2, 4.6). 3.43 (d. J = 9.58.
1H). 3.63 (dd, J= 11.9,6.44, 1H), 3.88 (dd, J = 11.8, 1.0, 1H), 3.95 (td, J= 9.0, 2.3, I H). 4.10
(t. .1 = 10.42. 1H). 4.12 (td, J = 10.34, 4.66, 1 H), 4.18 (d, J = 10.36, 1H), 4.24 (m, 211), 4.3 I
(t, .1=4.64, 1H), 4.35 (t, IH), 6.00 (d, J = 4.37, 1 H), 6.13 (d, J = 7.71, 1H), 7.98 (d, J=7.64,
1H). MS (ES); calc. for C21H32N5O11P ([M-H]), 629.47; found 627.9.
General procedures for Preparing CMP-SA-PEG using mPEG-(p-
nitrophenol)carbonate. Cytidine-5"-monophosphoryl-(5-glycylamido-3,5-dideoxy-ß-D
glycero-D-galacto-2-nonulopyranosuronate) (175 mg, 0.259 mMol) was dissolved in a
mixture of water. pH 8.5. and DMF or THE (in a ratio of 1:2). The mPEG-nitrophenol
carbonate (2 to 20 kDa mPEG's) (0.519 mMole) was added in several portions over 8 hr at
room temperature and the reaction mixture was stirred at room temperature for 3 days. When
complete, water (40 ml) and 1.5 ml of NH4OH (29% aqueous solution) were added. The
yellow reaction mixture was stirred for another 2 hr and then concentrated by rotary
evaporation. The reaction mixture was then diluted with water (pH 8.5) to about 500 ml
volume and was purified by reversed phase flash chromatography (Biotage 40M, CI8 silica
column) with a gradient of methanol/water. Appropriate fractions were combined and
concentrated to afford the products as white solids. Rf (silica; 1 -propanol / water /
29%NH4OH; 7 / 2 /l); (2 kDa PEG) = 0.31; (5 kDa PEG) = 0.33; (10 kDa PEG)= 0.36; (20
kDa PEG) = 0.38 (TLC silica. IPA/H2O/NH4OH 7/2/1): MS (MALDI), observe [M-
CMP+Na]: (2 kDa)- 2460: (5 kDa) = 5250; (10 kDa)= 10700; (20 kDa) = 22500.
Preparation of Cytidine-5'-monophosphoryl-[5-(N-
fluorenylmethoxycarboxamido)-glycylamido-3,5-dideoxy-ß-D-glycero-D-galacto-2-
nonulopyranosuronate]. Solium pyruvate (2.4 g, 218 mmol), HEPES buffer (0.25 M. pH
7.34) and 1.0 g (22 mmol) of Fmoc-glycylmannosamide were mixed in a 150 ml,
polycarbonate bottle. A neuraminic acid aldolase solution (19 ml„ ~ 600 U) was then added
and the reaction mixture was incubated at 30 °C on an orbital shaker. After 23 hours. Thin
layer chromatography (TLC) indicated that approximately 75% conversion to product had
occurred. The CTP (1.72 g, 33 mmol) and 0.1 M of MnCl2 (6 mL) were then added to the
reaction mixture. The pH was adjusted to 7.5 with 1 M NaOH (5.5 mL) and a solution
containing CMP-neuraminic acid synthetase (Neisseria) was added (25 mL, 386 U). The
reaction was complete after 24 hrs and the reaction mixture was chromatographed (C-18
silica, gradient from H2O (100%) to 10% MeOH/H20). Appropriate fractions were
recombined, concentrated and lyophilized to afford a white solid, Rt (IPA/ H2O/NH4OH,
7/2/1) = 0.52. 1HNMR(D2O,500MHz)d 1.64 (dt, 1H.J = 12.0, 6.0), 2.50 (dd, 1H.J=
13.2, 4.9), 3.38 (d, J - 9.67, 1H). 3.60 (dd, J=l 1.65, 6.64, 1H), 3.79 (d, J=4.11, 111), 3.87 (dd.
J= 12.24, 1.0, 1H), 3.97 (m,2H), 4.07 (td, J = 10.75, 4.84, 1H), 4.17 (dd, J = 10.68, 1.0, 1 H),
4.25 (s. 2H). 4.32 (t, J =4.4, 1H), 4.37 (t, J=5.8 1H), 4.6-4.7 (m, obscured by solvent peak).
5.95 (d. .1 = 4. 1 H). 6.03 (d, J = 7.4, 1H). 7.43-7.53 (m. 3H), 7.74 (m, 2H). 7.94 (q, .1 = 7, 3H)
. MS (ES); calc. for C35H42N5O18P ([M-H]-), 850.7; found 850.8.
Preparation ot'Cytidine-5'-monophosphoryl-[5-(N-methoxypolyoxyethylene-(1
kDa)-3-oxypropionamido)-glycylamido-3,5-dideoxy-ß-D-glycero-D-galacto-2-
nonulopyranosuronate]. Methoxypolyoxyethylene-( 1 kDa average molecular weight)-3-
oxypropionate-N-succinimidyl ester (52 mg, 52 µmol) dissolved in anhydrous DMF (450 µL)
and triethylamine (33 µL, 238 µmol). Cytidine-5'-monophosphoryl-(5-glycylamido-3.5-
dideoxy-ß-D-glycero-D-galacto-2-nonulopyranosuronate) (30 mg, 48 µmol) was added as a
solid. Water, pH 8 (330 µL) was added and after 30 min, an additional 28 mg of NHS-
activated PEG was added. After an additional 5 min, the reaction mixture was
chromatographed (C-18 silica, gradient of methanol/water), and appropriate fractions were
concentrated to afford 32 mg (40% yield) of a white solid, Rf = 0.31 (silica, IPA/H20/NH4OH
7/2/1); 1HNMR(D20, 500 MHz) 8 1.66 (td. 1H, J =5.3), 2.50 (dd, 1H,J= 13.2, 4.6), 2.64 (t.
.1=5.99. 3H) 3.43 (d. .1 = 9.58. 1H). 3.63 (m. 1H), 3.71 (s, 70H), 3.79 (m, obscured by 3.71
peak), 3.82 (t, J=6.19, IH) 3.88 (dd, J = 11.8, 1.0, 1H), 3.95 (td, J= 9.0, 2.3, 111), 3.98 (t, .1"
5.06, 1M), 4.12 (td, J - 10.34,4.66, 1 H), 4.18 (d, J = 10.36, 1H), 4.23 (d, J=4.85, 2H), 4.31
(I. J-4.64. 1 H). 4.35 (t. 1 H). 6.00 (d, J = 4.55, 1 H), 6.13 (d, J = 7.56. 1 H), 7.98 (d. J-7.54.
III). MS (MALDI), observe [(M-CMP)-H]; 1506.4, 1550.4, 1594.5, 1638.5. 1682.4, 1726.4.
1770.3. 1814.4, 1858.2.
Preparation of Cytidine-5'-monophosphoryl-{5-[N-(2,6-
dimethoxypolyoxyethylene-(20 kDa)-3oxypropionamidyl-lysylamido|-glycylamido-3,5-
dideoxy-ß-D-glycero-galacto-2-nonulopyranosuronate}. The 2,6-Di-
[methoxypolyoxyethylene-(20 kDa average molecular weight)-3-oxypropionamidyl]-
lysylamido-N-succinimidyl ester (367 mg. 9 µmol) was dissolved in anhydrous THF (7 mL)
and triethylamine (5 µL, 36 µmol). Cytidine-5"-monophosphoryl-(5-glycylamido-3,5-
didco.x.y-ß-D-glycero-D-galacto-2-nonulopyranosuronate) (30 mg, 48 µmol) was dissolved in
1.0 mL of water, and added to the reaction mixture. The reaction was stirred for 4 hours at
room temperature and was then chromotographed (HPLC, Waters Xterra RP8, gradient from
water/NH4OH. 100% to 20% methanol/water/NH4OH at 1 mL/min) to afford a white solid
with a R, - 22.8 min. MS (MALDI), observe [(M-CMP)-H]; 43027.01 (40,000 - 45.500).
3. Preparation of UDP-Gal-PEG.
This example sets forth the general procedure for making UDP-Gal-PEG.
Methoxypolyoxyethylenepropionate N-hydroxysuccinimide ester (mPEG-SPA, MW
1.000) 348 mg in THF (0.5 mL) was added to a solution of 25 mg of galactosamine-1-
phosphate in 1 ml of water, followed by the addition of 67 µL triethylamine. The resulting
mixture was stirred at room temperature for 17 hr. Concentration at reduce pressures
provided a crude reaction mixture which was purified by chromatography (C-18 silica, using
a step gradient of 10%. 20%, 30%, 40% aqueous MeOH) to afford 90 mg (74%) of product
after the appropriate fractions were combined and concentrated to dryness. R|-- 0.5 (silica,
Propanol/H20/NH.,OH 30/20/2); MS(MALDI), observed 1356, 1400, 1444, 1488, 1532.
1576, 1620.
[a-1-(Uridinc-5'-diphosphoryl)-1-deoxy-2-(methoxypolyoxyethylene-
propionoylamido-1 kDa)-a-D-galactosamine. The 2-deoxy-2-(methoxy-
polyoxyethylenepropionoylamido-1 kDa)-a-1-monophosphate-D-galactosamine (58 mg) was
dissolved in 6 mL of DMF and 1.2 mL of pyridine. UMP-morpholidate (60 mg) was then
added and the resulting mixture was stirred at 70°C for 48 hr. After concentration, the
residue was chromatographed (C18-silica, using a step gradient of 10%, 20%, 30%, 40%,
50% , 80% MeOH) to yield 50 mg of product after concentration of the appropriate fractions.
Rr= 0.54 (silica, propanol/H20/NH4OH 30/20/2). MS(MALDl); Observed 1485, 1529, 1618,
1706.
[a-1-(Uridinc-5'-diphosphoryl)-1-deoxy-6-(methoxypolyoxyethylene-amino-2
k.Da)-a-D-galactose. [a-1-(Uridine-5"-diphosphoryl)]-6-carboxaldehyde-a-D-galactose (10
mg) was disssolved in 2 mL of 25 mM sodium phosphate buffer (pH 6.0) and treated with
methoxypolyethyleneglycol amine (MW 2, 000, 70 mg) and then 25 µL of 1M NaBH3CN
solution at 0°C. The resulting mixture was frozen at -20°C for three days. The reaction
mixture was chromatographed (HPLC, Water Xterra P8) using 0.015 M NH4OH as mobile
phase A and MeOH as mobile phase B as eluent at the speed of 1.0 mL/min. The product
was collected, an concentrated to yield a solid; R, = 9.4 minutes. Rf= 0.27(silica, EtOH/H20
7/3).
[a-1-(Uridine-5'-diphosphoryl)]-6-amino-6-deoxy-a-D-galactose. Ammonium
acetate 15 mg was added to a solution of [a-1-(Uridine-5'-diphosphoryl)-6-carboxaldehyde-}
a-D-galactopyranosidc (10 mg) in sodium phosphate buffer (pH 6.0). A solution of (25 µL)
1M NaBH3CN was then added and the mixture was stirred for 24 hr. The solution was
concentrated and the residue was chromotographed (sephadex G10) to afford 10 mg of a white
solid, Rf= 0.62 (silica. EtOH/0.1 M NH4Ac).
[a-1-(Uridine-5'-diphosphoryl)]-6-deoxy-6-(methoxypolyoxyethylenepropionoyl
amido, ~2 kDa)-a-D-galactopyranoside. [a-1-(Uridine-5'-diphosphoryl)]-6-amino-6-
deoxy-a-D-galactopyranoside (5 mg) was dissolved in 1 ml. of H20. Then
methoxypolyetheneglycolpropionoyl-NHS ester (MW -2,000, 66 mg) was added, followed
by 4.6 L triethylamine. The resulting mixture was stirred at room temperature overnight,
and then purified on HPLC (C-8 silica) to afford the product, R, = 9.0 min.
[a-1-(Uridinc-5'-diphosphoryl)]-6-deoxy-6-
(methoxypolyoxycthylenecarboxamido, ~2 kDa)-a-D-galactopyranoside. [a-1-(Uridine-
5'-diphosphoryl)l-6-cimino-6-deoxy-(x-D-galactopyranoside (10 mg) was mixed with
methoxypolyethyleneglycoIcarboxy-HOBT (MW 2000, 67 mg) in 1 mL of H20. followed by
the addition of EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride 6.4 mg
and 4.6 jiL triethylamine. The resulting mixture was stirred at room temperature 24 hr. The
mixture was chromatographed (C-8 silica) to afford the product.
4. Preparation of UDP-GlcNAc-PEG
This example sets forth the general procedure for making UDP-GlcNAc-PEG. On
the left side of scheme 17, the protected amino sugar diphospho-nucleotide is oxidized to
form an aldehyde at the 6-position of the sugar. The aldehyde is converted to the
corresponding primary amine by formation and reduction of the Schiff base. The resulting
adduct is contacted with the p-nitrophenol carbonate of m-PEG, which reacts with the amine,
binding the m-PEG to the saccharide nucleus via an amide bond. On the right side of scheme
17 at the top. the protected amino sugar diphospho-nucleotide is treated with a chemical
oxidant to form a carboxyl group at the 6-carbon of the sugar nucleus. The carboxyl group is
activated and reacted with m-PEG amine, binding the m-PEG to the saccharide nucleus via
an amide bond. On the right side of scheme 17 at the bottom the reactions are substantially
similar to that on the top right, with the exception that the starting sugar nucleotide is
contacted with an oxidizing enzyme, such as a dehydrogenase, rather than a chemical
oxidant.
5. Preparation of UDP-GalNAc-PEG
This example (scheme 18) sets forth the general procedure for making UDP-GalNAc-
PEG. The reaction set forth above originates with a sugar diphospho-nucleotide. in which R
is either a hydroxyl 1 or a protected amine 2. In step a, the starting sugar is treated with a
mixture of an oxidase and a catalasc, converting the 6-postion of the sugar into an aldehyde
moiety (3 and 4). In step c, the aldehyde is converted to the corresponding amine (7 and 8)
by formation and reduction of a Schiffbase. In step e, the amine is optionally treated with an
activated m-PEG derivative, thereby acylating the amine to produce the corresponding m-
PEG amide (11 and 13). Alternatively, in step f, the amine is contacted with an activated m-
PEG species, such as a m-PEG active ester, thereby forming the corresponding m-PEG amide
(12 and 14). In step b. the starting material is also treated with a catalase and oxidase,
completely oxidizing the hydroxymcthyl moiety, forming a carboxyl group at the 6-position.
In step d, the carboxyl moiety is activated and subsequently converted to a m-PEG adduct (9
and 10) by reaction with a m-PEG amine intermediate. This is shown in scheme 18.
The aminu-sugar phosphate is contacted with a m-PEG N-hydroxy succinimide active
ester, thereby forming the corresponding sugar-PBG-amide. The amide is contacted with
UMP-morpholidate to form the corresponding active sugar diphospho-nucleotide.
6•.. Synthesis of CMP-SA-Levulinate
This example sets forth the procedure for the synthesis of CMP-SA-levulinate.
Preparation of 2-Ievulinamido-2-deoxy-D-mannopyranose. Isobutylchloroformate
(100 uL. 0.77 mmol) was added dropwise to a solution of levulinic acid (86 uL, 0.84 mmol),
anhydrous THF (3 ml.) and triethylamine (127 uL, 0.91 mmol). This solution was stirred for
3 hours at room temperature and was then added dropwise to a solution containing D-
mannosamine hydrochloride (151 mg, 0.7 mmol), triethylamine (127 uJL, 0.91 mmol), THF
(2 mL) and water (2 mL). The reaction mixture was stirred 15 hours and then concentrated to
dryness by rotary evaporation. Chromatography (silica, step gradient of 5-15%
MeOH/CH2Cl2) was used to isolate the product yielding 0.156 g (73% yield) of a white solid:
Rf-= 0.41 (silica, CHCl3/MeOH/water 6/4/1); 1HNMR (D20, 500 MHz) 8 2.23 (s. 3H), 2.24
(s, 3H), 2.57(td, J = 6.54, 3.68, 2H) 2.63 (t, J=6.71, 211), 2.86-2.90 (m, 4H), 3.42 (m, 1H).
3.53 (t, J=9.76, 1H). 3.64 (t, J=9.43. 1H), 3.80-3.91 (m, 4H). 4.04 (dd, J =9.79. 4.71, 1H).
4.31 (dd, J = 4.63.1.14. IH), 4.45 (dd. .1=4.16,1.13, 1H), 5.02 (d, J=1.29, 1H), 5.1 l(s, J=1.30,
1H). MS (ES): calculated for C11H19NO7, 277.27; found [M+l] 277.9.
Preparation of 5-levulinamido-3,5-dideoxy-D-glycero-D-galacto-2-
nonulopyranosuronate. Sodium pyruvate (0.616 g, 5.6 mmol) and N-acetylneuraminic acid
aldolase (50 U) was added to a solution of 2-levulinamido-2-deoxy-D-mannopyranose (0.156
g. 0.56 mmol) in 0.1 M HEPES (pH 7.5). The reaction mixture was heated to 37 °C for 20
hours and after freezing. The reaction mixture was then filtered through C18 silica, frozen
and freeze-dried. The crude solid was purified using flash chromatography (silica, first using
10-40% MeOH/CH2Cl2 and then CH2Cl2/MeOH/H20 6/4/0.5). Appropriate fractions were
combined and concentrated yielding 45 mg (80% yield) of a white solid: Rf = 0.15 (silica,
CHCl3/McOH/water 6/4/1); 'H NMR (D20, 500 MHz) 8 1.82 (t, J=l 1.9, 1H), 2.21 (dd. J =
13.76.4.84. 1H), 2.23 (s, 3H), 2.57 (app q, J = 6.6, 2H), 2.86-2.95 (m, 2H), 3.15-3.18 (m. 111).
3.28-3.61 (complex, IH), 3.60 (dd, J= 11.91,6.66, 1H), 3.75 (td, J = 6.65. 2.62, 1H), 3.84
(dd, J = 11.89,2.65, 1 II), 3.88-4.01 (complex, 2H), 4.04 (td, J = 11.18,4.67, 1H). MS (ES);
calculated for C14H23NO10. 365.33; found ([M-H), 363.97.
Preparation of cytidine-5'-monophosphoryl-(5-levulinamido-3,5-dideoxy-ß-D-
glycero-D-galacto-2-nonulopyranosuronate). 5-Levulinamido-3.5-dideoxy-D-glycera-D-
galacto--2-nonulopyranosuronate (50 mg, 137 µmol) was dissolved in 2 mL of 100 mM
11EPES pH 7.5 buffer and 1 M MnCl2 (300 uL. 300 µmol) was added. CTP-2Na+ (79 mg,
1.5 µmol) was dissolved in 5 mL HEPES buffer and was added to the sugar. The
sialyltransferase/CMP-ncuraminic acid synthetase fusion enzyme (11 U) was added and the
reaction mixture stirred at room temperature for 45 hours. The reaction mixture was filtered
through a 10,000 MWCO filter and the filtrate, which contained the product of the reaction,
was used directly without further purification: Rf = 0.35 (silica, IPA/water/NH4OH 7/2/1).
B. Glycoconjugation and GlycoPEGylation of Peptides
a-Protease Inhibitor (a-Antitrypsin)
7. Sialylation of Recombinant GlycoproteinsAntithrombin HI, Fetuin and al-
Antitrypsin
This example sets forth the preparation of sialylated forms of several recombinant
peptides.
Sialylatiun of Recombinant Glycoproteins Using ST3Gal III. Several
glycoproteins were examined for their ability to be sialylated by recombinant rat ST3Gal III.
for each of these glycoproteins, sialylation will be a valuable process step in the development
of the respective glycoproteins as commercial products.
Reaction Conditions. Reaction conditions were as summarized in Table 11. The
sialyltransferase reactions were carried out for 24 hour at a temperature between room
temperature and 37°. The extent of sialylation was established by determining the amount of
l4C-NeuAc incorporated into glycoprotein-1 inked oligosaccharides. See Table 1 1 for the
reaction conditions for each protein.

The results presented in Table 12 demonstrate that a remarkable extent of sialylation
was achieved in every case, despite low levels of enzyme used. Essentially, complete
sialylation was obtained, based on the estimate of available terminal galactose, fable 12
shows the relults of the sialylation reactions. The amount of enzyme used per mg of protein
(mU/mg) as a basis of comparison for the various studies. In several of the examples shown,
only 7-13 ml) ST3Gal III per mg of protein was required to give essentially complete
sialylation after 24 hours.
These results are in marked contrast to those reported in detailed studies with bovine
ST6Gal I where less than 50 mU/mg protein gave less than 50% sialylation, and 1070 mU/mg
protein gave approximately 85-90% sialylation in 24 hours. Paulson et al. (1977) .1. Biol.
Chem. 252: 2363-2371; Paulson et al. (1978) J. Biol. Chem. 253: 5617-5624. A study of rat
a2,3 and a2,6 sialyhransferases by another group revealed that complete sialylation of asialo-
AGP required enzyme concentrations of 150-250 mU/mg protein (Weinstein et al. (1982) J.
Biol. Chem. 257: 13845-13853). These earlier studies taken together suggested that the
ST6Gal I sialyltransferase requires greater than 50 mU/mg and up to 150 mU/mg to achieve
complete sialylation.
This Example demonstrates that sialylation of recombinant glycoproteins using the
ST3 Gal 111 sialyltransferase required much less enzyme than expected. For a one kilogram
scale reaction, approximately 7,000 units of the ST3GaI III sialyltransferase would be
needed, instead of 100,000-150,000 units that earlier studies indicated. Purification of these
enzymes from natural sources is prohibitive, with yields of only 1-10 units for a large scale
preparation after 1-2 months work. Assuming that both the ST6Gal I and ST3Gal III
sialyltransferases are produced as recombinant sialyhransferases, with equal levels of
expression of the two enzymes being achieved, a fermentation scale 14-21 times greater (or
more) would be required for the ST6Gal 1 sialyltransferase relative to the ST3Gal III
sialyltransferase. ['or the ST6Gal I sialyltransferase, expression levels of 0.3 U/I in yeast has
been reported (Borsig et al. (1995) Biochem. Biophys. Res. Commun. 210: 14-20).
Expression levels of 1000 U/liter of the ST3 Gal III sialyltransferase have been achieved in
Aspergillus niger. At current levels of expression 300-450,000 liters of yeast fermentation
would be required to produce sufficient enzyme for sialylation of 1 kg of glycoprotein using
the ST6Gal 1 sialyltransferase. In contrast, less than 10 liter fermentation of Aspergillus niger
would be required for sialylation of I kg of glycoprotein using the ST3Gal III
sialyltransferase. Thus, the fermentation capacity required to produce the ST3Gal 111
sialyltransferase for a large scale sialylation reaction would be 10-100 fold less than that
required for producing the ST6Gal I; the cost of producing the sialyltransferase would be
reduced proportionately.
Cri-IgG Antibody
8.Glyco-Remodeling of Cri-IgGl Antibodies
This example sets forth the procedures for in vitro remodeling of Cri-IgGl antibodies.
N-glycosylation at one conserved site at Asn 297 in the Fc domain of a monoclonal
antibody can modulate its pharmacokinetic behavior and effector functions (Dwek et al.,
1995, J. Anat. 187:279-292; Boyd et al.. 1995, Mol. Immunol. 32:1311-1318; Lund et al.,
1995. FASEBJ. 1995. 9:115-119; Lund et al., 1996. J. Immunol. 157:4963-4969; Wright &
Morrison. 1998, J. Immunol. 160:3393-3402; Flynn & Byrd, 2000, Curr. Opin. Oncol.
12:574-581). During cell culture fermentation or in certain pathological conditions,
significant heterogeneity arises in the glycosylation pattern at this site. The resulting
different patterns of glycosylation on the Fc domain are characterized by complex
biantennary structures with zero, one, and two terminal galactose residues (GO, Gl, and G2.
respectively, see Table 13). The observed glycoform variations, such as the variation in
terminal galactosylation, truncated N-glycoforms and bisecting modification, have been
shown to influence the antibody's therapeutic properties, especially its ability to mediate
targeted cell killing through complement binding and activation (Boyd et al., 1995, supra:
Wright & Morrison, 1998, supra. Mimura et al., 2000, Molec. Immunol. 37:697-706: Davies
et al., 2001, Biotechnol. Bioeng. 74:288-294).
In order to obtain different glycoforms of Cri-IgGl antibodies and test their l'c
effector functions. Cri-IgGl antibodies were trimmed back stepwise using exoglycosidases to
generate glycoforms lacking sialic acid (G2, Gl), glycoforms lacking sialic acid and
galactose (GO), and glycoforms lacking sialic acid, galactose and N-acetyl glucosamine
(M3N2F), as illustrated in Table 13. These molecules were subsequently modified using
different glycosyltransferases and appropriate sugars. Modification conditions were
developed that resulted in the conversion of the original antibody glycan structures into
different glycoforms: M3N2, GnT-I-M3N2 (the M3M2 glycoform with a GlcNAc moiety
added using GnT-I), GO, Bisecting-GO (the GO moiety with a bisecting GlcNAc added with
GnT-lII), galactosylatcd bisecting-GO (the bisecting-GO glycoform with terminal galactose
moieties added), G2, mono-sialylated Sl(a2,6)-G2 (the G2 glycoform with one terminal
sialic acid moiety added using a2,6-sialyltransferase), Sl(a2,3)-G2 (the G2 glycoform with
one terminal sialic acid moiety added using a2,3-sialyltransferase) and disialylated S2(a2,3)-
G2 (the G2 glycoform). After every glycoremodeling step, the glycan structures were
enzymatically released from the antibody protein and were analyzed by various methods,
including separation by capillary electrophoresis, 2-AA HPLC profiling and MALDI-TOF
mass spectrometry.

The materials ane methods used in these experiments are now described.
TheCri-IgGI Monoclonal Antibody. The Cri-lgGl antibody was obtained from R.
Jefferies, MRC Center for Immune Regulation, The Medical School, University of
Birmingham. UK. The antibody is a non-recombinant antibody, and is isolated from a human
myeloma. The antibody was prepared using three methods. In the first method, referred to as
"DEAL," the antibody was isolated under relatively mild conditions using a DEAE ion
exchange column. In the second method, referred to as "SPA," the antibody was purified on
a protein A column (Staphylococcus aureus protein A) with a low pH elution step. In the
third method, referred to as "Fc." the antibody was treated with a protease so that only the Fc
portion of the antibody remained and the antigen binding domains were removed. These
methods for antibody purification are well known to those of skill in the art and are not
repeated in detail here.
Affinity purification of remodeled antibodies. Antibody, modified either by
cxoglycosidase or glycosyltransferase, was affinity purified on a ProA-sepharose 4-fast How
column (Amersham Bioscience. Arlington Heights, IL ), eluted with 0.1 M glycine-HCl
buffer, pH 2.7, and immediately neutralized with 1 M Tris, pH 9.5. The eluates were buffer-
exchanged using a NAP-10 column (Amersham Bioscience, Arlington Heights, IL) to an
appropriate buffer for the next step of glycosylation, such as 100 mM MES, pH 6.5 or 50 mM
Tris-HCl. pH 7.2. The remodeled final products were dialyzed extensively against PBS at
4°C in Tube-O-Dialyzers™ (Chemicon International, Temecula, CA) with a MWCO of 8
kDa.
In vitro glycosidase treatment of Cri-antibodies. Antibody was buffer-exchanged into
50 mM Na phosphate/Citrate, pH 6.0 using NAP-10 column (Amersham Bioscience.
Arlington Heights. IL). In vitro trimming back of sugar moieties was carried out stepwise, by
contacting the antibody (5 mg/mL) with 20 mU/mg protein neuraminidase at 37°C overnight
(to remove terminal sialic acid moieties ). 20 mU/mg protein P-galactosidase at 37°C,
overnight (to remove terminal galactose moieties to result in the GO glycoform), and/or 2
U/mg p-N-acetylhexosaminidase (from Jack Bean, Seikagaku, Tokyo. Japan) at 37°C.
overnight (to remove terminal N-acetyl glucosamine to result in the M3N2 glycoform). The
samples were affinity purified as described above.
In vitro glycosvlation of Cri-antibodies. In vitro GnTI modification was performed
using 1 mg/ml of the M3N2 glycoform antibody as the substrate, and 25 mU/mg of
recombinant human ß1,2-mannosyl-UDP-N-acetylglucosaminosyltransferase in a buffer of
100 mM MES. pll 6.5. 5 mM MnCI2, 5 mM UDP-GlcNAc, and 0.02% NaN3 at 32°C for 24
hr. An aliquot was removed for glycan analysis, and the resulting products were affinity
purified as described above.
In vitro modification of the bisecting-glycoform was carried out using 1 mg/ml of the
M3N2 glycoform antibody as the substrate and 25 mU/mg of (31,2-recombinant human
mannosyl-UDP-N-acetylglucosaminosyltransferase I, 25 mU/mg of [31,2-recombinant human
mannosyl-UDP-N-acetylglucosaminosyltransferase II and 3.5 mU/mg of ß1,4-recombinant
mouse mannosyl-UDP-N-acetylglucosaminosyltransferase III in a buffer of 100 mM MES
pH 6.5. 10 mM MnCl2, 5 mM UDP-GlcNAc, and 0.02% NaTM3 at 32°C for 24 hrs. An aliquot
was removed for glycan analysis, and the remaining product was affinity purified as
described above.
In vitro galactosylation was performed using GO glycoform antibody or bisecting
glycoform antibody by contacting the antibody with 0.6 U/mg recombinant bovine milk ß1,4
galactosyltransferase in a buffer of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM UDP-
galactose. 5 mM MnCl2, at 32°C for 24 hrs. An aliquot was removed for glycan analysis, and
the remaining products were affinity purified as described above.
In vitro sialylation was carried out using the G2 glycoform antibody (1 mg/ml,) by
contacting it with 0.1 U/mg ST3Gal3 or 0.1 U/mg ST6Gall, 5 mM CMP-sialic acid, at 32°C
for 24 hr in a buffer of 50 mM Tris pH 7.4. 150 mM NaCl, and 3 mM CMP-SA. An aliquot
was removed for glycan analysis, and the remaining products were affinity purified as
described above.
Glycan Analysis:
Capillary Electrophoresis with Laser Induced Fluorescence Dectection. Buffer
components and nucleotide sugars were removed from an aliquot of the glycoremodeled
antibody by dilution and concentration in a Microcon™ YM-30 microconcentrator
(Millipore, Bedford, MA). N-linked oligosaccharides were released from the protein by
contacting it with PNGase F (Prozyme, San Leandro, CA) using the methodology provided
by the manufacturer. In brief, the sample was denatured in the buffer of 50 mM sodium
phosphate pH 7.5. 0.1% SDS, and 50 mM P-mercaptoethanol for 10 min at 100°C. TX100
was then added to 0.75% (v/v) as well as 10U PNGaseF/200 |ig protein. After 3 hours
incubation at 37°C, the protein was ethanol precipitated and the supernatant was dried down.
The released free oligosaccharides were then labeled with 8-aminopyrene-1.3,6-trisulfonic
acid and analyzed by capillary electrophoresis with a carbohydrate labeling and analysis kit
from Beckman-Coulter, Inc. (Fullerton, CA), as indicated by the manufacturer (see also. Ma
and Nashabeh, 1999. Anal. Chem. 71:5185-5192).
Capillary electrophoresis (CE) was carried out in an eCAP™ N-CHO coated
Capillary (50 urn I.D.. length to detector 40 cm; Beckman-Coulter, Inc., Fullerton, CA),
using a P/ACE™ MDQ Glycoprotein System (Beckman-Coulter, Inc. Fullerton, CA) with
Laser Induced Fluorescence Detector (Beckman-Coulter, Inc. Fullerton. CA). Samples were
introduced into the cartridge by 20 psi pressure for 10 sec. and separated under 25 kV with
reverse polarity for 20 min. Cartridge temperature was kept at 20°C. The electropherogram
was generated by laser-induced fluorescence detection at an excitation wavelength of 488 nm
and an emission wavelength of 520 nm.
Carbohydrate standards (Calbiochem®, EMD Biosciences, Inc., San Diego, CA).
including M3N2 (N-linked trimannosyl core without core fucose), GO (N-linked
oligosaccharide, asialo, agalacto, biantennary with core fucose), G2 (N-linked
oligosaccharide, asialo, biantennary with core fucose), and G2 without fucose, S1-G2 (mono-
sialylated. galactosylated biantennary oligosaccharide without core fucose) and S2-G2 (di-
sialylated, galactosylated biantennary oligosaccharide without core fucose), (from Glyko. see.
ProZyme, San Leandro. CA), M3N2F (N-linked trimannosyl core with core fucose) and
NGA2F (N-linked oligosaccharide asialo, agalacto, biantennary with core fucose and with
bisecting GlcNAc) were labeled with l-aminopyrene-3,6,8-trisulfonate (APTS. Beckman-
Coulter. Inc. Fullerton. CA) and used to identify the distribution of glycans released from the
antibody.
2-AA HPLC. PNGaseF released glycans were labeled with 2-AA (2-anthranilic acid)
according to the method described by Anumula and Dhume with slight modifications (1998.
Glycobiology 8:685- 694). Reductively-aminated N-glycans were analyzed using a Shodex
Asahipak NH2P-50 4D amino column (4.6 mm x 150 mm) (Showa Denko K.K., Tokyo.
Japan). The two solvents used for the separation are A) 2% acetic acid and 1 %
tetrahydrofuran in acetonitrile and B) 5% acetic acid, 3% triethylamine and 1%
tetrahydrofuran in water.
To separate neutral 2AA-labeled glycans, the column was eluted isocratically with
70% A for 5 minutes, followed by a linear gradient over a period of 60 minutes going from
70% to 50% B, followed by a steep gradient over a period of 5 minutes going from 50% to
5% B and a final isocratic elution with 5% B for 10 minutes. Eluted peaks were detected
using fluorescence detection with an excitation at 230 nm and detection wavelength at 420
nm. In this gradient condition, the GO glycoform will elute at about 30.5 minutes, the Gl
glycoform at about 34.0 minutes and the G2 glycoform at about 37.0 minutes. Under these
conditions, the presence of fucose does not change the elution time.
To separate anionic 2AA-labeled glycans, the column was eluted isocratically with
70% A for 2.5 minutes, followed by a linear gradient over a period of 97.5 min going from
70% to 5% A and a final isocratic elution with 5% A for 15 minutes. Eluted peaks were
detected using fluorescence detection with excitation at 230 nm and detection at 420 nm. In
this gradient, neutral glycans are expected to elute between 18.00 - 29.00 minutes, glycans
with one charge elute between 30.00 - 40.00 minutes, glycans with two charges elute
between 43.00 - 52.00 minutes, glycans with three charges elute between 54.00 - 63.00
minutes, and glycans with four charges elute between 65.00 - 74.00 minutes.
MALD1 analysis of reductively-aminated N-glycans. A small aliquot of the PNGase-
released N-glycans that were labeled with 2-anthranilic acid (2AA) were then dialyzed for 45
minutes on a MF-Millipore membrane filter (0.025 urn pore, 47 mm dia.), which was floating
on water. The dialyzed aliquot was dried in a Speedvac™ (ThermoSavant, Holbrook, NY),
redissolved in a small amount of water, and mixed with a solution of 2,5-dihydroxybenzoic
acid (10 g/L) dissolved in water/acetonitrile (50:50).
The mixture was dried onto a MALD1 target and analyzed using an Applied
Biosystems DH-Pro mass spectrometer (Applied Biosystems, Inc.. Foster City. CA) operated
in the linear/negative-ion mode. Oligosaccharide structures were assigned based on the
observed mass-to-chargc ratio and literature precedence. No attempt was made to fully
characterize isobaric structures.
SDS-PAGB. To determine the stability of the glycoremodeled antibody, all the
samples were analyzed by SDS-PAGE. The final products of the samples were run under
non-reducing conditions using 8-16% Tris-glycine gel (Invitrogen, Carlsbad, CA). Bovine
serum albumin was run under reducing condition as quantitative standards. The gel was
stained with GelCode Blue Stain Reagent (Pierce Chemical Co., Rockford, IL) for
visualization.
The results of the experiments are now described.
Native glycoforms of Cri expressed in human myeloma cells. Cri-IgGl antibody
purified from the serum of a patient having multiple myeloma contains variable glycoforms.
Figure 97A-97C shows the HPLC profiles of glycans enzymatically released from Cri-IgGl
antibody. Figure 98A-98C shows the MALDI profiles of glycans enzymatically released
from Cri-IgG 1 antibody expressed in human myeloma cells. The major forms are under-
galactosylated GO. Gl, while G2 and sialylated structures are relatively minor (Table 14 and
Figure 97C). To test the impact of modified glycans on the therapeutic properties of the
monoclonal antibody, Cri-IgGl antibody was modified by performing in vitro
exoglycosidases trimming and in vitro glycosylation remodeling to generate different
glycoforms of this antibody.
Initially, optimization of each step in exoglycosidases trimming and glyeosylation
was performed at small scale (100 |ug of each).
Trimannosyl core glycoform of Cri-lgGl Antibody (M3N2). M3N2 was created by
stepwise treatment of glycosidases, including neuraminidase, ß1,4-galactosidase and ß1-2, 3.
4. 6 N-acetylhexosaminidase. To assess the removal of terminal galactose and GlcNAc on
the glycoremodeled Cri-lgGl antibody samples, a quantitative capillary electrophoresis (CE)
method was used. The glycans were enzymatically released from the glycoremodeled
antibody with PNGase F and were derivatized with 8-aminopyrene-l,3,6-trisulfonic acid
(APTS) at the reducing terminus. The resulting products were analyzed by CE with on-
column laser-induced fluorescence detection (LIF) (Ma & Nashabeh, 1999, supra). Since the
separation of the glycans is based on the differences in hydrodynamic size, the APTS labeled
glycans migrate in order of increasing size (M3N2 Figures 99A-99D show the electropherograms indicating the glycans released from
glycoremodeled Cri-lgGl antibody as well as glycan standards derivatized with APTS
(Figure 99A). The glycoforms were identified by comparing their electrophoretic mobilities
to the standards. The relative amount of each glycan species was calculated from the relative
area percentage of each indicated peak, and the results are presented in Table 15. The
M3N2F glycoform represents 91% of the glycans of DEAE-Cri, 80% of the glycans of SPA-
Cri. and 100% of the glycans of Fc-Cri. Incomplete removal of GlcNAc moiety resulting in
the GnT-I-M3N2F glycoform (see. Table 15) was observed in the glycan structures from
DEAE-Cri (8.6%) and SPA-Cri (-20%). Glycoform GnT-I-M3N2F is the M3N2F glycoform
with one additional GlcNAc, such as would be added by GnT-I.
Degalactosylated glycoform (GO). Cri-IgG 1 antibody with GO glycoforms was
obtained by stepwise treatment the native Cri-IgG 1 antibody with neuraminidase and ß1,4-
galactosidase in for 24 hours for each reaction. The glycans released from the
glycoremodeled antibody were analyzed by CE, HPLC and MALD1. Figure 100A shows the
CE profile of the released glycans. In all three samples, only one peak was observed which
was designated as the GO glycoform based on comparison with the standards (Fig. 100A and
Tabic 16).

In addition to the glycan analysis provided by CE, a quantitative HPLC method was
also used to determine the percent of the GO glycoform represented by remodeled glycans of
the Cri-IgG 1 antibody. The glycan distribution on the glycoremodeled antibody was
monitored by enzymatically releasing the glycans with PNGase F and derivatizing the
released products with 2-anthranilic acid (2-AA) at the reducing terminus. The derivatized
mixture was separated by HPLC on a Shodex Asahipak NH2P-50 4D column with
fluorescence detection. Figures 101A-101C show the chromatograms obtained from the
released glycans. HPLC results confirmed CE analysis, as only one major peak was found in
all three samples. In agreement with CE and HPLC data, MALDI analysis also showed
almost complete glycoremodeling to the GO glycoform (Fig. 102A-102C).
Fully galactosylated G2 glycoform CG2). Cri-IgG antibodies were treated with
neuraminidase to yield asialo-glycoforms which were also under galactosylated. These
asialoglycoforms were then treated with 0.6 U/ml of bovine ß1.4 galactosyltransferase and a
galactose donor molecule to glycoremodel the antibody to have the G2 glycoform.
The extent of terminal galactosylation was determined by glycan analysis. Only one
major peak was observed in both CH and HPLC profiles (Figure 103A-103C and Fig. 104A-
104C). This peak corresponds to the G2 glycoform in each case. Calculation of the percent
total peak area showed almost complete (-90%) conversion to the G2 from the under
galactosylated glycoforms of the original samples (see, Table 14). These results are
summarized in Table 17. MALDI analysis of the glycans further supported the almost to
complete glycoremodcling to the G2 glycoform in all of the samples (Fig. 105A-105C).

GnT-1-glycoform (GnT-I-M3N2). The M3N2 glycoform Cri-IgG antibody was
glycoremodeled to the GnT-I-M3N2 glycoform by adding one GlcNAc moiety to the
molecule. The molecule was contacted with 25 mU GnT-I/mg antibody and an appropriate
GlcNAc donor molecule. CE, HPLC and MALDI analysis of released glycans (Figures
106A-106D, Figures 107A-107C and Fig. 108A-108C, respectively) indicated that the
original M3N2F glycoform was completely remodeled. However, only 40-60% of the
modified structures were the GnT-I-M3N2 glycoform, and about 30% were the GO
glycoform. The presence of the GO glycoform may be the result of incomplete GlcNAc
trimming when making the original M3N2 form.
Bisecting glycoform (NGA2F). The M3N2 glycoform Cri-IgG antibody was
glycoremodeled to the NGA2F glycoform by contacting it with a combination the three
transferases, GnT-I, GnT-II and GnT-III, and an appropriate N-acetylglucosamine donor
molecule. The reaction was completed in 24 hours. To determine the extent to which the
bisecting-GlcNAc moiety was added to the glycans, CE analysis was used to determine the
glycoforms present on the glycoremodeled antibody.

Figure 109A-109D shows the electropherograms obtained from CE analysis of the
glycans released from glycoremodeled Cri-IgGl antibody. Four peaks appeared after
remodeling. A major peak migrated at the same retention time as the NGA2F standard
glycoform. The three other minor peaks are likely to be the incompletely remodeled glycans.
For comparison, a quantitative HPLC method was also used, where the 2-AA labeled glycans
elulcd in order of increasing size (Gnl similar results were obtained from the CE analysis of the glycans. No M3N2F was found
using either the CE or HPLC analysis. NGA2F glycans were the major peaks 1 both CE and
HPLC analysis. The Gnl and GO glycans still remaining in the sample likely are the result of
incomplete modification. Most of the original M3N2F glycoforms were remodeled by three
GlcNAc moieties to the NGA2F glycoform (60-70%), about 15-18% were remodeled by the
addition of two GlcNAc moieties to the GO glycoform, and only small amount (~ 7%) were
remodeled by the addition of only one GlcNAc moiety. MALDI-MS analysis of the released
glycans (Figure 111 A.- 111 C) shows peaks of glycoforms with one, two or three terminal
GlcNAc moieties, in agreement with CE and HPLC analysis (Figures 109 and 110). The
relative amount of each glycan species was calculated from the relative area percentage of
each indicated peak, and is summarized in Table 18.
Galactosvlated Bisecting (Gal-NGA2F) glvcoforms. NGA2F glycoforms of Cri-lgGl
antibodies were glycoremodeled with bovine ß 1,4-galactosyltransferase and an appropriate
galactose donor. The terminal galactose moieties were added using 0.6 U/ml of ß 1.4
galactosyltransferase. Figure 112A-112D shows the electropherograms obtained using the 2-
AA HPLC method. In brief, the glycoforms terminating in GalNAc were almost 100%
galactosylated. Comparing Figure 112A to Figure 112B for DEAE Cri-lgGl, and Figure
112C to Figure 112D for Fc Cri-lgGl, the 2-AA HPLC profile of GnT-I. II and III modified
glycans (Figures 1 12A and 112C) is modified by GalTl so that all of the glycan peakes were
shifted to elute later due to the size increase from added galactose moieties (Fig. 112B and
112D). These results were further confirmed by MALDI-MS analysis.
Sialvlated (S2G2) glvcoforms of Cri-lgGl. The glycoremodeled G2 glycoforms of
Cri-lgGl antibody were further remodeled using both ST3Gal3 and ST6Gall. Figure 113A-
I 13C shows the IIPLC profile of the G2 glycoforms remodeled with ST3Gal3. Most of the
G2 glycoforms were converted into S2G2 glycoforms (the G2 glycoform with 2 additional
terminal sialic acid moieties; -70%, see, Table 19), and only small amounts were the S1G2
glycoform (the G2 glycoform with 1 additional terminal sialic acid moiety; 19). These results were further confirmed in the MALDI analysis shown in Figures 114A-
114C. MALDI data also shows that all the G2 glycoforms were sialylatcd to either S2G2 or
SIG2 glycoforms.
By comparison. ST6Gall remodeling of the GO glycoform did not reach the level of
completion found with ST3Gal3 remodeling. Figure 115A-115D and Figure 116A-116C
show the results obtained from CE and UPLC analysis, respectively. No S2G2 glycoforms
were seen in any of the glycoremodeled samples. However, all of the G2 glycoforms were
converted into S1-G2. Analysis from MALDI-MS also supports these data (Figures 117A-
117C).
Stability of remodeled glycans of Cri-IgGl. Lastly, the stability of the Cri-IgG 1
glycans remodeled by exoglycosidase treatment and glycosylation was investigated. Each
glycoremodeled Cri-IgGl antibody was stored at 4°C. and was checked by SDS-PAGE for
degradation at two weeks after remodeling. As shown in Figure 1 18A-118E, the remodeled
DEAE and SPA antibodies both retained a molecular weight of about 150 kDa, indicating
little to no degradation, regardless of the kind of glycoremodeling performed. The Fc Cri-
lgGl antibody retained a molecular weight of about 38 kDa, also indicating little to no
degradation, regardless of the kind of remodeling performed.
Effector Function Bioassay of Remodeled Cri-IgGl antibodies. The effector function
bioassay was derived from the procedure of Mimura et al. (2000, Molecular Immunology
37:697-706). The IC50 of the glycoforms of Cri-IgGl antibody was determined by inhibition
of the superoxide response of U937 cells elicited by red blood cells sensitized with native
anti-NIP antibody.
Monocytic U937 cells were cultured in the presence of 1000 units/mL interferon
gamma for 2 days to induce the differentiation of the cells and their capacity to generate
superoxide. The cells were then washed and resuspended at 2 x 106 cells/mL in Hanks
balanced salt solution without phenol red and containing 20 mM HEPES pH 7.4 and 0.15
mM BSA. The red blood cells were sensitized with anti-NIP (5-iodo-4-hydroxy-3-
nitrophenacetyl) antibody, in the absence or presence of the various glycoforms of Cri-IgGl
antibody, with incubation at 37°C for 30 minutes. The cells were then washed three times
with PBS and resuspended at 2.5 x 107 cells/mL in HBSS-BSA. The U937 cells (100 µl 2 x
106 cells/mL) were added to plastic tubes and lucigenin (20 µl, 2.5 mM) was added to the
tubes. The tubes were warmed in a 37°C water bath for 5 minutes. The sensitized red blood
Us (80 µl. 2.5 x 107/mL) were then added to the tubes. Superoxide anion production was
measured by lucigenin-enhanced chemiluminescence at 37°C over a 30 minute period using a
Berthold LV953 luminometer (Berthold Australia Pty Ltd. Bundoora, Australia).
The GO and M3N2 glycoforms Cri-IgGl antibody had relative inhibitory values ol"
92% and 85%, respectively, as compared with the native antibody. However, the native CR1-
IgG 1 antibody lacked core fucose. Shields et al. (2002, J. Biol. Chem. 277:26733-26740)
suggests that the lack of core fucose will improve inhibitory values 10 fold. Based on these
results, it is anticipated that inhibitory values of the galactosylated-bisecting-GO glycoform
will be greater than the bisecting-GO glycoform, which in turn will be much greater than the
G2 glycoform, which in turn will be approximately equal to the disialylated-G2 glycoform
and the monosialylated-G2 glycoform, which in turn will be greater than the native antibody
glycoform, which in turn will be greater than the GO glycoform, which in turn will be greater
than the M3N2 glycoform.
Complement Receptor-1
9. Sialylation and Fucosylation of TP10
This example sets forth the preparation of TP10 with sialyl Lewis X moieties and
analysis of enhanced biological activity.
Interrupting blood flow to the brain, even for a short time, can trigger inflammatory
events within the cerebral micro vasculature that can exacerbrate cerebral tissue damage. The
tissue damage that accrues is amplified by activation of both inflammation and coagulation
cascades. In a murine model of stroke, increased expression of P-selectin and ICAM-1
promotes leukocyte recruitment. sCRl is recombinant form of the extracellular domain of
Complement Receptor-1 (CR-1). sCR-1 is a potent inhibitor of complement activation.
sCRlsLex (CD20) is an alternately glycosylated form of sCRl that is alternately
glycosylated to display sialylated Lewisx antigen. Previously, sCR-lsLeX that was
expressed and glycosylated in vivo in engineered Lecl 1 CHO cells was found to correctly
localize to ischemic cerebral microvessels and Clq-expressing neurons, thus inhibiting
neutrophil and platelet accumulation and reducing cerebral infarct volumes (Huang et al..
1999. Science 285:595-599). In the present example, sCRlsLex which was prepared in vitro
by remodeling of glycans. exhibited enhanced biological activity similar to that of sCRsLe
glycosylated in vivo.
The TPIO peptide was expressed in DUK Bl 1 CHO cells. This CHO cell line
produces the TPIO peptide with the typical CHO cell glycosylation, with many but not all
glycans capped with sialic acid.
Sialylation of 66 mg of TPIO. TPIO (2.5 mg/mL), CMPSA (5 mM), and ST3Gal3
(0.1 U/mL) were incubated at 32°C in 50 mM Tris, 0.15M NaCl, 0.05% sodium azide, pH 7.2
for 48 hours. Radiolabeled CMP sialic acid was added to a small aliquot to monitor
incorporation. TP10 was separated from nucleotide sugar by SEC HPLC. Samples analyzed
at 24 hours and 48 hours demonstrated that the reaction was completed after 24 hours. The
reaction mixture was then frozen. The reaction products were subjected to Fluorophore
Assisted Carbohydrate Electrophoresis (FACE®; Glyko, Inc, Novato CA) analysis (Figure
119).
Pharmacokinetic studies. Rats were purchased with a jugular vein cannula. 10
mg/kg of either the pre-sialylation or post-sialylation TP10 peptide was given by tail vein
injection to three rats for each treatment (n=3). Fourteen blood samples were taken from 0 to
50 hours. The concentration in the blood of post-sialylation TP10 peptide was higher than
that of pre-sialylation TP10 at every time point past 0 hour (Figure 120). Sialic acid addition
doubled the area under the plasma concentration-time curve (AUC) of the pharmacokinetic
curve as compared to the starting material (Figure 121).
Fucosylation of sialylated TP10. 10 mL (25 mgTPIO) of the above sialylation mix
was thawed, and GDP-fucose was added to 5 mM, MnCI2 to 5 mM, and FTV1
(fucosyltransferase VI) to 0.05 U/mL. The reaction was incubated at 32°C for 48 hours. The
reaction products were subjected to Fluorophore Assisted Carbohydrate Electrophoresis
(FACE*; Glyko, Inc. Novato CA) analysis (Figure 122). To a small aliquot, radiolabeled
GDP-fucose was added to monitor incorporation. TP10 was separated from nucleotide sugar
by SEC HPLC. Samples analyzed at 24 hours and 48 hours demonstrated that the reaction
was completed at 24 hours. An in vitro assay measuring binding to E-selectin indicate that
fucose addition can produce a biologically-active E-selectin ligand (Figure 123).
Enbrel™
H). GlycoPEGylation of an antibody Enbrel™
This example sets forth the procedures to PEGylate the O-linked glycans of an
antibody molecule. Here, Enbrel™ is used as an example, however one of skill in the art will
appreciate that this procedure can be used with many antibody molecules.
Preparation of Enbrel™-SA-PEG (10 kDa). Enbrel™ (TNF-receptor-IgG,-
chimera), either with the O-linked glycans sialylated prior to PEGylation or not. is dissolved
at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl. 5 mM MnCI2, 0.05% NaN3, pH 7.2. The
solution is incubated with 5 mM UDP-galactose and 0.1 U/mL of galactosyltransferase at
32"C for 2 days to cap the undergalactosylatcd glycans with galactose. To monitor the
incorporation of galactose, a small aliquot of the reaction has 14C-galactose-UDP ligand
added; the label incorporated into the peptide is separated from the free label by gel filtration
on a Toso Haas G2000SW analytical column in methanol and water. The radioactive label
incorporation into the peptide is quantitated using an in-line radiation detector.
When the reaction is complete, the solution is incubated with 1 mM CMP-sialic acid-
linker-PEG (10 kDa) and 0.1 U/mL of ST3Gal3 at 32"C for 2 days. To monitor the
incorporation of sialic acid-linker-PEG, the peptide is separated by gel filtration on a Toso
Haas G3000SW analytical column using PBS buffer (pH 7.1). When the reaction is
complete, the reaction mixture is purified using a Toso Haas TSK-Gel-3000 preparative
column using PBS buffer (pH 7.1) and collecting fractions based on UV absorption. The
fractions containing product are combined, concentrated, buffer exchanged and then freeze-
dried. The product of the reaction is analyzed using SDS-PAGE and IEF analysis according
to the procedures and reagents supplied by Invitrogen. Samples are dialyzed against water
and analyzed by MALDI-TOF MS.
Erythropoietin (EPO)
11. Addition of GlcNAc to EPO
This example sets forth the addition of a GlcNAc residue on to a tri-mannosyl core.
Addition of GlcNAc to EPO. EPO was expressed in SF-9 insect cells and purified
(Protein Sciences, Meriden, CT). A 100% conversion from the tri-mannosyl glycoform of
Kpo to the "tri-mannosyl core + 2 GlcNAc" (Peak I. PI in Figure 124) was achieved in 24
hours of incubation at 32°C with 100mU/ml of GlcNAcT-I and 100mU/ml of GlcNAcT-ll in
the following reaction final concentrations:
100mM MES pH 6.5, or 100mM Tris pH 7.5
5mM UDP-GlcNAc
20mM MnCl2
100mU/mlGlcNAcT-I
100mU/mlGlcNAcT-II
1 mg/ml EPO (purified, expressed in Sf9 cells,
purchased from Protein Sciences).
Analysis of glycoforms. This assay is a slight modification on K-R. Anumula and ST
Dhume, Glycobiology 8 (1998) 685-69. N-glycanase (PNGase) released N-glycans were
reductively labeled with anthranilic acid. The reductively-aminated N-glycans were injected
onto a Shodex Asahipak NH2P-50 4D amino column (4.6 mm x 150 mm). Two solvents
were used for the separation: A) 5% (v/v) acetic acid, 1% tetrahydrofuran, and 3%
triethylamine in water, and B) 2% acetic acid and 1% tetrahydrofuran in acetonitrile. The
column was then eluted isocratically with 70% B for 2.5 minutes, followed by a linear
gradient over a period of 97.5 minutes going from 70 to 5% B and a final isocratic elution
with 5% B for 15 minutes. Eluted peaks were detected using fluorescence detection with an
excitation of 230 nm and emission wavelength of 420 nm.
Under these conditions, the trimannosyl core had a retention time of 22.3 minutes, and
the product of the GnT reaction has a retention time of 30 minutes. The starting material was
exclusively trimannosyl core with core GlcNAc (Figure 124).
12. Preparation of EPO with multi-antennary complex glycans
This example sets forth the preparation of PEGylated, biantennary F.PO, and
triantennary, sialylated EPO from insect cell expressed EPO.
Recombinant human erythropoietin (rhEPO) from the baculovirus/Sf9 expression
system (Protein Sciences Corp., Meriden, CT) was subjected to glycan analysis and the
resulting glycans were shown to be primarily trimannosyl core with core fucose, with a small
percentage of glycans also having a single GlcNAc.
Addition of N-acetylglucosamine with GnT-I and GnT-II. Two lots of rhEPO (1
mg/mL) were incubated with GnT-I and GnT-II, 5 mM UDP-glcNAc, 20 mM MnCl2. and
0.02% sodium azide in 100 mM MES pH 6.5 at 32°C for 24hr. Lot A contained 20 mg of
EPO. and 100 mU/mL GnT-I and 60 mU/mL GnT-II. Lot B contained 41 mg of EPO, and 41
mU/iriL GnT-I + 50 mU/ml. GnT-II. After the reaction, the sample was desalted by gel
filtration (PD10 columns. Pharmacia LKB Biotechnology Inc., Piscataway, NJ).
EPO glycans analyzed by 2-AA HPLC profiling. This assay is a slight
modification on Anumula and Dhume, Glycobiology 8 (1998) 685-69. Reductively-aminated
N-glycans were injected onto a Shodex Asahipak NH2P-50 4D amino column (4.6 mm x 150
mm). Two solvents were used for the separation. A) 5% (v/v) acetic acid, 1 %
tetrahydrofuran, and 3% triethylamine in water and B) 2% acetic acid and 1% tetrahydrofuran
in acetonitrile. The column was then eluted isocratically with 70% B for 2.5 min, followed
by a linear gradient over a period of 100 min going from 70 to 5% B, and a final isocratic
elution with 5% B for 20 min. Eluted peaks were detected using fluorescence detection with
an excitation of 230 nm and emission wavelength of 420 nm. Non-sialylated N-linked
glycans fall in the LC range of 23-34 min, monosialylated from 34-42 min. disialylated from
42-52 min, trisialylated from 55-65 min and tetrasialylated from 68 - 78 min.
Glycan profiling by 2AA HPLC revealed that lot A was 92% converted to a
biantennary structure with two GlcNAcs (the balance having a single GlcNAc. Lot B showed
97% conversion to the desired product (Figure 125A and 125B ).
Introducing a third antennary branch with GnT-V. EPO (1 mg/mL of lot B) from
the product of the GnT-I and GnT-II reactions, after desalting on PD-10 columns and
subsequent concentration, was incubated with 10 mU/mL GnT-V and 5 mM UDP-GlcNAc in
100 mM MES pH 6.5 containing 5 mM MnCI2 and 0.02% sodium azide at 32°C for 24 hrs.
2AA HPLC analysis demonstrated that the conversion occurred with 92% efficiency (Figure
126).
After desalting (PD-10) and concentration, galactose was added with rGalTl: EPO (1
mg/mL) was incubated with 0.1 U/mL GalTl, 5 mM UDP-galactose, 5 mM MnCI2 at 32°C
for 24 hrs.
MALDI analysis of reductively-aminated N-glycans from EPO. A small aliquot
of the PNGase released N-glycans from EPO that had been reductively labeled with
anthranilic acid was dialyzed for 45 min on an MF-Millipore membrane filter (0.025 µm
pore, 47 mm dia). which was floating on water. The dialyzed aliquot was dried in a
speedvac, redissolved in a small amount of water, and mixed with a solution of 2.5-
dihydroxybenzoic acid (10 g/L) dissolved in water/acetonitrile (50:50). The mixture was
dried onto the target and analyzed using an Applied Biosystems DE-Pro MALDI-TOF mass
spectrometer operated in the linear/negative-ion mode. Oligosaccharides were assigned
based on the observed mass-to-charge ratio and literature precedence.
Analysis of released glycans by MALDI showed that galactose was added
quantitatively to all available sites (Figure 127). Galactosylated EPO from above was then
purified by gel filtration on a Superdex 1.6/60 column in 50 mM Tris, 0.15M NaCl, pH 6.
Sialylation. After concentration and desalting (PD-10), 10 mg galactosylated KPO (1
mg/mL) was incubated with ST3Gal3 (0.05 U/mL), and CMP-SA ß mM) in 50 mM Tris.
150 mM NaCl, pH 7.2 containing 0.02% sodium azide. A separate aliquot contained
radiolabeled CMP-SA. The resulting incorporated label and free label was separated by
isocratic size exclusion chromatography/HPLC at 0.5mL/min in 45% MeOH, 0.1%TFA
(7.8mm x 30 cm column, particle size 5 µm, TSK G2000SWXL, Toso Haas, Ansys
Technologies, Lake Forest, CA). Using this procedure. 12% of the counts were incorporated
ß60 micromolar. at 33 micromolar EPO, or about 10.9 moles/mole). Theoretical ß N-linked
sites, tri-antennary) is about 9 moles/mole incorporation. These correspond within the limits
of the method. In an identical reaction with ST6Gall instead of ST3Gal3. 5.7% of the
radiolabel was incorporated into the galactosylated EPO, or about 48% compared with
ST3Gal3.
13. GlycoPEGylation of EPO produced in insect cells
This example sets forth the prepartion of PEGylated biantennary EPO from insect cell
expressed EPO.
Recombinant human erythropoietin (rhEPO) from the baculovirus/Sf9 expression
system (Protein Sciences Corp.. Meriden, CT) was subjected to glycan analysis and the
resulting glycans were shown to be primarily trimannosyl core with core fucose, with a small
percentage of glycans also having a single GlcNAc (Figure 128).
Addition of N-acetylglucosamine with GnT-I and GnT-II. Two lots of rhEPO (I
mg/mL) were incubated with GnT-I and GnT-II, 5 mM UDP-glcNAc, 20 mM MnCl2. and
0.02% sodium azide in 100 mM MES pH 6.5 at 32°C for 24hr. Lot A contained 20 mg of
HPO. and 100 mlJ/mL GnT-I and 60 mU/mL GnT-II. Lot B contained 41 mg of EPO. and 41
mU/µL GnT-I + 50 mU/mL GnT-II. After the reaction, the sample was desalted by gel
filtration (PD10 columns, Pharmacia LKB Biotechnology Inc., Piscataway, NJ).
Glycan profiling by 2AA HPLC revealed that lot A was 92% converted to a
biantennary structure with two GlcNAcs (the balance having a single glcNAc. Lot B showed
97% conversion to the desired product (Figure 125A and 125B ).
Galactosylation of EPO lot A. EPO (-16 mgs of lot A) was treated with GnT-II to
complete the addition of GlcNAc. The reaction was carried out in 50 mM Tris pH 7.2
containing 150 mM NaCl, EPO mg/ml, 1 mM UDP-GlcNAc, 5 mM MnCl2. 0.02% sodium
a/ide and 0.02 U/ml GnT-II at 32 C for 4 hrs. Then galactosylation of EPO was done by
adding UDP-galactose to 3 mM and GalTl to 0.5 U/ml and the incubation continued at 32° C
for 48 hrs.
Galactosylated EPO was then purified by gel filtration on a Superdex75 1.6/60
column in 50 mM Tris. 0.15M NaCl, pH 6. The EPO containing peak was then analyzed by
2AA HPLC. Based on the HPLC data -85% of the glycans contains two galactose and -15%
of the glycans did not have any galactose after galactosylation reaction.
Sialylation of galactosylated EPO. Sialylation of galactosylated EPO was carried
out in 100 mM Tris pH containing 150 mM NaCl, 0.5 mg/ml EPO, 200 mU/ml of ST3Gal3
and either 0.5 mM CMP-SA or CMP-SA-PEG (1 kDa) or CMP-SA-PEG (10 kDa) for 48 hrs
at 32 °C. Almost all of the glycans that have two galactose residues were fully sialylated (2
sialic acids / glycan) after sialylation reaction with CMP-SA. MALDI-TOF analysis
confirmed the HPLC data.
PEGylation of galactosylated EPO. For PEGylation reactions using CMP-SA-PEG
(1 kDa) and CMP-SA-PEG (10 kDa), an aliquot of the reaction mixture was analyzed by
SDS-PAGE (Figure 129). The molecular weight of the EPO peptide increased with the
addition of each sugar, and increased more dramatically in molecular weight after the
PEGylation reactions.
In vitro bioassay of EPO. In vitro EPO bioassay (adapted from Hammerling et al.
1996, J. Pharm. Biomcd. Anal. 14: 1455-1469) is based on the responsiveness of the TF-1
cell line to multiple levels of EPO. TF-1 cells provide a good system for investigating the
proliferation and differentiation of myeloid progenitor cells. This cell line was established b>
T. Kitamura et al. in October 1987 from a heparinized bone marrow aspiration sample from a
35 year old Japanese male with severe pancytopenia. These cells are completely dependent
on Interleukin 3 or Granulocyte-macrophage colony-stimulating factor (GM-CSF).
The TF-1 cell line (ATCC, Cat. No. CRL-2003) was grown in RPM1 + FBS 10% +
GM-CSF (12 ng/ml) and incubated at 37°C 5% C02. The cells were in suspension at a
concentration of 5000 cells/ml of media, and 200 µl were dispensed in a 96 well plate. The
cells were incubated with various concentrations of EPO (0.1 µg/ml to 10 µg/ml) for 48 hours.
A MTT Viability Assay was then done by adding 25 µl of MTT at 5 mg/ml (SIGMA
M5655), incubating the plate at 37°C for 20 min to 4 hours, adding 100 µl of
isopropanol/HCl solution (100 ml isopropanol + 333 µl HC1 6N), reading the OD at 570 nm.
and 630nm or 690nm. and subtracting the readings at 630 nm or 690 nm from the readings al
570 nm.
Figure 130 contains the results when sialylated EPO, and EPO glycoPEGylated with 1
kDa or 10 kDa PEG was subjected to an in vitro EPO bioactivity test. The EPO
glycoPEGylated with lkDa PEG had almost the same activity as the unglycoPEGylated EPO
when both were at a concentration of approximately 5 µg/ml. The EPO glycoPEGylated with
10 kDa PEG had approximately half the activity of the unglycoPEGylated EPO when both
were at a concentration of approximately 5 µg/ml.
14, GlvcoPEGvlation of O-Linked Glycans of EPO produced in CHO Cells
Preparation of O-linked EPO-SA-PEG (10 kDa). Asialo-EPO, originally produced
in CHO cells, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl. 0.05% NaN3. pH
7.2. The solution is incubated with 5 mM CMP-SA and 0.1 U/mL of ST3GaI3 at 32°C for 2
days. To monitor the incorporation of sialic acid onto the "N-linked glycans, a small aliquot
of the reaction had CMP-SA-14C added; the peptide is separated by gel filtration on a Toso
Haas G2000SW analytical column using methanol, water and the product detected using a
radiation detector. When the reaction is complete, the solution is concentrated using a
Centricon-20 filter. The remaining solution is buffer exchanged with 0.05 M Tris (pH 7.2),
0.15 M NaCl, 0.05% NaN3 to a final volume of 7.2 mL until the CMP-SA could no longer be
detected. The retentate is then resuspended in 0.05 M Tris (pH 7.2), 0.15 M NaCl, 0.05%
NaN3 at 2.5 mg/mL protein. The solution is incubated with 1 mM CMP-SA-PEG (10 kDa)
and ST3Gall. to glycosylate the O-linked site, at 32°C for 2 days. To monitor the
incorporation of sialic acid-PEG, a small aliquot of the reaction is separated by gel filtration
suing a Toso Haas TSK-gel-3000 analytical column eluting with PBS pH 7.0 and analyzing
by UV detection. When the reaction is complete, the reaction mixture is purified using a
Toso Haas TSK-gel-3000 preparative column using PBS buffer (pH 7.0) collecting fractions
based on UV absorption. The product of the reaction is analyzed using SDS-PAGE and IEF
analysis according to the procedures and reagents supplied by Invitrogen. Samples are
dialyzed against water and analyzed by MALD1-TOF MS.
15. EPO-Transferrin
This example sets forth the procedures for the glycoconjugation of proteins to O-
linked glycans, and in particular, transferrin is glycoconjugated to EPO. The sialic acid
residue is removed from O-linked glycan of EPO, and EPO-SA-linker-SA-CMP is prepared.
EPO-SA-linker-SA-CMP is glycoconjugated to asialotransferrin with ST3Gal3.
Preparation of O-linked asialo-EPO. EPO (erythropoietin) produced in CHO cells
is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, and is
incubated with 300 mU/mL sialidase (Vibrio cholera)-agarose conjugate for 16 hours at 32
°C. To monitor the reaction a small aliquot of the reaction is diluted with the appropriate
buffer and a IEF gel performed according to Invitrogen procedures. The mixture is
centrifuged at 10,000 rpm and the supernatant is collected. The supernatant is concentrated
to a EPO concentration of about 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3,
pH 7.2. The solution is incubated with 5 mM CMP-sialic acid and 0.1 U/mL of ST3Gal3 at
32°C for 2 days. To monitor the incorporation of sialic acid, a small aliquot of the reaction
had CMP-SA-fluorescent ligand added; the label incorporated into the peptide is separated
from the free label by gel filtration on a Toso Haas G3000SW analytical column using PBS
buffer (pH 7.1). When the reaction is complete, the reaction mixture is purified using a Toso
Haas G3000SW preparative column using PBS buffer (pH 7.1) and collecting fractions based
on UV absorption. The product of the reaction is analyzed using SDS-PAGE and IEF
analysis according to the procedures and reagents supplied by Invitrogen. Samples are
dialyzed against water and analyzed by MALD1-TOF MS.
Preparation of EPO-SA-linker-SA-CMP. The O-linked asialo-EPO 2.5 mg/mL in
50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3, pH 7.2. The solution is incubated with 1 mM
CMP-sialic acid-linker-SA-CMP and 0.1 U/mL of ST3Gall at 32°C for 2 days. To monitor
the incorporation of sialic acid-linker-SA-CMP, the peptide is separated by gel filtration on a
Toso Haas G3000SW analytical column using PBS buffer (pH 7.1).
After 2 days, the reaction mixture is purified using a Toso Haas G3000SW
preparative column using PBS buffer (pH 7.1) and collecting fractions based on UV
absorption. The product of the reaction is analyzed using SDS-PAGE and IEF analysis
according to the procedures and reagents supplied by Invitrogen. Samples are dialyzed
against water and analyzed by MALDI-TOF MS.
Preparation of Transferrin-SA-Linker-SA-EPO. EPO-SA-Linker-SA-CMP from
above is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3, pH 7.2.
The solution is incubated with 2.5 mg/mL asialo-transferrin and 0.1 U/mL of ST3Gal3 at
32"C for 2 days. To monitor the incorporation of transferrin, the peptide is separated by gel
filtration on a 1 oso I laas G3000S W analytical column using PBS buffer (pi 17.1) and the
product detected by UV absorption. When the reaction is complete, the solution is incubated
with 5 mM CMP-SA and 0.1 U/mL of ST3Gal3 (to cap any unreacted transferrin glycans) at
32°C for 2 days. The reaction mixture is purified using a Toso Haas G3000SW preparative
column using PBS buffer (pH 7.1) collecting fractions based on UV absorption. The product
of the reaction is analyzed using SDS-PAGE and IEF analysis according to the procedures
and reagents supplied by Invitrogen. Samples are dialyzed against water and analyzed by
MALDI-TOF MS.
16. EPO-GDNF
This example sets forth the procedures for the glycoconjugation of proteins, and in
particular, the preparation of EPO-SA-Linker-SA-GDNF.
Preparation of EPO-SA-Linker-SA-GDNF. EPO-SA-Linker-SA-CMP from above
is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3. pH 7.2. The
solution is incubated with 2.5 mg/mL GDNF (produced in NSO) and 0.1 U/mL of ST3Gal3
at 32°C for 2 days. To monitor the incorporation of GDNF, the peptide is separated by gel
filtration on a Toso Haas G3000SW analytical column using PBS buffer (pH 7.1) and the
product detected by UV absorption. When the reaction is complete, the solution is incubated
with 5 mM CMP-SA and 0.1 U/mL of ST3Gal3 (to cap any unreacted GDNF glycans) at
32"C for 2 days. The reaction mixture is purified using a Toso Haas G3000SW preparative
column using PBS buffer (pH 7.1) collecting fractions based on UV absorption. The product
of the reaction is analyzed using SDS-PAGE and IEF analysis according to the procedures
and reagents supplied by lnvitrogen. Samples are dialyzed against water and analyzed by
MALDI-TOF MS.
17. Mono-antennary GlycoPEGylation of EPO
This example sets forth the procedure for the preparation of glycoPEGylated mono-
antennary erythropoietin (EPO), and its bioactivity in vitro and in vivo.
When EPO (GenBank Accession No. P01588) is expressed in CHO cells, N-linked
glycans are formed at amino acid residues 24, 38 and 83, and an O-linked glycan is formed at
amino acid residue 126 (Fig. 131; Lai et al., 1986, J. Biol. Chem. 261:3116-3121). The
bioactivity of this glycoprotein is directly correlated with the level of NeuAc content.
Increased sialic acid decreases the binding of EPO to its receptor in vitro: however increased
sialic acid increases the bioactivity of EPO in vivo. The O-linked glycan has no impact on
the in vitro or in vivo activity of EPO, or the pharmacokinetics of the molecule (Wasley et al..
1991, Blood 77:2624-2632).
When EPO is expressed in insect cells, such as is accomplished using a
baculovirus/SP9 expression system (see also, Wojchowshi et al., 1987, Biochem. Biophys.
Acta 910:224-232; Quelle et al., 1989, Blood 74:652-657), N-linked glycans are formed at
amino acid residues 24. 38 and 83, but an O-linked glycan is not formed at amino acid
residue 126 (Fig. 132). This is because the insect cell does not have a glycosyl transferase
that recognizes the amino acid sequence around amino acid residue 126 of EPO. The
majority of the N-linked glycans are composed of GlcNAc2Man3Fuc. In the present example.
EPO expressed in insect cells was remodeled with high efficiency to achieve the complex
glycan SA2Gal2GlcNAc2Man3FucGlcNAc2 by contacting the protein with, in series, GnTI,2.
GalT-1. and ST in the presence of the appropriate donor molecules. These enzymatic
reactions were performed on insect cell expressed EPO using reaction conditions disclosed
herein, to yield the complex glycans herein with 92% total efficiency (Table 21). Optionally,
O-linked glycans can also be added (O'Conncll and Tabak, 1993, J. Dent. Res. 72:1554-
1558; Wang et al., 1993, J. Biol. Chem. .268:22979-22983).
Also in the present example, EPO expressed in insect cells was remodeled to form
mono-antennary, bi-anntenary and tri-antennary glycans, which were subsequently
glycoPEGylated with 1 kDa. 10 kDa and 20 kDa PEG molecules suing procedures described
elsewhere herein. The molecular weights of these EPO forms were determined, and were
compared to Epoetin™ having 3 N-linked glycans, and NESP (Aranesp™) having 5 N-linked
glycans (Fig. 133). Examples of the preparation of bi- and tri-antennary glycan structures are
given in Example 7. herein.
EPO having monoantennary PEGylated glycan structures is prepared by expressing
EPO peptide in insect cells, then contacting the EPO peptide with GnTI only (or alternatively
GnTII only) in the presence of a GlcNAc donor. The EPO peptide is then contacted with
Gall -I in the presence of a galactose donor. The EPO peptide is then contacted with ST in
the presence of SA-PEG donor molecules (Fig. 134A) to generate an EPO peptide having
three N-linked mono-antennary PEGylated glycan structures (Fig. 134B).
The in vitro bioactivity of EPO-SA and EPO-SA-PEG generated from insect cell
expressed EPO was accessed by measuring the ability of the molecule to stimulate the
proliferation of TF-1 erythroleukemia cells. Tri-antennary EPO-SA-PEG 1 kDa exhibited
almost all of the bioactivity of tri-antennary EPO-SA, and di-antennary EPO-SA-PEG 10
kDa exhibited almost all of the bioactivity of di-antennary EPO-SA over a range of EPO
concentrations (Fig. 135). Remodeled and glycoPEGylated EPO generated in insect cells
exhibited up to 94% of the in vitro bioactivity of Epogen™, which is EPO expressed in CHO
cells without further glycan remodeling or PEGylation (Table 22).

The in vivo pharmacokinetics of glycoPEGylated and non-glycoPEGylated EPO was
determined. GlycoPEGylated and non-glycoPEGylated [ll23]-labeled EPO was bolus injected
into rats and the pharmacokinetics of the molecules were determined. As compared with bi-
antennary EPO, the AUG of bi-antennary EPO-PEG 1 kDa was 1.8 times greater, and the
AUG of bi-antennary EPO-PEG 10 kDa was 11 times greater (Fig. 136). As compared with
bi-antennary EPO. the AUC of bi-antennary EPO-PEG 1 kDa was 1.6 times greater, and the
AUC of bi-antennary EPO-PEG 10 kDa was 46 times greater (Fig. 136). Therefore, the
pharmacokinetics of EPO was greatly improved by glycoPEGylation.
The in vivo bioactivity of glycoPEGylated and non-glycoPEGylated EPO was also
determined by measuring the degree to which the EPO construct could stimulate
reticulocytosis. Reticulocytosis is a measure of the rate of the maturation of red blood cell
precursor cells into mature red blood cells (erythrocyte). Eight mice per treatment group
were given a single subcutaneous injection of 10 µg protein/Kg, and the percent reticulocytes
was measured at 96 hours (Fig. 137). Tri- and bi-antennary PEGylated EPO exhibited greater
in vivo bioactivity than non-PEGylated EPO forms, including Epogen™.
Further determination of in vivo bioactivity of the EPO constructs was assessed by
measuring the hematocrit (the percent of whole blood that is comprised of red blood cells) of
CD-I female mice 15 days after intraperitoneal injection three times per week with 2.5 µg
peptide/kg body weight of the EPO construct. The hematocrit increment increased with the
size of the EPO form, with the 82.7 kDa mono-antennary EPO-PEG 20 kDa having a slightly
greater activity than the 35.6 kDa NESP (Aranesp™) and about two times the bioactivity of
28.5 kDa Epogen™ (Fig. 138).
This exarnple illustrates that the generation of a longer-acting glycoPEGylated EPO is
feasible. The pharmacokinetic profile of glycoPEGylated EPO can be customized by altering
the number of glycoPEGylation sites and the size of the PEG molecule added to alter the
half-life of the peptide in the bloodstream. Finally, glycoPEGylated EPO retains both in vitro
and in vivo bioactivity.
18. Preparation and Bioactivity of Sialylated and PEGylated Mono-. Bi- and
Tri-Antennary EPO
This example illustrates the production of glycoPEGylated EPO, in particular
PEGylated EPO having mono-antennary and bi-antennary glycans with PEG linked thereto.
The following EPO variants were produced: mono-antennary PEG (1 kDa), PEG (10 kDa)
and PEG (20 kDa); bi-antennary 2,3-sialic acid (SA), bi-antennary SA-PEG (1 kDa), bi-
antennary SA-PEG (10 kDa); tri-antennary 2,3-SA and tri-antennary 2,6-SA capped with 2,3-
SA.
Recombinant erythropoietin (rEPO) expressed in insect cells was obtained from
Protein Sciences (Lot # 060302. Meridan CT). The glycan composition of this batch of EPO
had approximately 98% trimannosyl core structure. Figure 139A depicts the HPLC analysis
of the released glycans from this EPO, with peak "P2" representing the trimannosyl core
glycan. Figure 139B shows the MALDI analysis of the released glycans with the structures
of the released glycans beside the peak they represent.
Mono-antennary branching
Several steps were performed to produce the mono-antennary branched structure. In
brief, the first step was a GnT-I/GalT-1 reaction followed by purification using Superdex-75
chromatography. This reaction adds a GlcNAc moiety to one branch of the tri-mannosyl
core, and a galactose moiety onto the GlcNAc moiety. Branching was extended with the
ST3Gal3 reaction to add the SA-PEG (10 kDa) moiety or the SA-PEG (20 kDa) moiety onto
the terminal galactose moiety. The final purification was accomplished using Superdex-200
chromatography (Amcrsham Biosciences, Arlington Heights, IL).
GnT-I/GalT-1 Reaction. The GnT-I and GalT-1 reactions were combined and
incubated at 32°C for 36 hours. The reaction contained 1 mg/mL EPO, 100 mM Tris-CI pH
7.2, 150 mM NaCL 5 mM MnCl2, 0.02% NaN3, 3 mM UDP-GlcNAc, 50 mU/mg GnT-I, 3
mM UDP-Gal, and 200 mU/mgGalT-1. Figure 140 depicts the MALDI analysis of glycans
released from EPO after the GnT-I/GalT-1 reaction. Glycan analysis showed approximately
90% of the glycans had the desired mono-antennary branched structure with a terminal
galactose moiety.
Superdex 75 Purification. After the GnT-I/GalTl reaction, EPO was purified from
the enzyme protein contaminants and nucleotide sugars using a 1.6 cm x 60 cm Superdex-75
gel filtration chromatography (Amersham Biosciences, Arlington Heights, IL) in PBS
containing 0.02% Tween 20 (Sigma-Aldrich Corp., St. Louis, MO).
ST3Gal3 Reaction. The ST3Gal3 PEGylation reaction was incubated at 32°C for 24
hours. The reaction contained 1 mg/mL EPO, 100 mM Tris-CI pH 7.2. 150 mM NaCl. 0.02%
NaN3. 200 mU/mg ST3Gal3, and 0.5 mM CMP-SA-PEG (10 kDa) or 0.5 mM CMP-SA-PEG
(20 kDa). Figure 141 depicts the SDS-PAGE analysis of EPO after this reaction. The
corresponding molecular weights of the protein bands indicate that the EPO glycans formed
by the GnT-I/GalT-1 reaction were completely sialylated with the PEG derivative.

Superdex 200 Purification. EPO then was purified from the contaminants of the
ST3Gal3 reaction by a 1.6 cm x 60 cm Superdex-200 gel filtration chromatography
(Amersham Biosciences, Arlington Heights, 1L) in PBS containing 0.02% Tween-20.
TF-1 Cell In Vitro Bioassay of Mono-antennary PEGylated EPO. The IF-1 cell
line is used to assess the activity of EPO in vitro. The TF-1 cells line is a myeloid progenitor
cell line available from the American Type Culture Collection (Catalogue No. CRL-2003.
Rockville. MD). The cell line is completely dependant on Interleukin-3 or Granulocyte-
Macrophage Colony-Stimulating Factor for viability. TF-1 cells provide a good system for
investigating the effect of EPO on proliferation and differentiation.
The TF-1 cells were grown in RPMI with 10% FBS and 12 ng/ml GM-CSF at 37°C in
5% C02. The cells were suspended at a concentration of 10,000 cells/ml of media. 200 µl
aliquots of cells were dispensed into a 96-well plate. The cells were incubated with 0.1 to 10
µg/ml EPO for 48 hrs.
The MTT viability assay was then performed by first adding 25 µl of 5 µg/ml MTT
ß-[4,5-dimethlythiazol-2-yl]-2,5-diphenyltetrazolium bromide, orthiazolyl blue; Sigma
Chemical Co., St. Louis, Mo., Catalogue No. M5655). The plate was incubated for 4 hrs at
37°C. 100 µl of isopropanol/HCl solution (100 ml isopropanol and 333 µl HCl 6N) was
added. The absorbency of the plates was read at 570 nm and either 630 or 690 nm, and the
reading at either 630 nm or 690 nm was subtracted for the reading at 570 nm.
Figure 142 depicts the results of the bioassay of EPO activity after PFXiylation of it
mono-antennary glycans. In this bioassay, the mono-antennary PEGylated EPO is much less
active that a non-PEGylated EPO (Epogen).
In Vivo Bioassay of Mono-antennary PEGylated EPO - Examination of
Hematological Parameters.
The effect of EPO-SA-PEG (10 kDa) and EPO-SA-PEG (20 kDa), the preparation of
which compounds is described in detail above, on hematological parameters of male Sprague
Dawley rats was examined. Sprague Dawley rats are a well-studied experimental model and
techniques for the use and care of such animals are well-known to those skilled in the art.
Therefore, Sprague Dawley rats provide a good system for studying the clinical and
physiological effects of EPO-SA-PEG (10 kDa) and EPO-SA-PEG (20 kDa) according to the
present invention.
Preparation, Care, and Handling of Test Subjects. In vivo testing in accordance
with the methods and compositions of the present invention was conducted at Therlmmune
Research Corporation (Gaithersburg, MD). Test subjects for the in vivo studies included 170
male Sprague Davvley rats, obtained from Harlan (Harlan Sprague Dawley, Inc., Indianapolis,
IN). Subjects were all at least 300 grams in weight and at least 70 days old at the time of
shipment. Subjects were cared for according to Therlmmune's Institutional Animal Care and
Use Committee, in accordance with provisions of the USDA Animal Welfare Act. the PI-IS
Policy on Humane Care and Use of Laboratory Animals, and the U.S. Interagency Research
Animal Committee Principles for the Utilization and Care of Research Animals. The study
was conducted in accordance with the FDA Good Laboratory Practice Standards, 21 CFR
Part 58. Subjects were housed individually in polycarbonate cages, under controlled
environmental conditions, and were provided fresh food weekly, ad libitum, and tap water
was made available to test subjects ad libitum via either an automatic watering system or
water bottles.
Subjects were randomly assigned to groups for testing, and the minimum number of
animals needed to give biologically meaningful data were used. Subjects were dosed via
subcutaneous injection, and dosing frequency and quantity is indicated in in Table 25, and
were observed prior to dose administration, and at least twice daily for signs of toxicity,
moribundity and mortality. Body weights were measured and physical examinations were
conducted on subjects pre- and post-study, as well as on a weekly basis during the study.
For hematology studies, a 0.25 ml (± 15%) sample of blood was collected from the
jugular vein of all surviving subjects prior to the first dose on study day one, as well as on
study days 3, 5, 8. 10, 12, 15, 17, 19, 22, 24, 26 and 29. Hematological analysis of the blood
of the test subjects included examination of cellular morphology, erythrocyte count,
hematocrit, hemoglobin, leukocyte, leukocyte differential, mean cell hemoglobin, mean cell
hemoglobin concentration, mean cell volume, mean platelet volume, platelet count, and
reticulocytes.
On study day 29, after clinical pathology blood collection, all surviving subjects were
euthanized via carbon dioxide inhalation. Blood was collected from the abdominal aorta.
Moribund subjects that were previously euthanized, as well as subjects found dead during the
study, were subjected to full necropsy.
Analysis of results and interpretation of data. Quantitative results were analyzed
using techniques known to those skilled in the relevant art. In particular, results were
analyzed using the Kolmogorov-Smirnov test for normality, the Levene Median test for equal
variance, and one-way Analysis of Variance (ANOVA). In instances where the normality or
equal variance test failed, the analysis was continued using the non-parametric Kruskal-
Wallis ANOVA on rank-transformed data. Parametric data was analyzed using the Dunnett's
t-test to delinieate which groups differed from the control. For non-parametric data, the
Dunns test was used to delineate which groups differed from the control. A probability value
of less than 0.05 (two-tailed) was used as the critical level of significance for all tests.
Based on one analysis of the data from the first fifteen days of the study, it was shown
that a single dose of 50 µg/kg of glycoPEGylated monoantennary EPO, either EPO-SA-PEG
(10 kDa) or EPO-SA-PEG (20 kDa) according to the present invention, gives an extended
rise in blood hemoglobin in rats, comparable to the effect of NESP given at the same dose
(Figure 192). Not wishing to be bound by any particular theory, Figure 192 illustrates that
EPO-SA-PEG (10 kDa) and EPO-SA-PEG (20 kDa) have unexpectedly enhanced biological
properties.
Bi-antennary Branching
Several reactions were performed to accomplish the bi-antennary branching of EPO.
Briefly, the first reaction combined the GnT-1 and GnT-II reactions to add GlcNAc moieties
to two of the tri-mannosyl core branches. The second reaction, the GalT-1 reaction, adds a
galactose moiety to each GlcNAc moieties. Superdex 75 chromatography (Amersham
Biosciences, Arlington Heights, 1L) was performed prior to the ST3Gal3 reaction. The bi-
antennary branching was further extended with the ST3Gal3 reaction to add either a 2,3-SA.
or SA-PEG (1 kDa). SA-PEG (10 kDa). Final purification was accomplished using Superdex
200 chromatagraphy (Amersham Biosciences, Arlington Heights, IL).
GnT-I/GnT-H Reaction. The GnT-I and GnT-11 reactions were combined and
incubated at 32°C for 48 hours. The reaction contained 1 mg/mL EPO, 100 mM MES pH
6.5. 150 mM NaCl, 20 mM MnCl2, 0.02% NaN3, 5 mM UDP-GlcNAc, 100 mU/mg GnT-I.
60 mU/mg GnT-II. The reaction achieved 92% completion of the addition of bi-antennary
GlcMAc moieties, with 8% mono-antennary GlcNAc moieties. Figure 143 A shows the
HPLC analysis of the released glycans, where peak "P3" represents the bi-antennary GlcNAc
glycan. Figure 143B depicts the MALDI analysis of the released glycans with the structures
of the glycans indicated beside the peak that they represent.
In order to further the reaction, an additional 20 mU/mg of GnT-II was added along
with 1 mM UDP-GlcNAc, 5 mM MnCl2, 0.02% NaN3, and the mixure was incubated for 4
hours at 32°C. Greater than 99% of this reaction achieved completion of the bi-antennary
GlcNAc glycan.
GalT-1 Reaction. The GalT-1 reaction was started immediately after the completion
of the second GnT-II reaction. Enzyme and nucleotide sugar were added to the completed
GnT-II reaction at concentrations of 0.5 U/mg GalT-1 and 3 mM UDP-Gal.
When the GalT-1 reaction was performed on a small scale, with about 100 µg EPO
per reaction, approximately 95% of the reaction produced EPO with bi-antennary terminal
galactose moiety. Figure 144A depicts the HPLC analysis of the released glycans where
peak "P2" is the bi-antennary glycan with terminal galactose moieties (85% of the glycans).
and peak "PI" is the bi-antennary glycan without the terminal galactose moieties (15% of the
glycans).
The GalT-1 reaction was also performed on a large scale with about 16 mg of EPO
per reaction. Figure 144B depicts the HPLC analysis of the release glycans from the large
scale GalT-1 reaction, where peak "P2" is the bi-antennary glycan with terminal galactose
moieties, and peak "PI" is the bi-antennary glycan without the terminal galactose moieties.
Superdex 75 Purification. EPO was then purified from the enzyme protein
contaminants and nucleotide sugars using a 1.6 cm x 60 cm Superdex-75 gel filtration
chromatography (Amersham Biosciences, Arlington Heights, 1L) in PBS containing 0.02%
Tween 20 after the GnT-1/GalTl reaction. Figure 145 depicts the chromatogram of the
Superdex 75 gel filtration, where peak 2 is EPO with bi-antennary glycans with terminal
galactose moieties. Figure 146 shows SDS-PAGE analysis of the products of each
remodeling step indicating the increase in the molecular weight of EPO with each remodeling
step.
ST3Gal3 Reaction. The ST3Gal3 reaction was incubated at 32°C for 24 hours. The
reaction contained 0.5 mg/mL EPO, 100 mM Tris-Cl pH 7.2, 150 mM NaCl, 0.02% NaN3.
100 mU/mg ST3Gal3. and 0.5 mM CMP-SA, 0.5 mM CMP-SA-PEG (1 kDa), or 0.5 mM
CMP-SA-PEG (10 kDa). Figure 147 shows the results of SDS-PAGE analysis of EPO before
and after the ST3Gal3 reaction. Based on this SDS-PAGE analysis, bi-antennary EPO
containing terminal Gal can no longer be visually detected after each ST3Gal3 reaction. All
sialylated EPO variants show an increase in size compared to non-sialylated EPO at the start
of the reaction.
Superdex 200 Purification. EPO was purified from the contaminants of the
ST3Gal3 reactions by a 1.6 cm x 60 cm Superdex-200 gel filtration chromatography
(Amersham Biosciences, Arlington Heights, IE) in PBS containing 0.02% Tween-20. Table
23 summaries the distribution of glycan structures at each remodeling step.
Tri-antennary Branching
Several reactions were performed to accomplish the tri-antennary branching of EPO.
Briefly, the first reaction combined the GnT-I and GnT-II reactions to add a GlcNAc moiety
to the two outer tri-mannosyl core branches of the glycan. The second reaction, GnT-V
reaction, adds a second GlcNAc moiety to one of the two outer trimannosyl core branches so
that there are now three GlcNAc moieties. The third reaction, GalT-1 reaction, adds a
galactose moiety to each terminal GlcNAc moiety. The EPO products were then separated
by Superdex 75 chromatography. The tri-antennary branching was further extended with the
ST3Gal3 reaction to add either a 2,3-SA moiety or a 2,6-SA moiety, and capped with a 2,3-
SA moiety. Final purification was accomplished using Superdex 75 chromatography.
GnT-I/GnT-H Reaction. The GnT-I and GnT-I 1 reactions were combined and
incubated at 32°C for 24 hours. The reaction contained 1 mg/mL EPO, 100 mM MES pH
6.5, 150 mM NaCl, 20 mM MnCl2, 0.02% NaN3, 5 mM UDP GlcNAc, 50 mU/mg GnT-I and
41 mlJ/mg GnT-II. The reaction achieved 97% completion of the addition of the bi-
antennary GlcNAc moiety, with 3% tri-mannosyl core remaining. Figure 148 depicts the
HPLC analysis of the glycans released from EPO after the GnT-I/GnT-II reaction.
GnT-V Reaction. The GnT-V reaction containing 100 mM MES pH 6.5. 5 mM
UDP-GlcNAc, 5 mM MnCI2, 0.02% NaN3, 10 mU/mg GnT-V and 1 mg/mL EPO, was
incubated at 32°C for 24 hours. This reaction adds a GlcNAc moiety to an outer mannose
moiety already containing a GlcNAc moiety. Figure 149 depicts the HPLC analysis of the
glycans released from EPO after the GnT-V reaction. Approximately 92% the glycans
released from EPO were the desired product, tri-antennary branched EPO with terminal
GlcNAc moieties, based on glycan and MALDI analysis. The remaining 8% of the glycans
were bi-antennary branched structures containing terminal GlcNAc moieties.
GalT-1 Reaction. The GalT-1 reaction containing 100 mM Tris pll 7.2. 150 mM
NaCl, 5 mM UDP Gal, 100 mU/mg GalT-1, 5 mM MnCI2, 0.02% NaN3 and 1 mg/mL EPO
was incubated at 32°C for 24 hours. Figure 150 depicts the HPLC analysis of the glycans
released from EPO after this reaction. Glycan and MALDI analysis indicates that 97% of the
released glycans had terminal galactose moieties on the tri-antennary branched structures.
The remaining 3% was a bi-antennary structure containing a terminal galactose.
Supcrdcx 75 Purification. After the GnT-I/GalTI reaction, EPO was purified from
the enzyme protein contaminants and nucleotide sugars using a 1.6 cm x 60 cm Superdex-75
gel filtration chromatography (Amersham Biosciences, Arlington Heights, IL) in PBS
containing 0.02% Tween 20. The purified material was divided into two batches to produce
the tri-antennary glycan with terminal 2.6-SA moieties and the tri-antennary glycan w ith
terminal 2,6-SA moieties capped with 2.6-SA moieties.
ST3Gal3 Reaction. The ST3Gal3 reaction was incubated at 32°C for 24 hours. The
reaction contained 1 mg/mL galactosylated EPO, 100 mM Tris-Cl pH 7.2, 150 mM NaCl,
0.02% NaN3, 50 mU/mg ST3Gal3, and 3 mM CMP-SA. Figure 151 depicts the HPLC
analysis of glycans released from EPO after this step. Based on glycan and MALD1 analysis,
approximately 80% of the released glycans were tri-antennary branched structures with
terminal 2,3-SA moieties. The remaining 20% of the released glycans were bi-antennary
structures with terminal 2.3-SA moieties.
ST6Gall sialylation Reaction following the ST3Gal3 Reaction. The ST6Gall
reaction was incubated at 32°C for 24 hours. The reaction contained 1 mg/mL sialylated
galactosylated EPO. 100 mM Tris-Cl pll 7.2. 150 mM NaCl, 0.02%NaN3. 50 mU/mg
ST6Gall, and 3 mM CMP-SA. Figure 152 depicts the results of HPLC analysis of the
glycans released from EPO after the ST6Gall reaction. Based on glycan and MALD1
analysis, approximately 80% of the tri-antennary branched glycans contained terminal 2,3-SA
moieties. The remaining 20% of the glycans were bi-antennary with terminal 2.3-SA
moieties.
Superdex 75 Purification. EPO was purified from the contaminants of the ST3Gal3
reactions by a 1.6 cm x 60 cm Superdex-75 gel filtration chromatography (Amersham
Biosciences. Arlington Heights, IL) in PBS containing 0.02% Tween-20.
Bioassay of Tri-antennary and Bi-antennary Sialylated or PEGylated EPO. The
activity of the tri-antennary and bi-antennary sialylated EPO glycoforms. and the PEG 10
kDa and 1 kDa bi-antennary glycoforms were assayed using the TF-1 cell line and the MTT
viability test, as described above. Figure 153 depicts the results of the MTT cell proliferation
assay. At 2 |ug/ml EDP. the bi-antennary sialylated EPO had nearly the activity of the control
Epogen, while the tri-antennary sialylated EPO had significanly less activity.
Factor IX
19. GlycoPEGylation of Factor IX produced in CHO cells
This example sets forth the preparation of asialoFactor IX and its sialylation with
CMP-sialic acid-PEG.
Desialylation of rFactor IX. A recombinant form of Coagulation Factor IX (rFactor
IX ) was made in CHO cells. 6000 1U of rFactor IX were dissolved in a total of 12 mL USP
HiO. This solution was transferred to a Centricon Plus 20, PL-10 centrifugal filter with
another 6 mL USP H20. The solution was concentrated to 2 mL and then diluted with 15 mL
50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCI2, 0.05% NaN3 and then reconcentraled.
The dilution/concentration was repeated 4 times to effectively change the buffer to a final
volume of 3.0 mL. Of this solution, 2.9 mL (about 29 mg of rFactor IX) was transferred to a
small plastic tube and to it was added 530 mU a2-3,6,8-Neuraminidase- agarose conjugate
(Vibrio cholerae, Calbiochem, 450 µL). The reaction mixture was rotated gently for 26.5
hours at 32 °C. The mixture was centrifuged 2 minutes at 10,000 rpm and the supernatant
was collected. The agarose beads (containing neuraminidase) were washed 6 times with 0.5
mL 50 mM Tris-HCl pH 7.12, 1 M NaCl, 0.05% NaN3. The pooled washings and
supernatants were centrifuged again for 2 minutes at 10,000 rpm to remove any residual
agarose resin. The pooled, desialylated protein solution was diluted to 19 mL with the same
buffer and concentrated down to ~ 2 mL in a Centricon Plus 20 PL-10 centrifugal filter. The
solution was twice diluted with 15 mL of 50 mM Tris-HCl pH 7.4, 0.15 MMaCI. 0.05%
NaN3 and reconcentrated to 2 mL. The final desialyated rFactor IX solution was diluted to 3
mL final volume (-10 mg/mL) with the Tris Buffer. Native and desialylated rFactor IX
samples were analyzed by IEF-Electrophoresis. Isoelectric Focusing Gels (pH 3-7) were run
using 1.5 µL (15 ug) samples first diluted with 10 µL Tris buffer and mixed with 12 µL
sample loading buffer. Gels were loaded, run and fixed using standard procedures. Gels
were stained with Colloidal Blue Stain (Figure 154), showing a band for desialylated Factor
IX.
Preparation of PEG (1 kDa and 10 kDa)-SA-Factor IX. Desialylated rFactor-IX
(29 mg. 3 mL) was divided into two 1.5 mL (14.5 mg) samples in two 15 mL centrifuge
tubes. Each solution was diluted with 12.67 mL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl,
0.05% NaN3 and either CMP-SA-PEG-1 k or 10k (7.25 µmol) was added. The tubes were
inverted gently to mix and 2.9 U ST3Gal3 ß26 µL) was added (total volume 14.5 mL). The
tubes were inverted again and rotated gently for 65 hours at 32 °C. The reactions were
stopped by freezing at -20 °C. 10 µg samples of the reactions were analyzed by SDS-PAGE.
The PEGylated proteins were purified on a Toso Haas Biosep G3000SW (21.5 x 30 cm. 13
µm) HPLC column with Dulbecco's Phosphate Buffered Saline, pH 7.1 (Gibco), 6 mL/min.
The reaction and purification were monitored using SDS Page and IEF gels. Novex Tris-
Glycine 4-20% 1 mm gels were loaded with 10 µL (10 µg) of samples after dilution with 2
uT of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% NaN3 buffer and mixing with 12 µL
sample loading buffer and 1 µL 0.5 M DTT and heated for 6 minutes at 85 °C. Gels were
stained with Colloidal Blue Stain (Figure 155) showing a band for PEG (I kDa and 10 kDa)-
SA-Factor IX.
20. Direct Sialyl-GlvcoPEGylation of Factor IX
This example sets forth the preparation of sialyl-PEGylation of Factor IX without
prior sialidase treatment.
Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(10 KDa). Factor IX (1100
IU), which was expressed in CHO cells and was fully sialylated, was dissolved in 5 mL of 20
mM histidine, 520 mM glycine, 2% sucrose, 0.05% NaN3 and 0.01% polysorbate 80. pH 5.0.
The CMP-SA-PEG-(10 kDa) (27 mg, 2.5 µmol) was then dissolved in the solution and 1 U of
ST3Gal3 was added. The reaction was complete after gently mixing for 28 hours at 32°C.
The reaction was analyzed by SDS-PAGE as described by lnvitrogen. The product protein
was purified on an Amersham Superdex 200 (10 x 300 mm, 13 µm) HPLC column with
phosphate buffered saline. pH 7.0 (PBS). 1 mL/min. Rt = 9.5 min.
Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(20 kDa). Factor IX (1100
IU), which was expressed in CHO cells and was fully sialylated, was dissolved in 5 mL of 20
mM histidine, 520 mM glycine, 2% sucrose, 0.05% NaN3 and 0.01% polysorbate 80, pH 5.0.
The CMP-SA-PEG-(20 kDa) (50 mg, 2.3 µmol) was then dissolved in the solution and CST-
II was added. The reaction mixture was complete after gently mixing for 42 hours at 32°C.
The reaction was analyzed by SDS-PAGE as described by lnvitrogen.
The product protein was purified on an Amersham Superdex 200 (10 x 300 mm. 13
µm) HPLC column with phosphate buffered saline, pH 7.0 (Fisher), 1 mL/min. Rt = 8.6 min.
21. Sialic Acid Capping of GlycoPEGylated Factor IX
This examples sets forth the procedure for sialic acid capping of sialyl-
glycoPEGylated peptides. Here, Factor-lX is the exemplary peptide.
Sialic acid capping of N-linked and O-linked Glycans of Factor-IX-SA-PEG (10
kDa). Purified r-Factor-IX-PEG (10 kDa) (2.4 mg) was concentrated in a Centricon® Plus 20
PL-10 (Millipore Corp., Bedford, MA) centrifugal filter and the buffer was changed to 50
mM Tris-HCl pH 7.2. 0.15 M NaCl, 0.05% NaN3 to a final volume of 1.85 mL. The protein
solution was diluted with 372 uJL of the same Tris buffer and 7.4 mg CMP-SA (12 µmol) was
added as a solid. The solution was inverted gently to mix and 0.1 U ST3Gall and 0.1 U
ST3Gal3 were added. The reaction mixture was rotated gently for 42 hours at 32 °C.
A 10 µg sample of the reaction was analyzed by SDS-PAGE. Novex Tris-Glycinc 4-
12% 1 mm gels were performed and stained using Colloidal Blue as described by Invitrogen.
Briefly, samples, 10 uT (10 ug). were mixed with 12 µL sample loading buffer and 1 uf 0.5
M DTT and heated for 6 minutes at 85 °C (Figure 156. lane 4).
Factor VIIa
22. GlycoPEGylation of Recombinant Factor VIIa produced in BHK cells
This example sets forth the PEGylation of recombinant Factor VIIa made in BHK
cells.
Preparation of Asialo-Factor VIIa. Recombinant Factor VIIa was produced in
BHK cells (baby hamster kidney cells). Factor VIIa (14.2 mg) was dissolved at 1 mg/ml in
buffer solution (pl I 7.4, 0.05 M Tris, 0.15 M NaCl, 0.001 M CaCI2, 0.05%) NaN3) and was
incubated with 300 mU/mL sialidase (Vibrio cholera)-agarose conjugate for 3 days at 32 °C.
To monitor the reaction a small aliquot of the reaction was diluted with the appropriate buffer
and an IEF gel performed according to Invitrogen procedures (Figure 157). The mixture was
centrifuged at 3,500 rpm and the supernatant was collected. The resin was washed three
times 3x2 mL) with the above buffer solution ( pH 7.4, 0.05 M Tris, 0.15 M NaCl. 0.05%
NaN3) and the combined washes were concentrated in a Centricon-Plus-20. The remaining
solution was buffer exchanged with 0.05 M Tris (pH 7.4), 0.15 M NaCl, 0.05% NaN3, to a
final volume of 14.4 mL.
Preparation of Factor VIIa-SA-PEG (1 kDa and 10 kDa). The dcsialylation
rFactor VIIa solution was split into two equal 7.2 ml samples. To each sample was added
either CMP-SA-5-PEG( 1 kDa) (7.4 mg) or CMP-SA-5-PEG( 10 kDa) (7.4 mg). ST3Gal3
(1.58U) was added to both tubes and the reaction mixtures were incubated at 32°C for 96 hrs.
The reaction was monitored by SDS-PAGE gel using reagents and conditions described by
Invitrogen. When the reaction was complete, the reaction mixture was purified using a Toso
Haas TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) and collecting fractions
based on UV absorption. The combined fractions containing the product were concentrated
at 4°C in Centricon-Plus-20 centrifugal filters (Millipore, Bedford. MA) and the concentrated
solution reformulated to yield 1.97 mg (bicinchoninic acid protein assay, BCA assay, Sigma-
Aldrich. St. Louis MO) of Factor VIIa-PEG. The product of the reaction was analyzed using
SDS-PAGE and 1EF analysis according to the procedures and reagents supplied by
Invitrogen. Samples were dialyzed against water and analyzed by MALDI-TOF. Figure 158
shows the MALDI results for native Factor VIIa. Figure 159 contains the MALD1 results for
Factor VIIa PEGyiated with 1 kDa PEG where peak of Factor VIIa PEGylated with IKDa
PEG is evident. Figure 160 contains the MALDI results for Factor VIIa PEGylated with 10
kDa PEG where a peak for Factor VIIa PEGylated with 10 kDa PEG is evident. Figure 161
depicts the SDS-PAGE analysis of all of the reaction products, where a band for Factor VIIa-
SA-PEG (10 kDa) is evident.
Follicle Stimulating Hormone (FSH)
23. GlycoPEGylation of human pituitary-derived FSH
This example illustrates the assembly of a conjugate of the invention. Follicle
Stimulating Hormone (FSH) is desialylated and then conjugated with CMP-(sialic acid)-PEG.
Desialylation of Follicle Stimulating Hormone. Follicle Stimulating Hormone
(FSH) (Human Pituitary. Calbiochem Cat No. 869001), 1 mg, was dissolved in 500 µL 50
mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCI2. This solution, 375 µL, was transferred to a
small plastic tube and to it was added 263 mU Neuraminidase II (Vibrio cholerae). The
reaction mixture was shaken gently for 15 hours at 32 °C. The reaction mixture was added to
N-(p-aminophenyl)oxamic acid-agarose conjugate, 600 µL, pre-equilibrated with 50 mM
Tris-HCl pH 7.4, 150 mM NaCl and 0.05% NaN3 and gently rotated 6.5 hours at 4 °C. The
suspension was centrifuged for 2 minutes at 14,000 rpm and the supernatant was collected.
The beads were washed 5 times with 0.5 mL of the buffer and all supernatants were pooled.
The enzyme solution was dialyzed (7000 MWCO) for 15 hours at 4 °C with 2 L of a solution
containing 50 mM Tris -HC1 pH 7.4, 1 M NaCl, 0.05% NaN3, and then twice for 4 hours at 4
°C into 50 mM Tris -HCl pH 7.4, 1 M NaCl, 0.05% NaN3. The solution was concentrated to
2 µg/ µL by Speed Vac and stored at -20 °C. Reaction samples were analyzed by 1EF gels
(pH 3-7) (Invitrogen) (Figure 162).
Preparation of human pituitary-derived SA-FSH and PEG-SA-Follicle
Stimulating Hormone. Desialylated FSH (100 µg, 50 µL) and CMP-sialic acid or CMP-SA-
PEG (I kDa or 10 kDa) (0.05 µmol) were dissolved in 13.5 µL H20 (adjusted to pH 8 with
NaOH) in 0.5 mL plastic tubes. The tubes were vortexed briefly and 40 mU ST3Gal3 36.5 µL) was added (total volume 100 µL). The tubes were vortexed again and shaken gently for
24 hours at 32 °C. The reactions were stopped by freezing at -80 °C. Reaction samples of 15 µg were analyzed by SDS-PAGE (Figure 163), IEF gels (Figure 164) and MALDI-TOF.
Native FSH was also analyzed by SDS-PAGE (Figure 165)
Analysis of SDS PAGE and IEF Gels of Reaction Products. Novex Tris-Glycine
8-16% 1 mm gels for SDS PAGE analysis were purchased from Invitrogen. 7.5 µL (15 ug)
of FSH reaction samples were diluted with 5 µL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl.
0.05% NaN3 buffer, mixed with 15 µL sample loading buffer and I µL 9 M µ-
mercaptoethanol and heated for 6 minutes at 85 °C. Gels were run as directed by Invitrogen
and stained with Colloidal Blue Stain (Invitrogen).
FSH samples (15 µg) were diluted with 5 µL Tris buffer and mixed with 15 µL
sample loading buffer (Figure 162). The samples were then applied to Isoelectric Focusing
Gels (pH 3-7) (Invitrogen) (Figure 165). Gels were run and fixed as directed by Invitrogen
and then stained with Colloidal Blue Stain.
24. GlycoPEGylation of recombinant FSH produced recombinantly in CHQ
cells
This example illustrates the assembly of a conjugate of the invention. Dcsialylated
FSH was conjugated with CMP-(sialic acid)-PEG.
Preparation of recombinant Asialo-Follicle Stimulation Hormone. Recombinant
Follicle Stimulation Hormone (rFSH) produced from CHO was used in these studies. The
7,500 IU of rFSH was dissolved in 8 mL of water. The FSH solution was dialyzed in 50 mM
Tris-HCl pH 7.4. 0.15 M NaCl, 5 mM CaCl2 and concentrated to 500 µL in a Centricon Plus
20 centrifugal filter. A portion of this solution (400 µL) (~ 0.8 mg FSH) was transferred to a
small plastic tube and to it was added 275 mU Neuraminidase II (Vibrio cholerae). The
reaction mixture was mixed for 16 hours at 32 °C. The reaction mixture was added to
prewashed N-(p-aminophenyl)oxamic acid-agarose conjugate (800 µL) and gently rotated for
24 hours at 4 °C. The mixture was centrifuged at 10,000 rpm and the supernatant was
collected. The beads were washed 3 times with 0.6 mL Tris-EDTA buffer, once with 0.4 mL
Tris-EDTA buffer and once with 0.2 mL of the Tris-EDTA buffer and all supematants were
pooled. The supernatant was dialyzed at 4 °C against 2 L of 50 mM Tris -HCl pH 7.4, 1 M
NaCl, 0.05% NaN3 and then twice more against 50 mM Tris -HCl pH 7.4. 1 M NaCl. 0.05%
NaN3. The dialyzed solution was then concentrated to 420 µL in a Centricon Plus 20
centrifugal filter and stored at -20 °C.
Native and desialylated rFSH samples were analyzed by SDS-PAGE and 1EF (Figure
166). Novex Tris-Glycine 8-16% 1 mm gels were purchased from Invitrogen. Samples (7.5 µL. 15 µg) samples were diluted with 5 µL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl.
0.05% NaN3 buffer, mixed with 15 uJL sample loading buffer and 1 \iL 9 M ß-
mercaptoethanol and heated for 6 minutes at 85 °C. Gels were run as directed by Invitrogen
and stained with Colloidal Blue Stain (Invitrogen). Isoelectric Focusing Gels (pH 3-7) were
purchased from Invitrogen. Samples (7.5 µL, 15 µg) were diluted with 5 µL Tris buffer and
mixed with 15 µL sample loading buffer. Gels were loaded, run and fixed as directed by
Invitrogen. Gels were stained with Colloidal Blue Stain. Samples of native and desialylated
FSH were also dialyzed against water and analyzed by MALDI-TOF.
Sialyl-PECiylation of recombinant Follicle Stimulation Hormone. Desialylated
FSH (100 µg, 54 µL) and CMP-SA-PEG (1 kDa or 10 kDa) (0.05 µmoL) were dissolved in 28
M-L 50 mM Tris-HCl. 0.15 M NaCl. 0.05% NaN3, pH 7.2 in 0.5 mL plastic tubes. The tubes
were vortexed briefly and 20 mU of ST3Gal3 was added (total volume 100 µL). The tubes
were vortexed again, mixed gently for 24 hours at 32 °C and the reactions stopped by freezing
at -80 °C. Samples of this reaction were analyzed as described above by SDS-PAGE gels
(figure 167). IFF gels (Figure 168) and MALDI-TOF MS.
MALD1 was also performed on the PEGylated rFSH. During ionization. SA-PEG is
eliminated from the N-glycan structure of the glycoprotein. Native FSH gave a peak at
13928; AS-rFSI I (13282); rcsialylated r-FSH (13332); PEG 1000-rFSH (13515;!4960 (1);
16455(2); 17796(3): 19321 (4)); and PEG 10000 (23560 (1); 34790 (2); 45670 ß); and
56760 (4)).
25. Pharmacokinetic Study of GlvcoPEGvlated FSH
This example sets forth the in vivo testing of the pharmacokinetic properties
glycoPEGylated Follicle Stimulating Hormone (FSH) prepared according to the methods of
the invention as compared to non-PFGylated FSH.
FSH, FSH-SA-PEG (1 kDa) and FSH-SA-PEG (10 kDa) were radioiodinated using
standard conditions (Amersham Biosciences, Arlington Heights, 1L) and formulated in
phosphate buffered saline containing 0.1% BSA. After dilution in phosphate buffer to the
appropriate concentration, each of the test FSH proteins (0.4 ^ig, each) was injected
intraveneously into female Sprague Dawley rats (250-300 g body weight) and blood drawn at
time points from 0 to 80 hours. Radioactivity in blood samples was analyzed using a gamma
counter and the pharmacokinetics analyzed using standard methods (Figure 169). FSH was
cleared from the blood much more quickly than FSH-PEG(1 kDa), which in tµm was clear
somewhat more quickly than FSH-PEG(10 kDa).
26. Sertoli Cell Bioassay for In Vitro Activity of GlycoPEGylated FSH
This example sets forth a bioassay for follicle stimulating hormone (FSH) activity
based on cultured Sertoli cells. This assay is useful to determine the bioactivily of FSHI after
glycan remodeling, including glycoconjugation.
This bioassay is based on the dose-response relationship that exists between the
amount of estradiol produced when FSH, but not lutenizing hormone (EH), is added to
cultured Sertoli cells obtained from immature old rats. Exogenous testosterone is converted
to 17ß-estradiol in the presence of FSH.
Seven to 10 days old Sprague-Dawley rats were used to obtain Sertoli cells. After
sacrifice, testes were decapsulated and tissue was dispersed by incubation in collagenase (1
mg/ml), trypsin (lmg/ml), hyaluronidase (1 mg/ml) and DNases (5 µg/ml) for 5 to 10 min.
The tubule fragments settled to the bottom of the flask and were washed in PBS (1 x). The
tubule fragments were reincubated for 20 min with a media containing the same enzymes:
collagenase (1 mg/ml). trypsin (lmg/ml). hyaluronidase (1 mg/ml) and DNases (5 µg/ml).
The tubule fragments were homogenized and plated into a 24 well plate in a serum
free media. 5x105 cells were dispersed per well. After 48h incubation at 37° C and 5%
CO2. fresh media was added to the cells. Composition of the serum free media: DMEM (1
vol), Ham's F10 nutrient mixture (1 vol), insulin 1 µg/ml. Transferrin 5 µg/ml, EGF 10
ng/ml. T4 20 pg/ml. Hydrocortisone 10-8M, Retinoic acid 10-6M.
The stimulation experiment consists of a 24 hour incubation with standard FSH or
samples at 37°C and 5% C02 The mean intra-assay coefficient of variation is 9% and the
mean inter-assay coefficient of variation is 11%.
The 17B-estradiol Elisa Kit DE2000 (R&D Systems, Minneapolis, MN) was used to
quantify the level of estradiol after incubation with FSH. FSH-SA-PEG (1 kDa) and FSH-
SA-PEG(lOkDa).
The procedure was as follows: 100 µl of Estradiol Standard (provided with kit and
prepared as per instructions with kit) or sample was pipetted into wells of 17B-estradiol Elisa
plate(s); 50 µl of 17B-estradiol Conjugate (provided with kit, prepared as per instructions
with kit) was added to each well; 50 µl of 17B-estradiol antibody solution (provided with kit
and prepared as per instructions with kit) was added to each well; plates were incubated for 2
hour at room temperature at 200 rpm; the liquid was aspirated from each well; the wells were
washed 4 times using the washing solution; all the liquid was removed from the wells; 200 µl
of pNPP Substrate (provided with kit and prepared as per instructions with kit) was added to
all wells and incubated for 45 min; 50 µl of Stop solution (provided with kit and prepared as
per instructions with kit) was added and the plates were read it at 405 nm (Figure 170).
While FSH-PEG( 10 kDa) exhibited a modest stimulation of Sertoli cells, at 1 µg/ml, FSII-
PEG(1 kDa) stimulated Sertoli cells up to 50% more than unPEGylated FSH.
27. Steelman-Pohley Bioassay of In Vivo Activity ot'GlycoPEGvlatcd FSH
In this example, the Steelman-Pohley bioassay (Steelman and Pohley. 1953,
Endocrinology 53:604-615) was used to determine the in vivo activity of glycoPEGylated
FSH. The Steelman-Pohley assay uses the change in ovary weight of a rat to measure the in
vivo activity of FSII that is coinjected with human chorionic gonadotropin.
The Steelman-Pohley bioassay was performed according to the protocol described in
Christin-Maitre et al. (2000, Methods 21:51 -57). Seventy female Sprague-Dawley Rats
(Charles River Laboratories, Wilmington, MA), aged 21 to 22 days, were housed in the
testing facility for at least 5 days before the beginning the assay procedure. Throughout the
procedure, the animal room was climate controlled at 18 to 26°C, 30 to 70% relative
humidity, and 12 hr. artificial light/12 hr. dark. All animals were fed Certified Rodent Chow
(Harlan Teklad, Madison WI) or the equivalent, and water, both ad libitum. Animal
procedures were performed at Calvert Preclinical Services, Inc. (Olyphant, PA).
Recombinant FSH was expressed in CHO cells, purified by standard techniques and
glycoPEGylated with PEG (1 kDa). The rats were divided into seven test groups, with ten
animals per group. On days -1 and 0, animals of all groups were subcutaneously injected
with 20 I.U. of human chorionic gonadotropin (HCG) in 0.5 ml of 0.9 % NaCl. On days 1, 2
and 3, the control animals were subcutaneously injected with a dose of 0.5 ml containing 20
I.U. HCG in 0.9% NaCl. while in the other groups, the HCG dose was augmented with either
rFSH or rFSH-SA-PEG (1 kDa) at either 0.14 µg, 0.4 µg or 1.2 µg per dose. On day 4, the
animals were euthanized by CO2 inhalation. The ovaries were removed, trimmed and
weighted. The average ovary weight was determined for each group.
Figure 171 presents the average ovary weight of the test groups on day 4. The groups
receiving HCG alone (control) or the low dose (0.14 µg) of either rFSH or rFSH-SA-PEG (1
kDa) had ovary weights that were roughly equivalent. The groups receiving the medium (0.4 µg) or high (1.2 µg) doses of rFSH or rFSH-SA-PEG (1 kDa) had ovary weights roughly
twice that of the control group. At the medium dose (0.4 µg), the glycoPEGylated rFSH had
roughly the same in vivo activity (as determined by ovary weight) as the unPEGylated rFSH.
At the high dose (1.2 µg), the glycoPEGylated rFSH had somewhat higher in vivo activity
than the unPEGylated rFSH.
G-CSF
2.8,. GlycoPEGylation of G-CSF produced in CHO cells
Preparation of Asialo-Granulocyte-Colony Stimulation Factor (G-CSF). G-CSF
produced in CHO cells is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4,
0.15 M NaCl. 5 mM CaCl2 and concentrated to 500 µL in a Centricon Plus 20 centrifugal
filter. The solution is incubated with 300 mU/mL Neuraminidase II (Vibrio cholerae) for 16
hours at 32 °C. To monitor the reaction a small aliquot of the reaction is diluted with the
appropriate buffer and a IFF gel performed. The reaction mixture is then added to prewashcd
N-(p-aminophenyl)oxamic acid-agarose conjugate (800 µL/mL reaction volume) and the
washed beads gently rotated for 24 hours at 4 °C. The mixture is centrifuged at 10,000 rpm
and the supernatant was collected. The beads are washed 3 times with Tris-EDTA buffer,
once with 0.4 mL Tris-EDTA buffer and once with 0.2 mL of the Tris-EDTA buffer and all
supernatants are pooled. The supernatant is dialyzed at 4 °C against 50 mM Tris -HO pH
7.4, 1 M NaCl, 0.05% NaN3 and then twice more against 50 mM Tris -HO pH 7.4. I M
NaCl, 0.05% NaN3,. The dialyzed solution is then concentrated using a Centricon Plus 20
centrifugal filter and stored at -20 °C. The conditions for the IEF gel were run according to
the procedures and reagents provided by Invitrogen. Samples of native and desialylated G-
CSF are dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of G-CSF-(alpha2,3)-Sialyl-PEG. Desialylated G-CSF was dissolved
at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3, pH 7.2. The solution is
incubated with 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST3Gall at 32°C for 2 days.
To monitor the incorporation of sialic acid-PEG, a small aliquot of the reaction had CMP-
SA-PEG-fluorescent ligand added; the label incorporated into the peptide is separated from
the free label by gel filtration on a Toso Haas G3000SW analytical column using PBS buffer
(pH 7.1). The fluorescent label incorporation into the peptide is quantitated using an in-line
fluorescent detector. After 2 days, the reaction mixture is purified using a Toso Haas
G3000SW preparative column using PBS buffer (pH 7.1) and collecting fractions based on
UV absorption. The product of the reaction is analyzed using SDS-PAGE and IEF analysis
according to the procedures and reagents supplied by Invitrogen. Samples of native and
PEGylated G-CSF arc dialyzed against water and analyzed by MALD1-TOF MS.
Preparation of G-CSF-(alpha2,8)-Sialyl-PEG. G-CSF produced in CHO cells,
which contains an alpha2,3-sialylated O-linked glycan, is dissolved at 2.5 mg/ml. in 50 mM
Tris-HCl, 0.15 M NaCl, 0.05% NaN3, pH 7.2. The solution is incubated with 1 mM CMP-
sialic acid-PEG and 0.1 U/mL of CST-1I at 32°C for 2 days. To monitor the incorporation of
sialic acid-PEG, a small aliquot of the reaction has CMP-SA-PFlG-fluorescent ligand added:
the label incorporated into the peptide is separated from the free label by gel filtration on a
Toso Haas G3000SW analytical column using PBS buffer (pH 7.1). The fluorescent label
incorporation into the peptide is quantitated using an in-line fluorescent detector. After 2
days, the reaction mixture is purified using a Toso Haas G3000SW preparative column using
PBS buffer (pH 7.1) and collecting fractions based on UV absorption. The product of the
reaction is analyzed using SDS-PAGE and IEF analysis according to the procedures and
reagents supplied by Invitrogen. Samples of native and PEGylated G-CSF are dialyzed
against water and analyzed by MALDI-TOF MS.
Preparation of G-CSF-(alpha2,6)-Sialyl-PEG. G-CSF, containing only O-linked
GalNAc, is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3, pH 7.2.
The solution is incubated with I mM CMP-sialic acid-PEG and 0.1 U/mL of ST6GalNAcI or
II at 32°C for 2 days. To monitor the incorporation of sialic acid-PEG, a small aliquot of the
reaction has CMP-SA-PEG-fluorescent ligand added; the label incorporated into the peptide
is separated from the free label by gel filtration on a Toso Haas G3000SW analytical column
using PBS buffer (pH 7.1). The fluorescent label incorporation into the peptide is quantitated
using an in-line fluorescent detector. After 2 days, the reaction mixture is purified using a
Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and collecting fractions
based on UV absorption. The product of the reaction is analyzed using SDS-PAGE and IEF
analysis according to the procedures and reagents supplied by Invitrogen. Samples of native
and PEGylated G-CSF are dialyzed against water and analyzed by MALDI-TOF MS.
G-CSF produced in CHO cells was treated with Arthrobacter sialidase and was then
purified by size exclusion on Superdex75 and was treated with ST3Gall or ST3 Gal2 and
then with CMP-SA-PEG 20Kda. The resulting molecule was purified by ion exchange and
gel filtration and analysis by SDS PAGE demonstrated that the PEGylation was complete.
This is the first demonstration of glycoPEGylation of an O-linkcd glycan.
Glucocerebrosidase
29. Glucocerebrosidase-mannose-6-phosphate produced in CHO cells
This example sets forth the procedure to glycoconjugate mannose-6-phosphate to a
peptide produced in CHO cells such as glucocerebrosidase.
Preparation of asialo-glucoceramidase. Glucocerebrosidase produced in CHO cells
is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, and is
incubated with 300 mlJ/mL sialidase-agarose conjugate for 16 hours at 32 °C. To monitor
the reaction a small aliquot of the reaction is diluted with the appropriate buffer and a IFF gel
and SDS-PAGE performed according to Invitrogen procedures. The mixture is centrifuged at
10,000 rpm and the supernatant is collected. The beads are washed 3 times with Tris-FDTA
buffer, once with 0.4 mL Tris-EDTA buffer, and once with 0.2 mL of the Tris-EDTA buffer.
All supernatants are pooled. The supernatant is dialyzed at 4 °C against 50 mM Tris-HCl pH
7.4. 1 M NaCl, 0.05% NaN3 and then twice more against 50 mM Tris-HCl pH 7.4, 1 M
NaCl, 0.05% NaN3. The dialyzed solution is then concentrated using a Centricon Plus 20
centrifugal filter. The product of the reaction is analyzed using SDS-PAGE and 1EF analysis
according to the procedures and reagents supplied by Invitrogen. Samples are dialyzed
against water and analyzed by MALDI-TOF MS.
Preparation of Glucocerebrosidase-SA-linker-Mannose-6-phosphate (procedure
1). Asialo-glucocerebrosidasefrom above is dissolved at 2.5 mg/mL in 50 mM Tris-HCl.
0.15 M NaCl, 0.05% NaN3. pH 7.2. The solution is incubated with 1 mM CMP-sialic acid-
linker-Man-6-phosphate and 0.1 U/ml. of ST3Gal3 at 32°C for 2 days. To monitor the
incorporation of sialic acid-linker-Man-6-phosphate, a small aliquot of the reaction had CMP-
SA-PEG-fluorescent ligand added; the label incorporated into the peptide is separated from
the free label by gel filtration on a Toso Haas TSK-Gel-3000 analytical column using PBS
buffer (pH 7.1). The fluorescent label incorporation into the peptide is quantitated using an
in-line fluorescent detector. When the reaction is complete, the reaction mixture is purified
using a Toso Haas TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) and
collecting fractions based on UV absorption. The product of the reaction is analyzed using
SDS-PAGE and IFF analysis according to the procedures and reagents supplied by
Invitrogen. Samples are dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of Glucocerebrosidase-SA-linker-Mannose-6-phosphate (procedure
2). Glucocerebrosidase. produced in CHO but incompletely sialylated, is dissolved at 2.5
mg/mL in 50 mM Tris-HCl. 0.15 M NaCl, 0.05% NaN3, pH 7.2. The solution is incubated
with 1 mM CMP-sialic acid-linker-Man-6-phosphate and 0.1 U/mL of ST3Gal3 at 32°C for 2
days. To monitor the incorporation of sialic acid-linker-Man-6-phosphate, a small aliquot of
the reaction had CMP-SA-PEG-fluorescent ligand added; the label incorporated into the
peptide is separated from the free label by gel filtration on a Toso Haas TSK-Gel-3000
analytical column using PBS buffer (pH 7.1). The fluorescent label incorporation into the
peptide is quantitated using an in-line fluorescent detector. When the reaction is complete,
the reaction mixture is purified using a Toso Haas TSK-Gel-3000 preparative column using
PBS buffer (pH 7.1) and collecting fractions based on UV absorption. The product of the
reaction is analyzed using SDS-PAGE and IEF analysis according to the procedures and
reagents supplied by Invitrogen. Samples are dialyzed against water and analyzed by
MALDI-TOF MS.
30. Glucocerebrosidase-transferrin
This example sets forth the procedures for the glycoconjugation of proteins, and in
particular, transferrin is glycoconjugated to glucocerebrosidase. The GlcNAc-ASN structures
are created on glucoceraminidase, and Transferrin-SA-Linker-Gal-UDP is conjugated to
GNDF GlcNAc-ASN structures using galactosyltransferase.
Preparation of GlcNAc-giucocerebrosidase (Cerezyme™). Cerezyme'M
(glucocerebrosidase) produced in CHO cells is dissolved at 2.5 mg/mL in 50 mM Iris 50 mM
Tris-HCl pH 7.4, 0.15 M NaCl, and is incubated with 300 mU/mL Fndo-II-agarose conjugate
for 16 hours at 32 °C. To monitor the reaction a small aliquot of the reaction is diluted with
the appropriate buffer and a IEF gel and SDS-PAGE performed according to Invitrogen
procedures. The mixture is centrifuged at 10,000 rpm and the supernatant is collected. The
beads are washed 3 times with Tris-EDTA buffer, once with 0.4 mL Tris-EDTA buffer and
once with 0.2 mL of the Tris-EDTA buffer and all supernatants are pooled. The supernatant
is dialyzed at 4 °C against 50 mM Tris -HC1 pH 7.4, 1 M NaCl, 0.05% NaN3 and then twice
more against 50 mM Iris -HC1 pH 7.4. 1 M NaCl. 0.05% NaN3. The dialyzed solution is
then concentrated using a Centricon Plus 20 centrifugal filter. The product of the reaction is
analyzed using SDS-PAGE and 1EF analysis according to the procedures and reagents
supplied by Invitrogcn. Samples are dialyzed against water and analyzed by MALD1-TOF
MS.
Preparation of Transferrin-SA-Linker-Gal-glucocerebrosidase. Transferrin-SA-
Linker-Gal-UDP from above is dissolved at 2.5 mg/mL in 50 mM Tris-HCl. 0.15 M NaCl. 5
mM MnCl2, 0.05% NaN3, pH 7.2. The solution is incubated with 2.5 mg/mL GlcNAc-
glucocerebrosidaseand 0.1 U/mL of galactosyltransferase at 32°C for 2 days. To monitor the
incorporation of glucocerebrosidase, the peptide is separated by gel filtration on a Toso Haas
G3000SW analytical column using PBS buffer (pH 7.1) and the product detected by UV
absorption. The reaction mixture is then purified using a Toso Haas G3000SW preparative
column using PBS buffer (pH 7.1) collecting fractions based on UV absorption. The product
of the reaction is analyzed using SDS-PAGE and IEF analysis according to the procedures
and reagents supplied by Invitrogen. Samples are dialyzed against water and analyzed by
MALDI-TOF MS.
GM-CSF
31. Generation and PEGylation of GlcNAc-ASN Structures: GM-CSF
produced in Saccharomyces
This example sets forth the preparation of Tissue-type Activator with PEGylated
GlcNAc-Asn structures.
Recombinant GM-CSF expressed in yeast is expected to contain 2 N-linked and 2 O-
linked glycans. The N-linked glycans should be of the branched mannan type. This
recombinant glycoprotein is treated with an endoglycosidase from the group consisting of
endoglycosidase H. endoglycosidase-Fl, endoglycosidase-F2, endoglycosidase-F3,
endoglycosidase-M either alone or in combination with mannosidases I, II and III to generate
GlcNAc nubs on the asparagine (Asn) residues on the peptide/protein backbone.
The GlcNAc-Asn structures on the peptide/protein backbone is then be modified with
galactose or galactosc-PFG using UDP-galactose or UDP-galactose-6-PEG. respectively, and
a galactosyltransferase such as GalTl. In one case the galactose-PEG is the terminal residue.
In the second case the galactose is further modified with SA-PEG using a CMP-SA-PKG
donor and a sialyltransferase such as ST3GalIII. In another embodiment the GlcNAc-Asn
structures on the pcptide/protein backbone can be galactosylated and sialylated as described
above, and then further sialylated using CMP-SA-PEG and an a2,8-sialyltranferase such as
the enzyme encoded by the Campylobacter jejuni cst-II gene.
Herceptin™
32. Glvcoconjugation of mithramycin to Herceptin™
This example sets forth the procedures to glycoconjugatc a small molecule, such as
mithramycin to Fc region glycans of an antibody molecule produced in mammalian cells.
Here, the antibody 1 Icrceptin™ is used, but one of skill in the art will appreciate that the
method can be used with many other antibodies.
Preparation of Herceptin™-Gal-linker-mithramycin. Herceptin™ is dissolved at
2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl2, 0.05% NaN3, pH 7.2. The
solution is incubated with I mM UDP-galactose-linker-mithramycin and 0.1 U/mL of
galactosyltransferase at 32°C for 2 days to introduce the mithramycin in the Fc region
glycans. To monitor the incorporation of galactose, a small aliquot of the reaction has l4C-
galactose-UDP ligand added; the label incorporated into the peptide is separated from the free
label by gel filtration on a Toso Haas G3000SW analytical column using PBS buffer (pH
7.1). The radioactive label incorporation into the peptide is quantitated using an in-line
radiation detector.
When the reaction is complete, the reaction mixture is purified using a Toso Haas
TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) and collecting fractions based
on UV absorption. The fractions containing product are combined, concentrated, buffer
exchanged and then freeze-dried. The product of the reaction is analyzed using SDS-PAGE
and IEF analysis according to the procedures and reagents supplied by Invitrogen. Samples
are dialyzed against water and analyzed by MALDI-TOF MS.
Interferon a and Interferon ß
33. GlycoPEGylation of Proteins expressed in Mammalian or Insect Systems:
EPO, Interferon a and Interferon ß
This example sets forth the preparation of PEGylated peptides that are expressed in
mammalian and insect systems.
Preparation of acceptor from mammalian expression systems. The peptides to be
glycoPEGylated using CMP-sialic acid PEG need to have glycans terminating in galactose.
Most peptides from mammalian expression systems will have terminal sialic acid that first
needs to be removed.
Sialidase digestion. The peptide is desialylated using a sialidase. A typical
procedure involves incubating a 1 mg/mL solution of the peptide in Tris-buffered saline. pH
7.2. with 5 mM CaCl2 added, with 0.2 U/mL immobilized sialidase from Vibrio cholera
(Calbiochem) at 32°C for 24 hours. Microbial growth can be halted either by sterile filtration
or the inclusion of 0.02% sodium azidc. The resin is then removed by centrifugation or
filtration, and then washed to recover entrapped peptide. At this point, EDTA may be added
to the solution to inhibit any sialidase that has leached from the resin.
Preparation from insect expression systems. EPO, interferon-alpha, and
inlerferon-beta may also be expressed in non-mammalian systems such as yeast, plants, or
insect cells. The peptides to be glycoPEGylated using CMP-sialic acid PEG need to have
glycans terminating in galactose. The majority of the N-glycans on peptides expressed in
insect cells, for example, are the trimannosyl core. These glycans are first built out to
glycans terminating in galactose before they are acceptors for sialyltransferase.
Building acceptor glycans from trimannosyl core. Peptide (1 mg/mL) in Tris-
buffered saline, pH 7.2, containing 5 mM MnCl2, 5 mM UDP-glcNAc, 0.05 U/mL
GLCNACT I, 0.05 U/mL GLCNACT II, is incubated at 32°C for 24 hours or until the
reaction is substantially complete. Microbial growth can be halted either by sterile filtration
or the inclusion of 0.02% sodium azide. After buffer exchange to remove UDP and other
small molecules,, UDP-galactose and MnCl2 are each added to 5 mM, galactosyltransferase is
added to 0.05 U/mL, and is incubated at 32°C for 24H or until the reaction is substantially
complete. Microbial growth can be halted either by sterile filtration or the inclusion of 0.02%
sodium azide. The peptides are then ready for glycoPEGylation.
Building O-linked glycans. A similar strategy may be employed for interferon alpha
to produce enzymatically the desired O-glycan Gal-GalNAc. If necessary, GalNAc linked to
serine or threonine can be added to the peptide using appropriate peptide GalNAc
transferases (e.g. GalNAc Tl, GalNAc T2, T3, T4, etc.) and UDP-GalNAc. Also, if needed,
galactose can be added using galactosytransferase and UDP-galactose.
GlycoPEGylation using sialyltransferase. The glycopeptides (1 mg/mL) bearing
terminal galactose in Iris buffered saline + 0.02% sodium azide are incubated with CMP-SA-
PEG (0.75 mM) and 0.4 U/mL sialyltransferase (ST3Gal3 or ST3Gal4 for N-glycans on KPO
and interferon beta: ST3Gal4, or ST3Gall for O-glycans on interferon alpha) at 32°C for 24
hours. Other transferases that may work include the 2,6 sialyltransferase from
Photobacterium damsella. The acceptor peptide concentration is most preferably in the range
of 0.1 mg/mL up to the solubility limit of the peptide. The concentration of CMP-SA-PEG
should be sufficient for there to be excess over the available sites, but not so high as to cause
peptide solubility problems due to the PEG, and may range from 50 µM up to 5 mM, and the
temperature may range from 2°C up to 40°C. The time required for complete reaction will
depend on the temperature, the relative amounts of enzyme to acceptor substrate, the donor
substrate concentration, and the pH.
34. GlycoPEGylation of Interferon a produced in CHO cells
Preparation of Asialo-Interferon a. Interferon alpha produced from CHO cells is
dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mM CaCl2
and concentrated to 500 µL in a Centricon Plus 20 centrifugal filter. The solution is
incubated with 300 mU/mL Neuraminidase II {Vibrio cholerae) for 16 hours at 32 °C. To
monitor the reaction a small aliquot of the reaction is diluted with the appropriate buffer and a
IEF gel performed. The reaction mixture is then added to prewashed N-(p-
aminophenyl)oxamic acid-agarose conjugate (800 |nL/mL reaction volume) and the washed
beads gently rotated for 24 hours at 4 °C. The mixture is centrifuged at 10.000 rpm and the
supernatant was collected. The beads are washed 3 times with Tris-EDTA buffer, once with
0.4 mL Tris-EDTA buffer and once with 0.2 mL of the Tris-EDTA buffer and all
supernatants were pooled. The supernatant is dialyzed at 4 °C against 50 mM Tris - HC1 pH
7.4. 1 M NaCl. 0.05% NaN3 and then twice more against 50 mM Tris -HCl pH 7.4. 1 M
NaCl, 0.05% NaN3. The dialyzed solution is then concentrated using a Centricon Plus 20
centrifugal filter and stored at -20 °C. The conditions for the IEF gel are run according to the
procedures and reagents provided by Invitrogen. Samples of native and desialylated G-CSF
are dialyzed against water and analyzed by MALD1-TOF MS.
Preparation of lnterferon-alpha-(alpha2,3)-Sialyl-PEG. Desialylated interferon-
alpha is dissolved at 2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3. pH 7.2.
The solution is incubated with 1 mM CMP-sialic acid-PEG and 0.1 U/mL of ST3Gall at
32ºC for 2 days. To monitor the incorporation of sialic acid-PEG, a small aliquot of the
reaction had CMP-SA-PEG-fluorescent ligand added; the label incorporated into the peptide
is separated from the free label by gel filtration on a Toso Haas G3000SW analytical column
using PBS buffer (pFI 7.1). The fluorescent label incorporation into the peptide is quantitated
using an in-line fluorescent detector. After 2 days, the reaction mixture is purified using a
Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and collecting fractions
based on UV absorption. The product of the reaction is analyzed using SDS-PAGE and IFF
analysis according to the procedures and reagents supplied by Invitrogen. Samples of native
and desialylated Interferon-alpha are dialyzed against water and analyzed by MALDI-TOF
MS.
Preparation of Interferon-alpha-(alpha2,8)-Sialyl-PEG. Interferon-alpha
produced in CHO, which contains an alpha2,3-sialylated O-linked glycan, is dissolved at 2.5
mg/mL in 50 mM Tris-HCl. 0.15 M NaCl, 0.05% NaN3, pH 7.2. The solution is incubated
with 1 mM CMP-sialic acid-PEG and 0.1 U/mL of CST-II at 32°C for 2 days. To monitor the
incorporation of sialic acid-PEG, a small aliquot of the reaction has CMP-SA-PEG-
fluorescent ligand added: the label incorporated into the peptide is separated from the free
label by gel filtration on a Toso Haas G3000SW analytical column using PBS buffer (pH
7.1). The fluorescent label incorporation into the peptide is quantitated using an in-line
fluorescent detector. After 2 days, the reaction mixture is purified using a Toso Haas
G3000SW preparative column using PBS buffer (pH 7.1) and collecting fractions based on
UV absorption. The product of the reaction is analyzed using SDS-PAGE and IEF analysis
according to the procedures and reagents supplied by Invitrogen. Samples of native and
PEGylated interferon-alpha are dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of lnterferon-alpha-(alpha2,6)-Sialyl-PEG. Interferon-alpha,
containing only O-linked GalNAc, was dissolved at 2.5 mg/mL in 50 mM Tris-HCl. 0.15 M
NaCl, 0.05% NaN3. pi I 7.2. The solution is incubated with 1 mM CMP-sialic acid-PEG and
0.1 U/mL of ST6GalNAcl or II at 32°C for 2 days. To monitor the incorporation of sialic
acid-PEG, a small aliquot of the reaction had CMP-SA-PEG-fluorescent ligand added; the
label incorporated into the peptide is separated from the free label by gel filtration on a Toso
Haas G3000SW analytical column using PBS buffer (pH 7.1). The fluorescent label
incorporation into the peptide is quantitated using an in-line fluorescent detector. After 2
days, the reaction mixture is purified using a Toso Haas G3000SW preparative column using
PBS buffer (pH 7.1) and collecting fractions based on UV absorption. The product of the
reaction is analyzed using SDS-PAGE and IEF analysis according to the procedures and
reagents supplied by Invitrogen. Samples of native and PEGylated interferon-alpha are
dialyzed against water and analyzed by MALDI-TOF MS.
35. GlycoPEGvlation of interferon-ß-1a with PEG (10 kDa) and PEG (20
kDa)
This example illustrates a procedure PEGylate Interferon-p with either PEG (10 kDa)
or PEG (20 kDa).
Briefly. Interferon-ß-la (INF-P) was obtained from Biogen (Avonex™). The 1FN-P
was first purified by Superdex-75 chromatography. The IFN-ß was then desialylated with
Vibrio cholerae sialidase. The INF-P was then PEGylated with SA-PEG (10 kDa) or SA-
PEG (20 kDa) and purified with Superdex-200 chromatography.
Superdcx-75 chromatography purification. INF-P (150 µg) was applied to a
Supcrdcx-75 column (Amersham Biosciences, Arlington Heights. IL) and eluted with PBS
with 0.5 M NaCl. 0.02 Tween-20, 20 mM histidine and 10% glycerol. The eluant was
monitored for absorbance at 280 nm (Figure 172A and 172B) and fractions were collected.
Peaks 4 and 5 were pooled, concentrated in an Amicon Ultra 15 spin filter (Millipore,
Billerica, MA), and the buffer was exchanged to TBS with 5 mM CaCI2, 0.02% Tween-20,
20 mM histidine and 10% glycerol.
Sialidase Reaction. The INF-ß was then desialydated with Vibrio cholera salidase
(70 mil/ml. CALBIOCHEM®, EMD Biosciences. Inc.. San Diego. CA) on agarose in TBS
with 5 mM CaCl2, 0.02% Tween-20, 20 mM histidine and 10% glycerol. The reaction was
carried out at 32°C for 18 hours. The INF-ß was removed from the agarose with a 0.22 µL
Spin-X™ filter (Corning Technology, Inc., Norcross, GA). Figure 173A depicts the MALD1
analysis of glycans released from native INF-ß. The native 1NF-ß has many glycoforms
containing terminal sialic acid moieties. Figure 173B depicts the MALD1 analysis of glycans
released from desialylated INF-ß. The desialylated INF-ß has primarily one glycoform
which is bi-antennary with terminal galactose moieties.
Lectin Dot-Blot Analysis of Sialylation. Samples of the INF-ß from the desialidase
reaction were dot-blotted onto nitrocellulose and then blocked with Tris buffered saline
(TBS: 0.05M Tris. 0.15M NaCl, pH 7.5) and DIG kit (glycan differentiation kit available
from Roche'#l 210 238) blocking buffer. Some of the blots were incubated with Mauckiu
amurensis agglutinin (MAA) labeled with digoxogenin (DIG) (Roche Applied Science.
Indianapolis, IL) to detect ot2,3-sialylation of INF-ß. These blots were washed with TBS
then incubated with anti-digitonin antibody labeled with alkaline phosphatase, then washed
again with TBS and developed withNBT/X-phosphate solution, wherein NBT is 4-nitro blue
tetrazolium chloride and X-phosphate is 5-bromo-4-chloro—3indoyl phosphate. The left side
of Figure 174 depicts the results of the MAA blot of INF-ß after the desialylation reaction.
The INF-ß is partially disialylatcd, as indicated by the decrease in dot development as
compared to native INF-ß in the desialylated samples.
Other blots were incubated with Erthrina cristagalli lectin (ECL) labeled with biotin
(Vector Laboratories. Burlingame, CA) to detect exposed galactose residues on INF-ß. After
incubation with 2.5 µg/ml ECL, the blots were washed in TBS and incubated with
streptavidin labeled with alkaline phosphatase. The blots were then washed again and
developed. The right side of Figure 174 depicts the ECL blot after development. The
increased intensity of the dot of desialylated INF-ß as compared to the native INF-ß indicate
more exposed galactose moieties and therefore extensive desialylation.
PEGylation of Desialylated INF-ß with SA-PEG (10 kDa). Desialylated INF-ß
(0.05 mg/ml) was PEGylated with ST3Gal3 (50 mlJ/ml) and CMP-SA-PEG (10 kDa) (250
µM) in an appropriate buffer of TBS + 5 mM CaCI2, 0.02% Tween 20, 20 mM histidine. 10%
glycerol for 50 hours at 32°C. Figure 175 depicts the SDS-PAGE analysis of the reaction
products showing PEGylated INF-ß at approximately 98 kDa.
PEGylation of Desialylated INF-ß with SA-PEG (20 kDa). Desialylated INF-ß
(0.5 mg/ml) was PHGylated with ST3Gal3 (170 mU/ml) and CMP-SA-PEG (20 kDa) in an
appropriate buffer of TBS + 5 mM CaCI2, 0.02% Tween 20, 20 mM histidine, 10% glycerol
for 50 hours at 32°C. Figure 176 depicts the SDS-PAGE analysis the products of the
PEGylation reaction. The PEGylated INF-ß has many higher molecular weight bands not
found in the unmodified INF-ß indicating extensive PEGylation.
Superdex-200 Purification of INF-ß PEGylated with PEG (10 kDa). The products
of the PEGylation reaction were separated on a Superdex-200 column (Amersham
Biosciences, Arlington Heights. IL) in PBS with 0.5 NaCl, 0.02 Tween-20, 20 mM histidine
and 10% glycerol at Iml/min and 30 cm/hr flow. The eluant was monitored for absorbance at
280 nm (Figure 177) and fractions were collected. Peaks 3 and 4 were pooled and
concentrated in an Amicon Ultra 15 spin filter.
Bioassay of INF-ß PEGylated with PEG (10 kDa).
The test is inhibition of the proliferation of the lung carcinoma cell line. A549. The
A549 cell line are lung carcinoma adherent cells growing in RPMI + 10% FBS at 37°C 5%
CO2. They can be obtained from ATCC # CCL-185. Wash the cells with 10 ml of PBS and
remove the PBS. Add 5 ml of trypsin, incubate for 5 minutes at room temperature or 2
minutes at 37°C. When the cells are detached resuspend into 25 ml of media and count the
cells. Dilute the cells at a concentration of 10000 cells/ml and add 200 µl / well (96 wells
plate). Incubate for 4 hours at 37°C 5% CO2. Prepare 1 ml of IFN B at a concentration of
0.1 µg/ml. Filter it under the hood with a 0.2 µm filter. Add 100 µl per well (8 replicates = I
lane). Incubate for 3 days (do not let the cells go to confluence). Remove 200 µl of media
(only lOOul per well left). Add 25 µl of MTT (Sigma) (5 mg/ml filtered 0.22um). Incubate
for 4 hours at 37°C and 5% CO2. Aspirate the media gently and add 100 µl of a mixture of
isopropanol (100 ml and 6N HC1. Aspirate up and down to homogenize the crystal violet.
Read OD 570nm (remove the background at 630 or 690 nm).
Figure 178 depicts the results of the bioassay of the peaks containing INF-ß
PEGylated with PEG (10 kDa) as eluted from the Superdex-200 column.
Superdex-200 Purification of INF-0 PEGylated with PEG (20 kDa). The products
of the PEG (20 kDa) PEGylation reaction were separated on a Superdex-200 column
(Amersham Biosciences. Arlington Heights, IL) in PBS with 0.5 NaCl, 0.02 Twcen-20, 20
mM histidine and 10% glycerol at 1 ml/min flow. The eluant was monitored for absorbance
at 280 nm (Figure 179) and fractions were collected. Peak 3 contained most of the INF-ß
PEGylated with PEG (20 kDa).

RemicadeTM
36. GIvcoPEGylation of Remicade™ antibody
This example sets forth the procedure to glycoPEGylate a recombinant antibody
molecule by introducing PEG molecules to the Fc region glycans. Here Remicade™. a TNF-
R:lgG Fc region fusion protein, is the exemplary peptide.
Preparation of Remicade™-Gal-PEG (10 kDa). Remicade™ is dissolved at 2.5
mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCI2, 0.05% NaN3, pH 7.2. The solution
is incubated with 1 mM UDP-galactose-PEG (10 kDa) and 0.1 U/mL of galactosyltransferase
at 32°C for 2 days to introduce the PEG in the Fc region glycans. To monitor the
incorporation of galactose, a small aliquot of the reaction has 14C-galactose-UDP ligand
added: the label incorporated into the peptide is separated from the free label by gel filtration
on a Toso Haas G3000SW analytical column using PBS buffer (pH 7.1). The radioactive
label incorporation into the peptide is quantitated using an in-line radiation detector.
When the reaction is complete, the reaction mixture is purified using a Toso Haas
TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) and collecting fractions based
on UV absorption. The fractions containing product are combined, concentrated, buffer
exchanged and then freeze-dried. The product of the reaction is analyzed using SDS-PAGH
and 1LT analysis according to the procedures and reagents supplied by Invitrogen. Samples
are dialyzed against water and analyzed by MALDI-TOF MS.
Rituxan™
37. Glycoconjugation of geldanamycin to Rituxan™
This example sets forth the glycoconjugation of a small molecule, such as
geldanamycin, to the Fc region glycans of an antibody produced in CHO cells, such as
Rituxan™. Here, the antibody Rituxan™ is used, but one of skill in the art will appreciate
that the method can be used with many other antibodies.
Preparation of Rituxan™-Gal-linker-geldanamycin. Rituxan™ is dissolved at 2.5
mg/mL in 50 mM Tris-HCl, 0.15 M NaCl, 5 mM MnCl2, 0.05% NaN3, pH 7.2. The solution
is incubated with 1 mM UDP-galactose-linker-geldanamycin and 0.1 U/mL of
galactosyltransferase at 32°C for 2 days to introduce the geldanamycin in the Fc region
glycans. To monitor the incorporation of galactose, a small aliquot of the reaction has 14C-
galactose-UDP ligand added; the label incorporated into the peptide is separated from the free
label by gel filtration on a Toso Haas G3000SW analytical column using PBS buffer (pH
7.1). The radioactive label incorporation into the peptide is quantitated using an in-line
radiation detector.
When the reaction is complete, the reaction mixture is purified using a Toso Haas
TSK-Gel-3000 preparative column using PBS buffer (pH 7.1) and collecting fractions based
on UV absorption. The fractions containing product are combined, concentrated, buffer
exchanged and then freeze-dried. The product of the reaction is analyzed using SDS-PAGE
and IEF analysis according to the procedures and reagents supplied by Invitrogen. Samples
arc dialyzed against water and analyzed by MALDI-TOF MS.
Rnase
38. Remodeling high mannose N-glycans to hybrid and complex N-glycans:
Bovine pancreatic RNase
This example sets forth the preparation of bovine pancreas RNase with hybrid or
complex N-glycans. The high mannose N-linked glycans of the RNase are enzymatically
digested and elaborated to create hybrid N-linked glycans. Additionally, the high mannose
N-linked glycans of the RNase are enzymatically digested and elaborated to create complex
N-linked glycans.
High mannose structures of AM inked oligosaccharides in glycopeptides can be
modified to hybrid or complex forms using the combination of a-mannosidases and
glycosyltransferases. This example summarizes the results in such efforts using a simple N-
Glycan as a model substrate.
Ribonuclease B (RNaseB) purified from bovine pancreas (Sigma) is a glycopeptide
consisting of 124 amino acid residues. It has a single potential N-glycosylation site modified
with high mannose structures. Due to its simplicity and low molecular weight (13.7 kDa to
15.5 kDa). ribonuclease B is a good candidate to demonstrate the feasibility of the Ar-Glycan
remodeling from high mannose structures to hybrid or complex N-linked oligosaccharides.
The MALDI-TOF spectrum of RNaseB (Figure 180A) and HPLC profile for the
oligosaccharides cleaved from RNaseB by N-Glycanase (Figure 180B) indicated that, other
than a small portion of the non-modified peptide, the majority of N-glycosylation sites of the
peptide are modified with high mannose oligosaccharides consisting of 5 to 9 mannose
residues.
Conversion of high mannose N-Glycans to hybrid N-Glycans. High mannose A -
Glycans were converted to hybrid N-Glycans using the combination of a1,2-mannosidase.
GlcNAcT-l (P-l,2-A'-acetyl glucosaminyl transferase), GalT-I (ßl,4-galactosyltransfease) and
(x2,3-sialyltransferasc /or a2,6-sialyltransferase as shown in Figure 181.
As an example, high mannose structures in RNaseB were successfully converted to
hybrid structures.
Man5GlcNAc:-R was obtained from Man5.9GlcNAc2-R catalyzed by a single al.2-
mannosidase cloned from Trichoderma reesei (Figure 182). RNase B (1 g. about 67 µmol)
was incubated at 30°C for 45 hr with 15 mU of the recombinant T. reesei a1,2-mannosidase
in MliS buffer (50 mM, pH 6.5) in a total volume of 10 mL. Man6..)GlcNAc2-protein
structures have been successfully converted to Man5GIcNAc2-protein with high efficiency by
the recombinant mannosidase.
Alternately. Man5GlcNAc2-R was obtained from Man5.9GlcNAc2-R catalyzed by a
single a1,2-mannosidase purified from Aspergillus saitoi (Figure 183). RNasc B (40 µg.
about 2.7 nmol) was incubated at 37°C for 42.5 hr with 25 µU of the commercial A. saitoi a1,2-mannosidase (Glyko or CalBioChem) in NaOAC buffer (100 mM, pH 5.0) in a total
volume of 20 µl. Man(,.gGlcNAc2-protein structures were successfully converted to
MansGlcNAc2-protein by the commercially available mannosidase. However, a new peak
corresponding to the GlcNAc-protein appears in the spectrum, indicating the possible
contamination of endoglycosidase H in the preparation. Although several mammalian alpha-
mannosidases were required to achieve this step, the fungal a1,2-mannosidase was very
efficient to remove all a1,2-linked mannose residues.
GlcNAcT-I then added a GlcNAc residue to the MansGlcNAc2-R (Figure 184). The
reaction mixture after the T. reesei a1,2-mannosidase reaction containing RNase B (600 µg.
about 40 nmol) was incubated with non-purified recombinant GlcNAcT-l ß4 mil) in MES
buffer (50 mM. pH 6.5) containing MnCl2 (20 mM) and UDP-GlcNAc (5 mM) in a total
volume of 400 µl. at 37°C for 42 hr. A GlcNAc residue was quantitatively added to
MansGlcNAcrprotein by the recombinant GlcNAcT-I.
A Gal residue was then added using GalT 1 (Figure 185). The reaction mixture after
the Gn 1-1 reaction containing RNase B (120 µg, about 8 nmol) was incubated at 37°C for 20
hr with 3.3 mU of the recombinant GalT-1 in Tris-HCl buffer (100 mM, pH 7.3) containing
UDP-Gal (7.5 mM) and MnCl2 (20 mM) in a total volume of 100 µl. A Gal residue was
added to about 98% of the GlcNAc-Man5GlcNAc2-protein by the recombinant GalT 1.
The next step was the addition of a sialic acid using an a2,3-sialyltransferase or an
a2.6-sialyltransferasc (Figure 186). As an example, ST3Gal 111, an a2,3-sialyltransferase was
used. The reaction mixture after the GalT-1 reaction containing RNase B (13 ( µg, about 0.87
nmol) was incubated at 37°C for 16 hr with 8.9 mil of recombinant ST3Gal III in Tris-HCl
buffer (100 mM, pH 7.3) containing CMP-Sialic acid (5 mM) and MnCl2 (20 mM) in a total
volume of 20 µl. A sialic acid residue was added to about 90% of the Gal-GlcNAc-
Man5GlcNAc2-protein by recombinant ST3Gal III using CMP-SA as the donor. The yield
can be further improved by adjusting the reaction conditions.
For convenience, no purification or dialysis step was required after each reaction
described above. More interesting. GalT 1 and ST3Gal III can be combined in a one-pot
reaction. Similar yields were obtained as compared with the separate reactions. The reaction
mixture after the GlcNAcT-l reaction containing RNase B (60 µg, about 4 nmol) was
incubated at 37°C for 20 hr with 1.7 mlJ of recombinant GalT 1. 9.8 mU of recombinant
ST3Gal III in Tris-HCl buffer (100 mM, pH 7.3) containing UDP-Gal (7.5 mM), CMP-sialic
acid (5 mM) and MnCl2 (20 mM) in a total volume of 60 µl.
As shown in Figure 187, SA-PEG (10 kDa) was successfully added to the RNaseB.
The reaction mixture after the GalT-1 reaction containing RNase B (6.7 µg. about 0.45 nmol)
was dialyzed against H20 for 1 hour at room temperature and incubated at 37°C for 15.5
hours with 55 mU of the recombinant ST3Gal III in Tris-HCl buffer (50 mM, pH 7.3)
containing CMP-SA-PBG (10 kDa) (0.25 mM) and MnCl2 (20 mM) in a total volume of 20
µl. PEG-modified sialic acid residues were successfully added to the Gal-GlcNAc-
Man.sGlcNAc2-peplide by the recombinant S'DGal III. The yield can be further improved by
adjusting the reaction conditions.
Conversion of high mannose N-Glycans to complex N-Glycans. To achieve this
conversion, a GlcNAcß1,2Man3GlcNAc2-peptide intermediate is obtained. As shown in
Figure 188, there are at least four feasible routes to carry out the reaction from
Man-iGlcNAc2-peptide to this intermediate:
Route I: The Man5GlcNAc2-peptide produced by the fungal a1,2 mannosidase is a
substrate of GlcNAc transferase I (GlcNAcT-I, enzyme 2) which adds one GlcNAc. The
terminal al,3- and a1,6-linked mannose residues of GlcNAcMan5GlcNAc2-peptide is
removed by Golgi a-mannosidase II (Manll, enzyme 5). This route is a part of the natural
pathway for the processing of N-linked oligosaccharides carried out in higher organisms.
Route II: Two mannose residues are first removed by an a-mannosidase (enzyme 6),
then a GlcNAc is added by GlcNAcT-I (enzyme 2). Other than its natural acceptor
Man5GlcNAc2-R, GlcNAcT-I can also recognize Man3GlcNAc2-R as its substrate and add
one GlcNAc to the mannose core structure to form GlcNAcMan3GlcNAc2-peplide.
Route III: The al,6-linked mannose is removed by an al,6-mannosidase, followed
by the addition of GlcNAc by GlcNAcT-I and removal of the terminal al,3-linked mannose
by an a1,3-mannosidase. From the experimental data obtained, GlcNAcT-I can recognize
this ManiGlcNAc2-peptide as acceptor and add one GlcNAc residue to form
GlcNAcMan4GlcNAc2-peptide.
Route IV: Similar to Route III, a1,3-linked mannose is removed by an al,3-
mannosidase, followed by GlcNAcT-I reaction. Then the terminal a1,6-linked mannose can
be removed by an a1,6-mannosidase.
After the function of GlcNAcT-I (responsible for the addition of the GlcNAc ß1.2-
linked to the a1,3-mannose on the mannose core) and GIcNAcT-II (responsible for the
addition of a second GlcNAc ß1,2-linked to the al,6-mannose on the mannose core), the
GlcNAc2Man3GlcNAc2-peptide can be processed by GalT 1 and sialyltransferase to form bi-
antennary complex N- Glycans. Other GlcNAc transferases such as GlcNAcT-IV, GlcNAcT-
V. and/or GlcNAcT-VI (Figure 188 and Figure 189) can also glycosylate the
GlcNAc2Man-,GlcNAc2-peptide. Additional glycosylation by the GalT 1 and
sialyltransferases will form multi-antennary complex N-glycans. The enzyme GlcNAcT-lII
catalyzes the insertion of a bisecting GlcNAc, thus preventing the actions of Manll and
subsequent action of transferases GlcNAcT-II, GlcNAcT-IV and GlcNAcT-V.
Tissue-Type Plasminogen Activator (TPA)
39. Fucosvlation of TPA to create Sialyl Lewis X
This example sets forth the preparation of Tissue Tissue-type Plasminogen Activator
(TPA) with N-linkcd sialyl Lewis X antigen.
Sialylation. TPA expressed in mammalian cells will often contain a majority of the
glycans terminating in sialic acid, but to ensure complete sialylation, it would be beneficial to
first perform an in vitro sialylation. TPA in a suitable buffer (most preferably between pH
5.5 and 9, for example Tris buffered saline, pH 7.2) is incubated with CMP sialic acid and
sialyltransferase for a time sufficient to convert any glycans lacking sialic acid to sialylated
species. Typical conditions would be I mg/mL TPA, 3 mM CMP sialic acid. 0.02 U/mL
ST3Gal3, 32°C for 24 hours. Microbial growth can be halted either by sterile filtration or the
inclusion of 0.02% sodium azide. The TPA concentration is most preferably in the range 0.1
mg/mL up to the solubility limit of the peptide. The concentration of CMP-SA should be
sufficient for there to be excess over the available sites, and might range from 50 µM up to 50
mM. and the temperature from 2°C up to 40°C. The time required for complete reaction will
depend on the temperature, the relative amounts of enzyme to acceptor substrate, the donor
substrate concentration, and the pH. Other sialyltransferases that may be capable of adding
sialic acid in 2,3 linkage include ST3Gal4; microbial transferases could also be used.
Fucosylation. Typical conditions for fucosylation would be 1 mg/mL TPA. 3 mM
GDP-fucose. 0.02 U/mL FTVI, 5 mM MnCl2, 32°C for 24H in Tris buffered saline.
Microbial growth can be halted either by sterile filtration or the inclusion of 0.02% sodium
azide. The TPA concentration is most preferably in the range 0.1 mg/mL up to the solubility
limit of the peptide. The concentration of GDP-fucose should be sufficient for there to be
excess over the available sites, and might range from 50 µM up to 50 mM, and the
temperature from 2°C up to 40°C. The time required for complete reaction will depend on
the temperature, the relative amounts of enzyme to acceptor substrate, the donor substrate
concentration, and the pH. Other fucosyltransferases that may be capable of making sialyl
Lewis x include FTVII, FTV, FTIII, as well as microbial transferases could also be used.
40. Trimming of high mannose to tri-mannose core structure: Tissue-type
Plasminogen Activator produced in CHO
This example sets forth the preparation of Tissue-type Plasminogen Activator with a
trimannose core by trimming back from a high mannose glycan.
Tissue-type plasminogen activator (TPA) is currently produced in Chinese Hamster
Ovary (CHO) cells and contains a low amount of high mannose N-linked oligosaccharide.
The mannoses can be trimmed down using a variety of the specific mannosidascs. The first
step is to generate Man5GlcNAc2(Fuc0-l) from Man9GlcNAc2(Fuc0-l). This can be done
using mannosidasc 1. Then either GlcNAcTl (GlcNAc transferase I) is used to make
GlcNAclMan5GlcNAc2(Fuc0-l) or Mannosidase III is used to make Man3GlcNAc2(FucO-
1). From Man3GlcNAc2(FucO-l), GlcNAclMan3GlcNAc2(Fuc0-l) can be produced using
GlcNAcTl or from GlcNAclMan5GlcNAc2(Fuc0-1), GlcNAclMan3GlcNAc2(Fuc0-1) can
be produced using Mannosidase II. GlcNAc I Man3GlcNAc2(Fuc0-l) is then converted into
GlcNAc2Man3GlcNAc2(FucO-l) using GlcNAcTransfcrase II (GlcNAcTIl). Ihe two
terminal GlcNAc residues are then galactosylated using GalTI and then sialylated with SA-
PkG using ST3GalIII.
Conversely. TPA can be produce in yeast or fungal systems. Similar processing
would be required for fungal derived material.
41. Generation and PEGylation of GlcNAc-ASN structures: TPA produced in
Yeast
This example sets forth the preparation of PEGylated GlcNAc-Asn structures on a
peptide such as TPA expressed in yeast.
Yeast expression is expected to result in a TPA which contains a single N-linked
mannan-type structure. This recombinant glycoprotein is first treated with endoglycosidasc
11 to generate GlcNAc structures on the asparagine (Asn) residues on the peptide.
The GlcNAc-Asn structures on the peptide/protein backbone are then be modified
with galactose or galactose-PEG using UDP-galactose or UDP-galactose-6-PEG.
respectively, and a galactosyltransferase such as GalTI. In one case, the galactose-PEG is
the terminal residue. In the second case, the galactose is further modified with SA-PEG
using a CMP-SA-PEG donor and a sialyltransferase such as ST3GalIII. In another
embodiment, the GlcNAc-Asn structures on the peptide/protein backbone may be
galactosylated and sialylated as described above, and then further sialylated using CMP-SA-
PEG and an a2,8-sialyltransferase such as the enzyme encoded by the Campylobacter jejuni
cst-ll gene.
Transferrin
42. GlycoPEGylation of Transferrin
This example sets forth the preparation of asialotransferrin and its sialylation with
PEG-CMP-sialic acid.
Preparation of Asialo-transferrin. Human-derived holo-Transferrin. (10 mg) was
dissolved in 500 µL of 50 mM NaOAc, 5 mM CaCl2, pH 5.5. To this solution was added
500 mU Neuraminidase II {Vibrio cholerae) and the reaction mixture was shaken gently for
20.5 hours at 37 °C. The reaction mixture was added to the prewashed N-(p-
aminophenyl)oxamic acid-agarose conjugate (600 µL) and the washed beads gently rotated
for 24 hours at 4 °C. The mixture was centrifuged at 10.000 rpm and the supernatant was
collected. The reaction mixture was adjusted to 5 mM EDTA by addition of 100 µL of 30
mM EDTA to the washed beads, which were gently rotated for 20 hours at 4 °C. The
suspension was centrifuged for 2 minutes at 10,000 rpm and the supernatant was collected.
The beads were washed 5 times with 0.35 mL of 50 mM NaOAc, 5 mM CaCl2, 5 mM
EDTA, pH 5.5 and all supernatants were pooled. The enzyme solution was dialyzcd twice at
4 ºC into 15 mM Tris-HCl, 1 M NaCl, pH 7.4. 0.3 mL of the transferrin solution )(3.3 mL
total) was removed and dialyzed twice against water. The remainder was dialyzed twice
more at 4 °C against phosphate buffered saline. The dialyzed solution was stored at -20 " C.
Protein samples were analyzed by 1EF Electrophoresis. Samples (9 µL, 25 µg) were diluted
with 16 µL Tris buffer and mixed with 25 µL of the sample loading buffer and applied to
Isoelectric Focusing Gels (pH 3-7). Gels were run and fixed using standard procedures. Gels
were stained with Colloidal Blue Stain.
Sialyl-PEGylation of asialo-Transferrin. Desialylated transferrin (250 µg) and
CMP-sialic acid or CMP-SA-PEG (1 kDa or 10 kDa)(0.05 µmol) were dissolved in 69 µL 50
mM Tris-HCl, 0.15 M NaCl, 0.05% NaN3. pH 7.2 in 1.5 mL plastic tubes. The tubes were
vortexed briefly and 100 mU ST3Gal3 (90 u. L) were added (total volume 250 u L). The
tubes were vortexed again and mixed gently for 24 hours at 32 °C. The reactions were
stopped by freezing at -80 °C. Novex Tris-Glycine 8-16% 1 mm gels were used for SDS
PAGE analysis (Figure 190). Samples (25 µL, 25 µg) were mixed with 25 µL of sample
loading buffer and 0.4 µL of P-mercaptoethanol and heated for 6 minutes at 85 °C. Gels
were run using standard conditions and stained with Colloidal Blue Stain. 1EF gels were also
performed as described above Figure 191). Samples were also dialyzed against water
analyzed by MALDI-TOF.
Results. MALDI was also performed. Native transferrin (78729); asialotransferrin
(78197); resialylated transferrin (79626/80703); with SA-PEG Ik (79037 (1); 80961 (2);
82535 ß); 84778 (4)); with SA-PEG 5k (90003 (2): 96117 (3); 96117 (4)): with SA-PEG 10k
(100336 (2); 111421 (3): 122510 (4)).
43. Transferrin-GDNF
this example sets forth the procedures for the glycoconjugation of proteins, and in
particular, transferrin is glycoconjugated to GDNF. Transferrin-SA-Linker-Gal-UDP is
prepared from transferrin. The galactose residue is removed from GNDF glycans, and
Transferrin-SA-Linker-Gal-UDP is conjugated to GNDF glycans using a
galactosyltransferasc.
Preparation of agalacto-GDNF. GDNF produced in NSO cells (NSO murine
myeloma cells) is dissolved at 2.5 mg/mL in 50 mM Tris 50 mM Tris-HCl pH 7.4. 0.15 M
NaCl, and is incubated with 300 mU/mL beta-galactosidase-agarose conjugate for 16 hours at
32°C. To monitor the reaction a small aliquot of the reaction is diluted with the appropriate
buffer and a IEF gel performed according to Invitrogen procedures. The mixture is
centrifuged at 10.000 rpm and the supernatant is collected. The supernatant is dialyzed at 4
°C against 50 mM Tris -HCl pH 7.4. 1 M NaCl, 0.05% NaN3 and then twice more against 50
mM Tris -HCl pH 7.4. 1 M NaCl, 0.05% NaN3. The dialyzed solution is then concentrated
using a Centricon Plus 20 centrifugal filter and stored at -20 °C. The conditions for the IEF
gel are run according to the procedures and reagents provided by Invitrogen. Samples are
dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of Transferrin-SA-Linker-Gal-UDP. Asialo-transferrin is dissolved at
2.5 mg/mL in 50 mM Tris-HCl, 0.15 M NaCl. 0.05% NaN3. pH 7.2. The solution is
incubated with CMP-sialic acid-linker-Gal-UDP (molar amount to add 1 molar equivalent of
nucleotide sugar to transferrin) and 0.1 U/mL of ST3Gal3 at 32°C for 2 days. To monitor the
incorporation of sialic acid, a small aliquot of the reaction has l4C-SA-UDP ligand added; the
label incorporated into the peptide is separated from the free label by gel nitration on a Toso
Haas G3000SW analytical column using PBS buffer (pH 7.1). The radioactive label
incorporation into the peptide is quantitated using an in-line radiation detector.
The solution is incubated with 5 mM CMP-sialic acid and 0.1 U/mL of ST3Gal3 (to
cap any unreacted transferrin glycans) at 32°C for 2 days. The incorporation into the peptide
is quantitated using an in-line UV detector. After 2 days, the reaction mixture is purified
using a Toso Haas G3000SW preparative column using PBS buffer (pH 7.1) and collecting
fractions based on UV absorption. The product of the reaction is analyzed using SDS-PAGL
and IFF analysis according to the procedures and reagents supplied by Invitrogcn. Samples
arc dialyzed against water and analyzed by MALDI-TOF MS.
Preparation of Transferrin-SA-Linker-Gal-GDNF. The transfcrrin-SA-Linkcr-
Gal-UDP prepared as described above is dissolved at 2.5 mg/mL in 50 mM Tris-HCl. 0.15 M
NaCl. 5 mM MnCl2. 0.05% NaN3. pH 7.2. The solution is incubated with 2.5 mg/mL
agalacto-GDNF and 0.1 U/mL of galactosyltransferase at 32°C for 2 days. To monitor the
incorporation of galactose, a small aliquot of the reaction has ,4C-galactosc-UDP ligand
added; the label incorporated into the peptide is separated from the free label by gel filtration
on a Toso Haas G3000SW analytical column using PBS buffer (pH 7.1). The radioactive
label incorporation into the peptide is quantitated using an in-line radiation detector.
When the reaction is complete, the solution is incubated with 5 mM UDP-Gal and 0.1
U/mL of galactosyltransferase (to cap any unreacted transferrin glycans) at 32°C for 2 days
followed by addition of 5 mM CMP-SA and 0.1 U/mL of ST3Gal3. After 2 additional days,
the reaction mixture is purified using a Toso Haas G3000SW preparative column using PBS
buffer (pH 7.1) collecting fractions based on UV absorption. The product of the reaction is
analyzed using SDS-PAGE and IEF analysis according to the procedures and reagents
supplied by lnvitrogen. Samples are dialyzed against water and analyzed by MALDI-TOF
MS.
The disclosures of each and every patent, patent application, and publication cited
herein are hereby incorporated herein by reference in their entirety.
While this invention has been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of this invention may be devised by others
skilled in the art without departing from the true spirit and scope of the invention. The
appended claims are intended to be construed to include all such embodiments and equivalent
variations.
We claim:
1. A glycoPEGylated EPO peptide comprising an recombinant EPO peptide
and atleast one glycan and atleast one poly(ethylene glycol) molecule
covalently attached to said glycan, wherein said poly(ethyfene glycol)
molecule is added to said EPO peptide using a glycosyltransferace.
2. The grycoPEGylated EPO peptide as claimed in claim 1, comprising
atleast one mono-antennary glycan.
3. The glycoPEGylated EPO peptide as claimed in clarm 1, wherein each of
said atleast one glycan is N-linked and is mono-antennary.
4. The grycoPEGylated EPO peptide as claimed in claim \, wherein each of
said atleast one glycan is N-linked and atleast one of said glycans
comprise said poly(ethylene glycol)
5. The glycoPEGylated EPO peptide as claimed in claim 4, wherein more
than one of said, atleast one glycan comprises said poly(ethylene glycol).
6. The glycoPEGylated EPO peptide as claimed in claim 1, wherein each of
said atleast one glycan are N-linked and all of said glycans comprise said
poly(ethylene glycol).
7. The grycoPEGylated EPO peptide as claimed in claim I, comprising
atleast three mono-antennary glycans having said poly(ethylene glycol)
covalently attached thereto.
8. A glycoPEGylated EPO peptide, wherein said EFO peptide comprises
three or more glycans.
9. The glycoPEGylated EPO peptide as claimed in claim 8, wherein atleast
one of said glycans comprises a poly(ethylene glycol) covalently attached
thereto,
10. The glycoPEGylated EPO peptide as claimed in claim 8, wherein more
than one of said comprises said poly(ethylene glycol) covalently attached
thereto.
11. The glycoPEGylated EPO peptide as claimed in claim 8, wherein all of
said glycans comprise said poly(ethylene glycol) covalently attached
thereto.
12. The glycoPEGyiated EPO peptide as claimed in claim 8, wherein said
poly(ethylene glycol) is linked to atleast one sugar moiety selected from
the group consistmg of a fucose (Fuc), a N-acetylgracosamine (GIcN Ac), a
galactose (Gal) and a sialic acid (SA) .
13. The glycoPEGylated EPO peptide as claimed in claim 12, wherein said
sialic acid is N-acetyIneuraminic acid.
14. The glycoPEGylated EPO peptide as claimed in claim 8, wherein said EPO
peptide does not comprise an O-linked glycan.
15. The glycoPEGyiated EPO peptide as claimed in claim 8, wherein said EPO
peptide comprises atleast one O-linked glycan.
16. The glcoPEGvlated EPO peptide as claimed in claim 15, wherein said O
linked peptide comprises said poly(ethylene glycol) covalently attached
thereto.
17, The glycoPEGylated EPO peptide as claimed in claim 16, wherein said
EPO peptide is recombinantly expressed in a cell.
18, The glycoPEGylated EPO peptide as claimed in claim 17, wherein said cell
is selected from the group consisting of an insect cell, a fingal cell and a
mammalian cell.
19. The glycoPEGylated EPO peptide as claimed in claim 18, wherein said
fungal cell is a yeast cell.
20, The glycoPEGylated EPO peptide as claimed in claim 18, wherein said
mammalian cell is a CHO cell.
21. The glycoPEGylated EPO peptide as claimed in claim 8, wherein said
poly(ethylene glycol has a molecular weight selected from the group
consisting of about 1 kDa, 2 kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, and 40
kDa.
22. The glycoPEGylated EPO peptide as claimed in claim 8, wherein said EPO
peptide is selected from the group consisting of a naturally occurring EPO
peptide and a mutated EPO peptide.
23, The glycoPEGylated EPO peptide as claimed in claim 22, wherein said
mutated EPO peptide comprises an amino acid sequence of SEQ ID NO:
73 having at least onemutation selected from the group consisting of
Argw 139 Ala139, to Arg143 to Ala143 and Lys154 to Alais154.
24. A method for making a glycoPEGylated EPO peptide, said method
comprising the step of:
(a) contacting an EPO peptide with a mixture comprising a nucleotide
sugar covalently linked to a poly(ethylene glycol) and a
glycosyltransferase underconditions sufficient to transfer said
poly(ethylene glycol) to said EPO peptide.
25. The method as claimed in claims 24, wherein the sugar of said nucleotide
sugar is selected from the group consisting of a fucose (Fuc), a a N-
acetylglucosamine (GIcNAc), a galactose (Gal) and a sialic acid (SA).
26. The method as claimed in claim 25, wherein said sialic acid is N-
acetylceueaminic acid (NAN).
27. The method as claimed in claim 24, wherein said poly(ethylene glycol) has
a molecular weight selected from the group conmsisting of about 1 kDa, 2
kDa, 5 kDa, 10 kDa, 20 kDa, 30 kDa, and 40 kDa.
28. The method as claimed in claim 24, wherein said EPO peptide is
recombinantly expressed in a cell.
29. The method as claimed in claim 28, wherein said cell is selected from the
group consisting of an insect cell, a fungal cell and a mammalian cell.
30. The method as claimed in claim 28, wherein said cell is said yeast cell.
31. The method as claimed in claim 29, wherein said mammalian cell is a
CHO cell.

32. The method as claimed in claim 24, wherein said EPO peptide is selected
from the group consisting of a naturally occurring EPO peptide and a
mutated EPO peptide.
33. The method as claimed in claim 32, wherein said mature EPO peptide has
the sequence of SEQ ID NO:73.
34. The method as claimed in claim 32, wherein said mutated EPO peptide
comprises the amino acid sequence SEQ ID NO:73 haying atleast one
mutation selected from the group consisting of Arg139 to Ala139, to Arg143
to Ala143 and Lys154 to Ala154.
35. The method as claimed in claim 24, further comprising before step (a): (b)
contacting said EPO peptide with a mixture comprising a nucleotide-N-
acetyfglucosaumine (OcNAc) molecule and an N-acetylglucosamine
transferase (GnT) for which the nucleotide-GlNAc is a substrate under
conditions sufficient to form a bond between said GlcNAc and said RPO,
wherein said GnT selected from the group consisting of GnT I, GnT II,
GnT III, GnT XV, GnT V, and GnT VI,
36. The method as claimed in claim 35, wherein said mixture comprises one
GnT selected from the group consisting of GnT I, GnT II, GnT IV, GnT V,
and GnT VI.
37. The method as claimed in claim 36, wherein said GnT is GnT I.
38. The method as claimed in claim 36, wherein said GnT is GnT II.
39. The method as claimed in claim 24, wherein said glycoPEGylated EPO
peptide comprises atleast one mono-antennary glycan.
40. The method as claimed in claim 24, wherein the sugar of said nucleotide
sugar is galactose and said glycosyitransferase is galactosyl transferase I
(GaITI),
41. The method as claimed in claim 34, further comprising before step (a) but
after step (b): (c) contacting said EPO peptide with a mixture comprising a
nucleotide galactose (Gal) and galactosyl transfarase I (GalT I) under
conditions sufficient to transfer galactose to said EPO peptide.
42. The method as claimed in claim 24, wherein in step (a), the sugar of said
nucleotide sigar is a sialic acid and said glycosyitransferase is a
sialytransferase.
43. The method as claimed in claim 42, wherein said sialic acid is N-
acetylneuxaminic acid (NAN).
44. The method as claimed in claim 42, wherein said sialyltransferase is
selected from the group consisting of a(2,3) sialyltransferase, a(2,6)
sialyltransferase and (2,3) sialyltransferase.
45. A glycoPEGylated EPO peptide made by the method as claimed in claim
24.
46. A glycoPEGylated EPO peptide, said EPO peptide comprising the
sequence of SEQ ID NO:73.
47. A glycoPEGylated EFO peptide, said EFO peptide comprising the
sequence of SEQ ID NO:73 and further comprising a mutation in said
sequence.
48. A method of making a glycoPEGylated EPO peptide, said method
comprising the steps of:
(a) contacting an EFO peptide with a mixture comprising a nucleotide
sugar covalently linked to poly(ethylene glycol) and a
glycosyltransferase under conditions sufficient to transfer said
poly(ethylene glycol) to said EPO peptide, wherein said
glycosyltransferase is a fucosyltransferase.
49. The method as claimed in claim 48, wherein said fucosyltransferase is
selected from the group consisting of fucosyltransferase I,
fucosyltransferase III, fucosyltransferase IV, fucosyltransferase V,
fucosyltransferase VI and fucosyltransferase VII.
50. A glycoPEGylated EFO peptide made by the method as claimed in claim
48.
51. The method as claimed in claim 48, wherein said EPD peptide is
expressed in a CHO cell.

A glycoPEGylated EPO peptide comprising an recombinant EPO peptide and
atleast one glycan and atleast one poly(ethylene glycol) molecule covalently
attached to said glycan, wherein said poIy(ethylene glycol) molecule is added to
said EPO peptide using a glycosyltransferace.

Documents:

836-KOLNP-2005-ASSIGNMENT.pdf

836-KOLNP-2005-CORRESPONDENCE 1.1.PDF

836-KOLNP-2005-CORRESPONDENCE 1.2.pdf

836-KOLNP-2005-CORRESPONDENCE 1.3.pdf

836-KOLNP-2005-CORRESPONDENCE-1.1.pdf

836-KOLNP-2005-CORRESPONDENCE.pdf

836-KOLNP-2005-FORM 16.pdf

836-KOLNP-2005-FORM 27-1.1.pdf

836-KOLNP-2005-FORM 27.pdf

836-KOLNP-2005-FORM-27.pdf

836-kolnp-2005-granted-abstract.pdf

836-kolnp-2005-granted-claims.pdf

836-kolnp-2005-granted-correspondence.pdf

836-kolnp-2005-granted-description (complete).pdf

836-kolnp-2005-granted-drawings.pdf

836-kolnp-2005-granted-examination report.pdf

836-kolnp-2005-granted-form 1.pdf

836-kolnp-2005-granted-form 13.pdf

836-kolnp-2005-granted-form 18.pdf

836-kolnp-2005-granted-form 2.pdf

836-kolnp-2005-granted-form 26.pdf

836-kolnp-2005-granted-form 3.pdf

836-kolnp-2005-granted-form 5.pdf

836-kolnp-2005-granted-reply to examination report.pdf

836-kolnp-2005-granted-sequence listing.pdf

836-KOLNP-2005-OTHERS.pdf

836-KOLNP-2005-PA.pdf


Patent Number 233936
Indian Patent Application Number 836/KOLNP/2005
PG Journal Number 17/2009
Publication Date 24-Apr-2009
Grant Date 22-Apr-2009
Date of Filing 09-May-2005
Name of Patentee NEOSE TECHNOLOGIES, INC.
Applicant Address 102 WITMER ROAD, HORSHAM, PA
Inventors:
# Inventor's Name Inventor's Address
1 SHAWN DEFREES 126 FILLY DRIVE NORTH WALES, PA 19454
2 DAVID JAMES HAKES 14 FERN AVENUE WILLOW GROVE, PA 19090
3 XI CHEN 107 WHITNEY PLACE LANSDALE, PA 19446
4 DAVID A. ZOPF 560 WEST BEECHTREE LANE WAYNE, PA 19087
5 ROBERT J. BAYER 6105 DIRAC STREET SAN DIEGO, CA 92122
6 CARYN BOWE 276 CHERRY LANE DOYLESTOWN, PA, 18901
PCT International Classification Number C07K 14/505, 14/435
PCT International Application Number PCT/US2003/031974
PCT International Filing date 2003-10-08
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10/287,994 2002-11-05 U.S.A.
2 10/360,770 2003-01-06 U.S.A.
3 10/360,779 2003-02-19 U.S.A.
4 10/410,945 2003-04-09 U.S.A.
5 PCT/US2002/32263 2002-10-09 U.S.A.