Title of Invention

"POLYMER CONJUGATES OF MUTATED NEUBLASTIN"

Abstract A dimer comprising a mutated neublastin polypeptide coupled ID a polymer is disclosed. Such dimers exhibit prolonged bioavailability and, in preferred embodiments, prolonged biological activity relative to wild-type forms of neublastin.
Full Text POLYMER CONJUGATES OF MUTATED NEUBLASTIN
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from International application PCTAJS02/02319, filed
January 25,2002, which claims priority from United States provisional application Serial No.
60/266,071, filed February 1,2001 (abandoned).
FIELD OF THE INVENTION
The invention relates to protein chemistry, molecular biology, neurobiology,
neurology, and pain management.
BACKGROUND OF THE INVENTION
Neurotrophic factors are naturally-occurring proteins that regulate neuronal survival
during development and regulate plasticity and structural integrity of the adult nervous
system. Neurotrophic factors can be isolated from neural tissue and from non-neural tissue.
During the last twenty years, many neurotrophic factors have been discovered. These
neurotrophic factors can be classified into superfamilies, families, subfamilies and individual
species based on their structure and function.
Neurotrophic factor superfamilies include the fibroblast-growth factor {FGF)
superfamily, the neurotrophin superfamily, and the transforming growth factor-J5 (TGF-P)
superfamily. The glial cell line-derived neurotrophic factor'(GDKF)-related ligands are a
family of proteins within the TGF-p superfamily. GDNF-related ligands include GDNF,
persephin (PSP), neurturin (NTN) and neublastin (NBN; known as artemin orenovin).
Members of the GDNF-related ligand family are distinguished by, among other things, their
seven conserved cysteine residues. These residues form intramolecular and intermolecular
disulfide bridges and give rise to the tertiary and quaternary structure of the dimerized
polypeptide ligand. Members of the family also share the ability to induce signaling through
a multicomponent receptor complex consisting of a glycosylphosphatidylinositol
anchored co-receptor of the GFRa family, a member of the GDNF-related ligand subfamily,
and the RET tyrosine kinase receptor.
Activated RET initiates a signal transduction cascade that is responsible, at least in
part, for the downstream effects of GDNF-related ligands. Accordingly, activation of RET
may represent one desirable aspect of a therapy which acts through a GFRa receptor pathway
to affect downstream cellular processes.
Neublastin is classified within the GDNF family because it shares regions of
homology with other GDNF ligands including the seven cysteine motif (eg., as described in
EP02/02691, PCT publications US02/02319 and US02/06388), and because of its ability Jo
bind to, and activate, the RET receptor as part of a GFRa complex. Specifically, neublastin
is highly selective for binding to the GFRa3-RET receptor complex. In that respect,
neublastin contains unique sub regions in its amino acid sequence as compared with other
members of the GDNF-related ligand family.
Current data suggest that neublastin may have a protective and regenerative role in the
peripheral and central nervous systems and, as a result, may be useful as a therapeutic agent
for neurodegenerative disorders. For example, data suggest that neublastin may have survival
promoting effects on cultured sensory neurons from dorsal root ganglia and from trigeminal
ganglia, and on cultured substantia nigra dopaminergic neurons (Baloh et al., Neuron 21:
1291-1302 (1998)). It therefore appears that neublastin may promote survival of neuronal
populations including sensory and dopaminergic neurons. This is important because the
degeneration and dysfunction of neurons has been associated with disease states. For
example, sensory and dopaminergic neuron pathologies underlie peripheral neuropathy and
Parkinson's disease, respectively..
Therefore, administration of neublastin may be useful, for example, in the treatment
of diseases associated with neuronal degeneration and dysfunction. However, neublastin is
rapidly cleared by the body, which may affect the neublastin dosing paradigm required in
therapeutic applications. Thus, a need exists for modified neublastin polypeptides with
enhanced bioavailability. Accordingly, it is an object of the present invention to identify
modified forms of neublastin which exhibit enhanced bioavailability.
SUMMARY OF THE INVENTION
The invention provides polymer-conjugated, mutated neublastin dimers. Each dimer
contains a first polypeptide comprising a first amino-terminal amino acid and a second
polypeptide comprising a second amino-terminal amino acid. Each polypeptide individually
contains: (a) an amino acid sequence characterized by at least 70%, £0%, 90%, or 95%
sequence identity with amino acids 8-113 of SEQ ID N0:l; (b) a cysteine residue at each of
positions 16,43,47,80,81,109, and 111 (numbering according to SEQ ID NO:1); acid residues as follows: C at position 16, L at position 18, V at position 25, L at position 28, G
at position 29, L at position 30, G at position 31, E at position 36, F at position 40, R at
position 41, F at position 42, C at position 43, G at position 45, C at position 47, C at position
80, C at position 'SI, R at position 82, P at position 83, F at position 91, D at position 93, S at
position 105, A at position 106, C at position 109 and C at position 111; and (d) an LGLG
repeat, an FRFC motif, a QPCCRP motif, and aSATACGC motif. The dimer includes at feast
one amino acid substitution (with respect to SEQ ID NO: 1), which provides an internal
polymer conjugationisite to which a polymer is conjugated.
The invention also provides a polymer-conjugated, mutated neublastin dimer
containing a first polypeptide and a second polypeptide, wherein each polypeptide contains
90-140, e.g., 95-120 or 100-110, ammo acids of SEQ ID NO:6 with 1-6 amino acid
substitutions, each substitution providing a polymer conjugation site to which a polymer is
conjugated. Specific examples of polypeptides of the invention include NBN113 (SEQ ID
NO:2), NBNl 40 (SEQ ID N0:6), NBN116 {SEQ JD N0:7), NBNl 12 (SEQ ED NO:8),
NBNl11 (SEQ ID NO:9), NBNl 10 (SEQ ID NO:10), NBN109 (SEQ ID NO:11), NBN108
(SEQ ID N0:12), NBNl 07 (SEQ ID NO:13), NBNl 06 (SEQ ID KO:14), NBN105 £SEQ 3D
NO:15), NBN104 (SEQ ID NO:16), NBN103 (SEQ ID N0:17), NBNl 02 (SEQ ID NO:18),
NBN101 (SEQ ID NO:19), NBN100 (SEQ ID NO:2G) and NBN99 {SEQ ID NO:21).
Preferably, at least one of the two amino-terminal amino acids in the dimer is
conjugated to a polymer. Preferred amino acid substitutions include replacement of an
arginine residue with a lysine residue (Raa#K; where aa# is the amino acid number based on
SEQ ID NO:1), and replacement of an asparagine residue with a lysine residue {Naa#K) or an
aspartate residue (Naa#D). Specific-examples of such substitution are R14K,.R39K, R68K,
N95D, and N95K (numbering based on 13EQ ID NO: 1). A particularly preferred substitution
isN95K.
Preferably, the total combined molecular weight of the polymers on atlimer is
20,000-40,000 Da. Preferably, the average molecular weight ofeach polymer is 2,000-
100,000 Da; more preferably, 5,000-50,000 Da; and most preferably, about 10,000 to 20,000
Da. The polymer can be linear or branched. Preferably, the polymer is a polyalkyiene glycol
moiety, e.g., a polyethylene glycol (PEG) moiety. In some embodiments, at least one
polypeptide is glycosylated.
In some embodiments of the invention, the polymer-conjugated dimer contains a first
polypeptide and a second polypeptide, wherein: (a) each polypeptide individuallyxjomprises
100 to 110 amino acids of SEQ 3D NO:1, (b) each polypeptide comprises an asparagine-tolysine
substitution at amino acid number 95 in SEQ ID NO:1, (c) and the dimer comprises 3
or 4 PEG moieties, wherein the molecular weight of each PEG moiety is about lt),000 Da,
and each PEG moiety is conjugated at an ammo-terminus or at lysine 95. A preferred
embodiment is a homodimer containing a pair of monomers designated 3(,4)x 104cDa PEG
NBN106-N95K.
The invention includes a pharmaceutical-composition comprising a dimer according
to the invention. In some embodiments, the composition contains two or mor-e different
dimers according to the invention.
The invention includes a nucleic acid, e.g., a DNA expression vector that encodes a
polypeptide for incorporation into a dimer of the invention. The invention also includes a
host cell transformed with the nucleic acid. ,
The invention includes a method for beating neuropathic pain in a mammal. The
method includes administering to the mammal a therapeutically effective amount of the dimer
of the invention. In some embodiments, the therapeutically •effective amount is from 0.1
ug/kg to 1000 ug/kg, from 1 ug/kg to 100 ug/kg, or from 1 ug/kg to30 .ug/kg. Administration
of the dimer can be by various routes, e.g., intramuscular, subcutaneous or intravenous. In
some methods according to the invention, the dimer is administered three times per week.
The invention also provides a method -of activating the RET receptor in a mammal. The
method includes administering to the mammal an effective amount of the dimer.
Other features and advantages of the invention will be apparent from the -following
detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a scatter plot summarizing data from a KIRA ELIS A analysis of 3,(4)X10K
PEGylated NBN106-N95K, compared to wild-type NBN113.
FIG. 2 is a scatter plot summarizing data from a KIRA ELIS A analysis of 3X1 OK
PEGylated NBN106-N95K and 4X10K PEGylated NBN106-N95K, -compared to wM-type
NBN113. FIG. 3 is abroken line plot illustrating near complete reversal of fully -established
tactile allodynia by 10 ng/kg of a mixture of 3xlOK PEGylated NBN106-N95K and 4xlOK
PEGylated NBN106-N95K (i.e., "3(,4)x 10 kDa PEG NBN106-N95K") in rats with spinal
nerve ligation.
FIG. 4 is a broken line plot illustrating near complete reversal of fully-established
thermal hyperalgesia by 10 ug/kg of 3(,4)x 10 kDa PEGylated NBN106-N95K in rats with
spinal nerve Jigation.
DETAILED DESCRIPTION OF THE INVENTION
Unless otherwise stated, any reference to a neublastin amino acid position number will refer to the numbering illustrated in SEQ ID NO: 1.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will -control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
As used herein, "wild-type neublastin polypeptide" means a naturally-occurring neublastin polypeptide sequence. A wild-type neublastin polypeptide may be further defined by the source of the neublastin, for example, human, mouse, or rat neublastin (see, e.g., SEQ ID NO: 2, 3, or 4). A consensus neublastin polypeptide sequence is provided as SEQ ID NO:l.
As used herein, "mutated neublastin polypeptide," means a polypeptide that-contains at least a specified minimum level of sequence identity with respect to a wild-type neublastin polypeptide, contains at least one amino acid substitution, insertion or fusion, with respect to the

wild-type neublastin polypeptide, and displays neublastin activity fag., as described in
International application No. PCT/US02/02319 (WO 02/060929).)
As used herein, "internal polymer conjugation site" means a non-terminal amino acid
residue in a mutated neublastin polypeptide, which residue provides a side chain suitable for
conjugation of a polymer.
As used herein, "modified neublastin polypeptide," means a polypeptide that contains at
least one attached polymer.
As used herein, "fusion" means a co-linear, covalent linkage of two or more
polypeptides through their respective peptide backbones through-genetic expression of a
polynucleotide molecule encoding those proteins in the same reading frame.
As used herein, "identity" refers to the sequence similarity between two polypeptides,
molecules or between two nucleic acids. When a position in both of the two compared
sequences is occupied by the same base or amino acid monomer subunit (for instance, if a
position in each of the two DNA molecules is occupied by adenine, or a position in each t>f
two polypeptides is occupied by a lysine), then the respective molecules are homologous at
that position. The "percentage identity" between two sequences is a function of the number
of matching positions shared by the two sequences divided by the number of positions
compared x 100. For instance, if 6 of 10 positions in two sequences are matched, then the two
sequences have 60% identity. By way of example, the DNA sequences CTGACT and
CAGGTT share 50% homology (3 of the 6 total positions are matched). Generally, a
comparison is made when two sequences are aligned to give maximum homology. Such
alignment can be provided using, for instance, the method of Needleman et al., J. Mol Biol.
48: 443-453 (1970), implemented conveniently by computer programs such as the Align
program (DNAstar, Inc.). "Similar" sequences are those which, when aligned, share identical
and similar amino acid residues, where similar residues are conservative substitutions for, or
"allowed point mutations" of, corresponding amino acid residues in an aligned reference
sequence. In this regard, a "conservative substitution" of a residue in a reference sequence is a
substitution by a residue that is physically or functionally similar to the corresponding
reference residue, e.g., that has a similar size, shape, -electric charge, chemical properties,
including the ability to form covalent or hydrogen bonds, or the like. Thus, a conservative
substitution mutated" sequence is one that differs from areference sequence or a wild-type
sequence in that one or more conservative substitutions or allowed point mutations are
present. The "percentage positive" between two sequences is a function of the number of
positions that contain matching residues or conservative substitutions shared by the two
sequences divided by the number of positions compared x 100. For instance, if 6 of 10
positions in two sequences are matched and 2 of 10 positions contain conservative
substitutions, then the two sequences have 80% positive homology.
Mutated Neublastin Polypeptides
The mutated neublastin polypeptides of the invention retain neurotrophic activity and
have enhanced bioavailability as compared to the wild-type neublastin polypeptide. For
example, the mutated neublastins of this invention activate the RET gene product hi assays in
which the wild-type neublastin activates RET. hi general, the mutated neublastin polypeptide
will retain at least one of the following features but will additionally comprise at least one
modification, such that an internal polymer conjugation site is created:
(i) seven conserved cysteine residues at positions 16,43,47, SO, 81,109, and 111
when numbered in accordance with SEQ ID NO: 1-4;
(ii) amino acid residues as follows:
C at position 16, L at position 18, V at position 25, L at position 28,
G at position 29, L at position 30, G at position 31, E at position 36, F at
position 40, R at position 41, F at position 42, C at position 43, G at position
45, C at position 47, C at position 80, C at position 81, R at position 82, P at
position 83, F at position 91, D at position 93, S at position 105, A at position
106, C at position 109 and C at position 111, each when numbered in
accordance with SEQ ID NO: 1-4;
(iii) an LGLG repeat, an FRFC motif, a QPCCRP motif, and a SATACGC motif.
In some embodiments, the invention provides a truncated mutated neublastin
polypeptide, wherein the amino terminus of the truncated neublastin polypeptide lacks one or
more amino-terminal amino acids of a mature neublastin polypeptide but is mutated to
possess an internal polymer attachment. Preferably, the truncated mutated neublastin
polypeptide, when dimeriaed, activates a RET polypeptide. In some embodiments the
mutated neublastin polypeptide induces dimenzation of the RET polypeptide. "Such induction
may require additional polypeptides or co-factors, as would be apparent to one of skill in the
art.
Amino acid sequences of human and mouse neublastin polypeptides are disclosed in
PCTpublicationWOOO/01815. Examples of wild-type neublastin polypeptides according to
the invention are presented in Table 1A. A neublastin consensus sequence (•consensus with
respect to human, mouse and rat) is set forth in Table IB.
(Table Removed)
In some embodiments, at least one of the arginuie (Arg or R) or asparagine (Asn orN)
residues shown in bold in Table 1A is substituted with a diiferent amino acid residue. In a
preferred embodiment, the mutated neublastin polypeptide has a lysine (Lys or K) residue
substituted for the asparagine at amino acid position 95, indicated by an asterisk in Table 1 A,
and is referred to as NBN-N95K. In general, the N95K substitution results in improved
solubility. This facilitates formulation at high concentrations.
Table IE. I^NllS Consensus Sequence
(Table Removed)
The invention includes a polymer-conjugated mutated neublastin polypeptide
comprising an amino acid sequence that is, for example, at least 70% identical to amino acids
8-113 of SEQ ID NO:1 (also shown in Table 1). In one embodiment, one or more of the
arginines at position 14, position 39, position 68, or the asparagine at position 95 is replaced
by an amino acid other than arginine or asparagine. In one embodiment, the wild-type amino
acid is substituted with lysine or cysteine.
The substituted residues in the mutated neublastin polypeptide can be chosen to
facilitate coupling of a polymer, e.g., a polyalkylene glycol polymer, at the substituted amino
acid. Advantageous sites of modification are those at solvent accessible regions in the
neublastin polypeptide. Such sites can be chosen based on inspection of the crystal structure
of the related neurotrophic factor, such as GDNF, whose crystal structure is described in
Eigenbrot et al., Nat. Struct. Biol. 4:435-38,1997. Sites also can be chosen basedtm the
crystal structure of neublastin, whose crystallization and structure determination is described
below. Also, sites can be chosen based on structural-functional information provided for
persephin/neublastin chimeric proteins. These chimeras are described in Baloh et al., J. Biol.
Chem. 275:3412-20,2000. An exemplary listing of solvent accessible or surface exposed
neublastin amino acids identified through this methodology is set forth in Table 2.
Table 2 provides a list of residues and numbers in human neublastin that are expected,
to be surface exposed. The first column refers to surface exposed residues determined by
examining the structure of the rat GDNF dimer formed by chains A and B (PDB code 1AGQ)
and determining whether a residue was on the surface of the structure. This -structure was
then compared to a sequence alignment of GDNF and neublastin in Baloh et al, Neuron
21:1291-1302,1998 to determine the proper residues in neublastin. The second and third
columns, respectively, refer to the surface exposed residues determined by examining the
structure of the human neublastin dimer formed by chains'A and B. The numbering-scheme
(Table Removed)
indicates that the residues are not present in the structures of GDNF or neublastin. This is
either because of construct design, flexible regions, or inserts in neublastin relative to
GDNF (residues 68-71).
indicates the residues are buried and not on the surface or are cysteine residues involved
in disulfide bonds. As this protein is a cysteine knot, a great majority of the residues ate
on the surface.
indicates that this residue is surface exposed in the GDNF structure or in the neublastin
structure, although the loop containing residues 66-75 is visible in only one of the GDNF
monomers (presumably flexible). This loop also contains a 4 residue insert in neublastin
relative to GDNF.
Insome embodiments, the neublastin poly'peptide retains the seven conserved Cys
residues that are characteristic of the GDNF subfamily and of the TGF-beta super family.
The sequence of the human full-length prepro NBN polypeptkle (SEQ ID NO:5) is
shown in Table 3. Three mature forms of neublastin polypeptides were identified. These
forms include:
(i) the 140 AA polypeptide designated herein as NBN140, which possesses the amino
acid sequence designated as SEQ ID N0:6;
(ii) the 116 AA polypeptide designated herein as NBN116, which possesses the amino
acid sequence designated as SEQ ID N0:7; and
(Hi) the 113 AA polypeptide designated herein as NBN113, which possesses the
amino acid sequence designated as SEQ ID NO:2.
Table 3 illustrates the relationship between the disclosed prepro neublastin
polypeptide sequences of the invention. Line 1 provides the polypeptide of SEQ ID NO:5,
line 2 provides the polypeptide of SEQ ID NO:6, line 3 provides the polypeptide of SEQ ID
N0:7 and line 4 provides the polypeptide of SEQ ID NO:2. The seven conserved cysteine
residues are designated by symbols ("*", "#","+" and "|") to indicate the intramolecular (*
with *, # with #, and + with +) and intermotecular ("J") disulfide bridges formed in the
mature dimerized neublastin ligand. The caret mark ("A") indicates the asparagine residue at
amino acid position 95 that is substituted with a lysine in NBN106-N95K.
(Table Removed)
In alternative embodiments, the sequence of the above identified neublastin
polypeptides have been truncated at their amino-teniiinal amino acid sequence. Examples of
these include:
(iv) the 112AA polypeptide sequence designated herein as NBM112. which possesses
the 112 carboxy terminal amino acids of a neublastin polypeptide, e.g., amino
acids 29-140 of SEQ ID NO:6 (SEQ ID NO:S) or amino acids 2-113 of SEQ ID
NOs:l,3or4.
(v) the 111AA polypeptide sequence-designated herein as NBN111, which possesses
the 111 carboxy terminal amino acids of a neublastin polypeptide, e.g., amino
acids 30-140 of SEQ ID NO:6 (SEQ ID NO:9) or amino acids 3-113 of SEQ ID
NOs:l,3or4.
(vi) the 110AA polypeptide sequence designated herein as NBN110, which possesses
the 110 carboxy terminal amino acids of a neublastin polypeptide, e.g., amino
acids 31-140 of SEQ ID NO:6 (SEQ ID NO:10) or amino acids 4-113 of SEQ ID
NOs:l,3or4.
(vii) the 109AA polypeptide sequence designated herein as NBN109, which possesses
the 109 carboxy terminal amino acids of a neublastin polypeptide, e.g., amino
acids 32-140 of SEQ ID NO:6 (SEQ ID NO:11) or amino acids 5-113 of SEQ ID
NOs:l,3or4.
(viii) the 108AA polypeptide sequence designated herein as NBN108, which
possesses the 108 carboxy terminal amino acids of a neublastin polypeptide, e.g..,
amino acids 33-140 of SEQ ID N0:6 (SEQ ID NO:12) or amino acids €-113 of
SEQ ID NOs: 1,3 or 4.
(ix) the 107AA polypeptide sequence designated herein as NBN107, which possesses
the 107 carboxy terminal amino acids of a neublastin polypeptide, e.g., amino
acids 34-140 of SEQ ID NO:6 (SEQ ID NO:13) or amino acids 7-113 ofSEQ ID
NOs: 1,3 or 4.
(x) the 106AA polypeptide sequence designated herein alternatively as NBN106 or
N-7, which possesses the 106 carboxy terminal amino acids of a neublastin
polypeptide, e.g., amino acids 35-140 of SEQ ID N0:tf (SEQ ID NO: 14) or amino
acids 8-113 of SEQ ID NOs:l, 3 or 4.
(xi) the 105AA polypeptide sequence designated herein as MBN1-05, which possesses
the 105 carboxy terminal amino acids of a neublastin polypeptide, e.g., amino
acids 36-140 of SEQ ID NO:6 (SEQ ID NO:15) or amino acids 9-113of SEQ ED
NOs:l,3or4.
(xii) the 104AA polypeptide sequence designated herein alternatively as NBK104 or
N-9, which possesses the 104 carboxy terminal amino acids of a neublastin
polypeptide, e.g., amino acids 37-140 of SEQ ID NO:6 (SEQ ID NO: 16) or ammo
acids 10-113 of SEQ ID NOs:l, 3 or 4.
(xiii) the 103AApolypeptide sequence designated herein as NBN103, which
possesses the 103 carboxy terminal amino acids,of a neublastin polypeptide, e.g.,
amino acids 38-140 of SEQ ID NO:6 (SEQ ID N0:17) or amino acids 11-1J3 of
SEQ ID NOs: 1,3 or 4.
(xiv) the 102AA polypeptide sequence designated herein as NBN102, which
possesses the 102 carboxy terminal amino acids of a neublastin polypeptide, e.g.,
amino acids 39-140 of SEQ ID N0:6 (SEQ ID NO:18) or amino acids 12-113 of
SEQ ID NOs: 1,3 or 4.
(xv) the 101AA polypeptide sequence designated herein as NBN101, which possesses
the 101 carboxy terminal amino acids of a neublastin polypeptide, e.g., amino
acids 40-140 of SEQ ID NO:6 (SEQ ID NO.19) or amino acids 13-113 of SEQ ID
NOs: 1,3 or 4.
(xvi) the 100AA polypeptide sequence designated herein as NBN100, which
possesses the 100 carboxy terminal amino acids of a neublastin polypeptide, e.g.,
amino acids 41-140 of SEQ ID NO:6 (SEQ ID N0:20) or amino acids 14-113 of
SEQ ID NOs: 1,3 or 4.
(x\>ii) the 99AA polypeptide sequence designated herein alternatively as NBN99 or
N-14, which possesses the 99 carboxy terminal amino acids of a neublastin
polypeptide, e.g., amino acids 42-140 of SEQ ID NO:6 (SEQ ID NO:21) or amino
acids 15-113 of SEQ ID NOs:l, 3 or 4.
The polypeptide sequences of these truncated neublastin polypeptides are shown in
Table 4 for NBN113 through NBN99. Disulfide bridge formation is as described for (Table Removed)

A mutated neublastin polypeptide according to the invention can be, e.g., at least
80%, 85%, 90%, 95%, 98% or 99% identical to amino acids 8-113 of SEQ ID NO:1. In
some embodiments, the amino acid sequence of the mutated neublastin polypeptide includes
the amino acid sequence of a naturally occurring rat, human or mouse neublastin polypeptide
at amino acids 1-94 and 96-113 of the mutated neublastin polypeptide, e.g., the polypeptide
has the amino acid sequence of SEQ ID NOs: 2, 3, or 4 at these positions.
A mutated neublastin polypeptide differing in sequence from those disclosed in SEQ
ID NOs:l-4 may include one or more conservative amino acid substitutions. Alternatively,
or in addition, the mutated neublastin polypeptide may differ by one or more non
conservative amino acid substitutions, or by deletions or insertions. Preferably, the
substitutions, insertions or deletions do not abolish the isolated protein's biological activity.
A conservative substitution is the substitution of one amino acid for another with
similar characteristics. Conservative substitutions include substitutions within the following
groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic
acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and
phenylalanine and tyrosine. The non-polar hydrophobic amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral
amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
The positively charged (basic) amino acids include arginine, lysine and histidine. The
negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any
substitution of one member of the above-mentioned polar, basic or acidic groups by another
member of the same group can be deemed a conservative substitution.
Other substitutions can be readily identified by those of ordinary skill in the art. For
example, for the amino acid alanine, a substitution can be taken from any onet>f D-alanine,
glycine, beta-alanine, cysteine and D-cysteine. For lysine, a replacement can be any one of
D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, omithine, or
D-ornithine. Generally, substitutions in functionally important regions .mat may be expected
to induce changes in the properties of isolated polypeptides are those in which: (i) a polar
residue, e.g., serine or threonine, is substituted for (or b}') a hydrophobic residue, e.g.,
leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by)
any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or
histidine, is substituted for (or by) a residue having an electronegative-side chain, e.g.,
glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine,
is substituted for (or by) one not having such a side chain, e.g., glycine. The likelihood that
one of the foregoing non-conservative substitutions may alter functional properties of the
protein is also correlated to the position of the substitution with respect to functionally
important regions of the protein. Some non-conservative substitutions may accordingly have
little or no effect on biological properties.
In many cases, a polymer-conjugated mutated neublastin polypeptide has a longer
serum half-life relative to the half-life of the wild-type polypeptide or mutated polypeptide in
the absence of the polymer. In some embodiments, the polymer conjugated mutated
neublastin polypeptide has significantly increased potency in vivo relative to the potency of
the polypeptide or glycosylated-polypeptide in the absence of the polymer.
The polymer-conjugated neublastin polypeptide can be provided as a dimer that
includes at least one polymer-conjugated neublastin polypeptide. hi some embodiments, the
dimer is a homodimer of polymer-conjugated mutated neublastin polypeptides. In other
embodiments, the dimer is a homodimer of polymer-conjugated mutated truncated neublastin
polypeptides. In other embodiments, the dimer is a heterodimer that includes one polymerconjugated
mutated neublastin polypeptide and one wild-type neublastin polypeptide. hi
other embodiments, the dimer is a heterodimer that includes one polymer-conjugated mutated
neublastin polypeptide, and one polymer-conjugated wild-type neublastin polypeptide where
the polymer conjugation is at the amino-terminus, and where the polypeptides may or may
not be truncated. Other dimers include heterodirners or homodimers of polymer-conjugated
mutated neublastin polypeptide forms that may or may not be truncated.
Provided in the invention are mature and truncated mutated polypeptide sequences
comprising the carboxy-terminal-most amino acid residues of the preproNBN polypeptide,
such as provided in SEQ ID NO:5, and which are designated herein as NBN#, where #
represents the number of carboxy-terminal residues remaining in the referenced neublastin
polypeptide. Polymer-conjugated neublastin polypeptides present in the bioactive neublastin
dimers may be products of a protease cleavage reaction or a chemical cleavage reaction, or
may be expressed from recombinant DMA construct, or may be synthesized. Example
neublastin polypeptides include, e.g., NBN140, NBN116, and NBN113. Additional
neublastin polypeptides of the invention include NBN112, NBN111, NBN110, NBN109,
NBN108, NBN107, NBN106, NBN105, NBN104, NBN103, NBN102, NBN101, NBNKX)
and NBN99 1SEQ ID NOS:S-21).
A preferred polymer-conjugated neublastin polypeptide is a homodimer of NBN106-
N95K conjugated either to three 10 kDa PEG moieties ("3x10 kDa PEG NBN106-N95K")or
to four 10 kDa PEG moieties ("4x10 kDa PEG NBN106-N95K"). Also pr-e-ferred is a mixed
population of NBN106-N95K homodimers conjugated either to three 10 kDa PEG moieties
or to four 10 kDa PEG moieties, referred to herein as "3(,4)xlO kDa PEG NBN1-06-N95K".
Also preferred is a 3(,4)xlO kDa PEG NBN106-N95K homodimer, wherein the two aminoterminal
amino acids are covalently linked to PEG moieties and the third and/or fourth PEG
moiety is covalently linked to one or both substituted N95K residue(s).
In some embodiments, the polymer-conjugated neublastin polypeptide is based on the
consensus sequence of SEQ ED NO:1. In certain embodiments, a polymer-conjugated
neublastin polypeptide includes amino acids 1-7 of SEQ ID NO:1 in addition to amino acids
8-113.
hi some embodiments, the polymer-conjugated neublastin polypeptide, when
dimerized, binds GFRct3. In some embodiments, the polymer-conjugated neublastin
polypeptide, when dimerized, stimulates tyrosine phosphorylation of a RET polypeptide,
either on its own or when bound to GFRa3.
In some embodiments, the polymer-conjugated neublastin polypeptide, when
dimerized, enhances neuron survival, e.g., enhances survival of a sensory neuron.
hi some embodiments, the polymer-conjugated neublastin polypeptide, when
dimerized, reduces or reverses pathological changes of a neuron, such as a sensory neuron.
hi some embodiments, the polymer-conjugated neublastin polypeptide, when
dimerized, enhances survival of a neuron, e.g., an autonomic neuron, or a dopaminergic
neuron.
hi some embodiments, the polymer-conjugated neublastin polypeptide includes one,
two, three, four or more of the amino acid substitutions selected from the group consisting of
an amino acid other than arginine at position 14 in the amino acid sequence of the polymerconjugated
polypeptide, an amino acid other than arginine at position 39 in the amino acid
sequence of the polymer-conjugated polypeptide, an amino acid other than arginine at
position 68 of the polymer-conjugated polypeptide, and an amino acid other than asparagine
at position 95 of the polymer-conjugated polypeptide. In some embodiments, the amino acid
at one or more of the amino acid at positions 14,39,68, and 95 is lysine. Preferably, amino
acids 8-94 and 96-113 of the polymer-conjugated neublastin polypeptide are at least 90%
identical to amino acids 8-94 and 96-113 ofSEQ ID NO:1. More preferably, the amino acids
sequences are at least 95% identical thereto. Most preferably, the amino acid-sequence of the
polymer-conjugated neublastin polypeptide includes the amino acid sequence of a naturally
occurring human, mouse or rat neublastin polypeptide at amino acids 8-94 and 945-113 of the
polymer-conjugated neublastin polypeptide. For example, amino acids 8-94 and 96-113 of
the polymer-conjugated neublastin polypeptide can include the amino acid sequence of amino
acids 8-94 and 96-113 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO: 4. In
the above embodiments, the preferred residue at amino acid position 95 is a lysine or a
cysteine.
The invention includes a construct that is a heterodimer or a homodimer containing
polymer-conjugated neublastin fusion proteins, e.g., the polyhistidine (His)-tagged neublastin
provided in SEQ ID NO:36, or a neublastin fusion protein where the fusion moiety is an
immunoglobulin (Ig) polypeptide,serum albumin polypeptide or a replicase-derived
polypeptide. Neublastin fusion proteins can have enhanced pharmacokmetic and
bioavailability properties in vivo.
The invention provides a nucleic acid molecule encoding a mature or truncated
neublastin polypeptide, with a mutated polypeptide sequence. The nucleic acid molecule
encoding a provided neublastin polypeptide is preferably provided in a vector, e.g., an
expression vector. A mutated neublastin nucleic acid molecule, or a vector including the
same, can be provided in a cell. The cell can be, e.g., a mammalian cell, fungal cell, yeast
cell, insect cell, or bacterial cell. A preferred mammalian cell is a Chinese hamster ovary cell
("CHO cell").
Also provided by the invention is a method of making a polymer-conjugated
neublastin polypeptide, by culturing a cell containing a nucleic acid encoding a neublastin
polypeptide under conditions allowing for expression of a neublastin polypeptide. hi some
embodiments, the neublastin is conjugated to a naturally occurring moiety. In specific
embodiments, the naturally occurring moiety is a glycosyl moiety. In certain embodiments,
the glycosylated neublastin is expressed, e.g., in a CHO cell. The invention further includes a
neublastin polypeptide expressed in a cell. Similar nucleic acids, vectors, iost cells,, and
polypeptide production methods are disclosed herein for the fusion proteins {such as the
neublastin-serum albumin fusion proteins) of this invention.
hi some embodiments, a neublastin polypeptide that is expressed in a cell is recovered
and conjugated to a. polymer. In some embodiments, the polymer is a polyalkylene glycol
moiety. In particular embodiments, the polymer is a PEG moiety.
Specifically provided by the invention is a composition that includes a mutated
neublastin polypeptide coupled to a non-naturally occurring polymer. The mutated
neublastin polypeptide in the composition preferably includes an amino acid sequence at least
70% identical to amino acids 8-113 of SEQ ID NO: 1, provided that the polymer-conjugated
neublastin polypeptide includes one or more of the amino acid substitutions selected from the
group consisting of an amino acid other than arginine at position 14 in the amino acid
sequence of the polymer-conjugated polypeptide, an amino acid other man arginine at
position 39 in the amino acid sequence of the polymer-conjugated polypeptide, an amino acid
other than arginine at position 68 of the polymer-conjugated polypeptide, and an amino acid
other than asparagine at position 95 of the polymer-conjugated polypeptide, wherein the
positions of the amino acids are numbered in accordance with the polypeptide sequence of
SEQIDNO:!.
The invention includes a stable, aqueous soluble conjugated neublastin polypeptide or
mutated neublastin polypeptide complex comprising a neublastin polypeptide or mutated
neublastin polypeptide coupled to a PEG moiety, wherein the neublastin polypeptide or
mutated neublastin polypeptide is coupled to the PEG moiety by a labile bond. In some
embodiments, the labile bond is cleavable by biochemical hydrolysis, proteolysis, or
sulfhydryl cleavage, hi some embodiments, the labile bond is cleavable under in vivo
conditions.
Also provided by the invention is a method for making a modified neublastin
polypeptide that has prolonged serum half-life relative to a wild-type neublastin. The method
included providing a neublastin polypeptide or mutated neublastin polypeptide, and coupling
the polypeptide or mutatedneublastin polypeptide to a non-naturally occurring polymer
moiety, thereby forming a coupled polymer neublastin polypeptide composition.
The polymer-conjugated mutated neublastin polypeptides of this invention include
one or more amino acid substitutions in which, for example, an amino acid other than
arginine occurs at position 14 in the amino acid sequence of the polymer-conjugated
polypeptide, an amino acid other than arginine at position 39 occurs in the amino acid
sequence of the polymer-conjugated polypeptide, an amino acid other than arginine at
position 68 occurs in the polymer-conjugated polypeptide, or an amino acid other than
asparagine at position 95 occurs in the polymer-conjugated polypeptide, when the positions
of the amino acids are numbered in accordance with the polypeptide sequence of SEQ
IDNOrl.
Synthesis and Isolation of Wild-Type and Mutated Neublastin Polypeptides
Neublastin polypeptides can be isolated using methods known in the art. Naturally
occurring neublastin polypeptides can be isolated from cells or tissue sources by an
appropriate purification scheme using standard protein purification techniques. Alternatively,
mutated neublastin polypeptides can be synthesized chemically using standard peptide
synthesis techniques. The synthesis of short amino acid sequences is well established in the
peptide art. See, e.g., Stewart, et al., Solid Phase Peptide Synthesis (2d ed., 1984).
In some embodiments, mutated neublastin polypeptides are produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding a mutated neublastin
polypeptide can be inserted into a vector, e.g., an expression vector, and the nucleic acid can
be introduced into a cell. Suitable cells include, e.g., mammalian cells (such as human cells
or CHO cells), fungal cells, yeast cells, insect cells, and bacterial cells. When expressed in a
recombinant cell, the cell is preferably cultured under conditions allowing for expression of a
mutated neublastin polypeptide. The mutated neublastin polypeptide can be recovered from a
cell suspension if desired. As used herein, "recovered" means that the mutated polypeptide is
removed from those components of a cell or culture medium in which it is present prior to the
recovery process. The recovery process may include one or more refolding or purification
steps.
Mutated neublastin polypeptides can be constructed using any of several methods
known in the art. One such method is site-directed mutagenesis, in which a specific
nucleotide (or, if desired a small number of specific nucleotides) is changed in order to
change a single amino acid (or, if desired, a small number of predetermined amino acid
residues) in the encoded neublastin polypeptide. Those skilled in the art recognize that sitedirected
mutagenesis is a routine and widely used technique. In fact, many site-directed
mutagenesis kits are commercially available. One such kit is the "Transformer Site Directed
Mutagenesis Kit" sold by Clontech Laboratories (Palo Alto, Calif.).
Practice of the present invention will employ, unless indicated otherwise,
conventional techniques of cell biology, cell culture, molecular biology, microbiology,
recombinant DNA, protein chemistry, and immunology, which are within the skill of the art.
Such techniques are described in the literature. See, for example, Molecular Cloning: A
Laboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.), Cold Spring Harbor
Laboratory Press, 1989; DNA Cloning, Volumes I and fl (D.N. Glover, ed), 1985;
Oligonucleotide Synthesis, (M.J. Gait, ed.), 1984; U.S. Patent No. 4,683,195 (Mullis et a/
Nucleic Acid Hybridization (B.D. Haines and SJ. Higgins, eds.), 1984; Transcription and
Translation (B.D. Hames and SJ. Higgins, eds.), 1984; Culture of Animal Cells (R.I.
Freshney, ed). AlanR. Liss, Inc., 1987; Immobilized Cells and Enzymes, IRL Press, 1986; A
Practical Guide to Molecular Cloning (13.Perbal), 1984; Methods in Enzymology, Volumes
154 and 155 (Wu et al, eds), Academic Press, New York; Gene Transfer Vectors for
Mammalian Cells (J.H. Miller and M.P. Calos, eds.), 1987, Cold Spring Harbor Laboratory;
Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker,-eds.),
Academic Press, London, 1987; Handbook of Experiment Immunology, Volumes I-IV (D.M.
Weir and C.C. Blackwell, eds.), 1986; Manipulating the Mouse Embryo, Cold Spring Harbor
Laboratory Press, 1986.
Polymer Conjugation of Neublastin Polypep tides
Chemically modified neublastin polypeptides may be prepared by one of-skill in the
art based upon the present disclosure. The chemical moieties preferred for conjugation to a
neublastin polypeptide are water-soluble polymers. A water-soluble polymer is advantageous
because the protein to which it is attached does not precipitate in an aqueous environment,
such as a physiological environment. Preferably, the polymer will be pharmaceutically
acceptable for the preparation of a therapeutic product or composition.
If desired, a single polymer molecule may be employed for conjugation with a
neublastin polypeptide, although more than one polymer molecule can be attached as well.
Conjugated neublastin compositions of the invention may find utility in both hi vivo as well
as non-in vivo applications. Additionally, it will be recognized that the conjugating polymer
may utilize any other groups, moieties, or other conjugated species, as appropriate to the end
use application. By way of example, it may be useful in some applications to covalently
bond to the polymer a functional moiety imparting UV-degradation resistance, or
antioxidation, or other properties or characteristics to the polymer. As a further example, it
may be advantageous in some applications to functionalize the polymer to render it reactive
or cross-linkable in character, to enhance various properties or characteristics of the overall
conjugated material. Accordingly, the polymer may contain any functionality, repeating
groups, linkages, or other constituent structures that do not preclude the efficacy of the
conjugated neublastin composition for its intended purpose.
One skilled in the art will be able to select the desired polymer based on such
considerations as whether the polymer/protein conjugate will be used therapeutically, and if
so, the desired dosage, circulation time, resistance to proteolysis, and other considerations.
The effectiveness of the derivatization may be ascertained by administering the derivative, in
the desired form {e.g., by osmotic pump, or, more preferably, by injection or infusion, or,
further formulated for oral, pulmonary or other delivery routes), and determining its
effectiveness.
Suitable water-soluble polymers include, but are not limited to, PEG, copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1, 3,6-trioxane, ethylene/maleic anhydride
copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or
poly(n-vinyl pyrrolidone) PEG, propropylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl
alcohol, and mixtures thereof.
The polymer may be of any suitable molecular weight, and may be branched or
unbranched.
For PEG, suitable average molecular weight is between about 2 kDa and about 100
kDa. This provides for ease hi handling and manufacturing. Those of skill in the art will
appreciate that in preparations of PEG, some molecules will weigh more, some less, than the
stated molecular weight. Thus, molecular weight is typically specified as "average molecular
weight." Other molecular weights (sizes) may be used, depending on the desired therapeutic
profile (e.g., the duration of sustained release desired; the effects, if any, on biological
activity; the ease in handling; the degree or lack of antigenicity and other known effects of
PEG on a therapeutic protein). In various embodiments, the molecular weight is about 2 kDa,
about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about
40 kDa or about 100 kDa. In certain preferred embodiments, the average molecular weight of
each PEG chain is about 20 kDa. In certain preferred embodiments, the average molecular
weight is about 10 kDa.
The number of polymer molecules so attached may vary, and one skilled in the art
v/ill be able to ascertain the effect on function. One may mono-derivatize, or may provide for
a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical
moieties (e.g., polymers, such as different weights of PEGs). The proportion of polymer
molecules to protein (or polypeptide) molecules will vary, as will their concentrations in the
reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that
there is no excess unreacted protein or polymer) will be determined by factors-such as the
desired degree of derivatization (e.g., mono, di-, tri-, etc.), the molecular weight of the
polymer selected, whether the polymer is branched or unbranched, and the reaction
conditions.
The PEG molecules {or other chemical moieties) should be attached to the protein
with consideration of effects on functional or antigenic domains of the protein. There are a
number of attachment methods available to those skilled in the art. See, e.g., EP 0 4013S4
(coupling PEG to G-CSF); Malik et al., Exp. Hematol. 20:1028-1035,1992 (reporting
PEGylation of GM-CSF using tresyl chloride).
For example, PEG may be covalently bound (PEGylation) through amino acid
residues via a reactive group, such as, a free amino or carboxyl group. The amino acid
residues having a free amino group include lysine residues and the ammo-terminal amino
acid residue. Those having a free carboxyl group include aspartic acid residues, glutamic
acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as
a reactive group for attaching the PEG molecule(s). For therapeutic purposes, attachment can
be at an amino group, e.g. at the N- terminus or lysine group. One may specifically desire an
amino-terminal chemically modified protein.
Using PEG as an illustration of the present compositions, one may select from a
variety of PEG molecules (by molecular weight, branching, etc.), the proportion of PEG
molecules to protein (or peptide) molecules in the reaction mix, the type of PEGylation
reaction to be performed, and the method of obtaining the selected amino-terminally
PEGylated protein. The method of obtaining the amino-terminal PEGylated preparation (i.e.,
separating this moiety from other monoPEGylated moieties if necessary) may be by
purification of the amino-terminal PEGylated material from a population of PEGylated
protein molecules. Selective amino-terminal chemical modification may be accomplished by
reductive alkylation that exploits differential reactivity of different types of primary amino
groups (lysine versus the amino-terminal) available for derivatization in a particularprotein.
Under the appropriate reaction conditions, substantially selective derivatization .of the protein
at the ammo-terminus with a carbonyl group containing polymer is achieved. For example,
one may selectively PEGylate the ammo-terminus of the protein by performing the reaction
at a pH which allows one to take advantage of the pKa differences between the epsilon
(8)-amino group of the lysine residues and that of the alpha (a)-amino group of the aminoterminal
residue of the protein. By such selective derivatization, attachment of a water
soluble polymer to a protein is controlled: the conjugation with the polymer takes place
predominantly at the amino-terminus of the protein and no significant modification of other
reactive groups, such as the lysine side -chain amino groups, occurs.
Using reductive alkylation, the water-soluble polymer may be of the type described
above, and should have a single reactive aldehyde for coupling to the protein. PEG
propionaldehyde, containing a single reactive aldehyde, may be used.
The present invention includes mutated neublastin polypeptides that are expressed in
prokaryotes or eukaryotes or made synthetically. In some embodiments, the neublastin is
glycosylated. In some specific embodiments, the neublastin dimer is polymer-conjugated at
each amino-terminus and glycosylated at each internal Asn95 residue. Li other embodiments,
the mutated neublastin dimer is polymer-conjugated at each amino-terminus and polymerconjugated
at one or both internal Lys95 residues.
PEGylation maybe carried out by any suitable PEGylation reaction. Various
PEGylation chemistries are known in the art. See, e.g., Focus on Growth Factors, 3 (2): 4-10,
1992; EP 0 154 316; EP 0 401 384; and the other publications cited herein that relate to
PEGylation. The PEGylation may be carried out via an acylation reaction or an alkylation
reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
PEGylation by acylation generally involves reacting an active ester derivative of PEG.
Any known or subsequently discovered reactive PEG molecule may be used to carry out the
PEGylation. A preferred activated PEG ester is PEG esterified to N-hydroxysuccinimide
(NHS). As used herein, "acylation" includes without limitation the following types of
linkages between the therapeutic protein and a water soluble polymer such as PEG: amide,
carbamate, urethane, and the like. See, Bioconjugate Chem. 5: 133-140,1994. Reaction
conditions may be selected from any of those known in the PEGylation art or those
subsequently developed, but should avoid conditions such as temperature, solvent, and pH
that would inactivate the neublastin protein or polypeptide to be modified.
PEGylation by acylation will generally result in a poly-PEGylated neublastin protein
product. Preferably, the connecting linkage will be an amide. Also preferably, the resulting
product will be substantially only (e.g., > 95%) mono, di- or tri-PEGylated. However, some
species with higher degrees of PBGylation may be formed in amounts depending on the
specific reaction conditions used. If desired, more purified PEGylated species may be
separated from the mixture, particularly unreacted species, by standard purification
techniques, including, among others, dialysis, salting-out, ultrafiltration, ion- exchange
chromatography, gel filtration chromatography and electrophoresis.
PEGylation by alkylation generally involves reacting a terminal aldehyde derivative
of PEG with neublastin in the presence of a reducing agent. PEGylation by alkylation can
also result in poly-PEGylated neublastin protein products. In addition, one can manipulate
the reaction conditions to favor PEGylation substantially only at the cc-amino .group of the
amino-terminus of neublastin (i.e., a mono-PEGylated protein). In either ease of mono-
PEGylation or poly-PEGylation, the PEG groups are preferably attached to the protein via a
-CH2-NH- group. With particular reference to the -CH2- group, this type of linkage is
referred to herein as an "alky!" linkage.
Derivatization via reductive alkylation to produce a mono-PEGylated product exploits
differential reactivity of different types of primary amino groups (lysine versus the aminoterminal)
available for derivatization. The reaction is performed at a pH that allows one to
take advantage of the pKa differences between the e-amino groups of the lysine residues and
that of the a-amino group of the amino-terminal residue of the protein. By such selective
derivatization, attachment of a water soluble polymer that contains a reactive group such as
an aldehyde, to a protein is controlled: the conjugation with the polymer takes place
predominantly at the amino-terminus of the protein and no significant modification of other
reactive groups, such as the lysine side chain amino groups, occurs.
The polymer molecules used in both the acylation and alkylation approaches may be
selected from among water-soluble polymers as described above. The polymer selected
should be modified to have a single reactive group, such as an active ester for acylation or an
aldehyde for alkylation, preferably, so that the degree of polymerization may be controlled as
provided for in the present methods. An exemplary reactive PEG aldehyde is PEG
propionaldehyde, which is water stable, or mono Cl-Cl 0 alkoxy or aryloxy derivatives
thereof (see, U.S. Patent 5,252,714). The polymer may be branched or unbranched. For the
acylation reactions, the polymer(s^) selected should have a single reactive ester .group. For the
present reductive alkylation, the polymer(s) selected should have a single reactive aldehyde
group. Generally, the water-soluble polymer will not be selected from naturally-occurring
glycosyl residues since these are usually made more conveniently by mammalian
recombinant expression systems. The polymer may be of any molecular weight, and maybe
branched or unbranched.
An exemplar}' water-soluble polymer for use herein is PEG. As used herein,
polyethylene glycol encompasses any of the forms of PEG that have been4ised other proteins, including but not limited to, e.g., mono-(Cl-ClO) alkoxy- or aryloxy-PEG.
In general, chemical derivatization may be performed under any suitable condition
used to react a biologically active substance with an activated polymer molecule. Methods
for preparing a PEGylated neublastin will generally comprise the steps of (a) reacting a
neublastin protein or polypeptide with PEG (such as a reactive-ester or aldehyde derivative of
PEG) under conditions whereby the molecule becomes attached to one or more PEG groups,
and (b) obtaining the reaction produces). In general, the optimal reaction conditions for the
acylation reactions will be determined case by case based on known parameters and the
desired result. For example, the larger the ratio of PEGrprotein, the greater the percentage of
poly-PEGylated product.
Reductive alkylation to produce a substantially homogeneous population of monopolymer/
neublastin will generally comprise the steps of: (a) reacting a neublastin protein or
polypeptide with a reactive PEG molecule under reductive alkylation conditions, at a pH
suitable to pen-nit selective modification of the a-amino group at the amino terminus of
neublastin; and (b) obtaining the reaction product(s).
For a substantially homogeneous population of mono-polymer/ neublastin, the
reductive alkylation reaction conditions are those that permit the selective attachment of the
water-soluble polymer moiety to the ammo-terminus of neublastin. Such reaction conditions
generally provide for pKa differences between the lysine amino groups and the a-amino
group at the ammo-terminus (the pKa being the pH at which 50% of the amino groups are
protonated and 50% are not). The pH also affects the ratio of polymer to protein to be used.
In general, if the pH is lower, a larger excess of polymer to protein will be desired {z.e., the
less reactive the amino-terminal a-amino group, the more polymer needed to achieve optimal
conditions). If the pH is higher, the polymenprotein ratio need not be as large (i.e., more
reactive groups are available, so fewer polymer molecules are needed). For purposes of the
present invention, the pH will generally fall within the range of 3-9, preferably 3-6.
Another important consideration is the molecular weight of the polymer. In general,
the higher the molecular weight of the polymer, the fewer polymer molecules may be
attached to the protein. Similarly, branching of the polymer should be taken into account
when optimizing these parameters. Generally, the higher the molecular weight (or the more
branches) the higher the polymenprotein ratio, hi general, for the PEGylation reactions
included herein, the preferred average molecular weight is about 2 kDa to about 1T)0 kDa.
The preferred average molecular weight is about 5 kDa to about 50 kDa, particularly
preferably about 10 kDa to about 20 kDa. The preferred total molecular weight is about
lOkDa to about 40 kDa.
In some embodiments, the neublastin polypeptide is linked to the polymer via a
terminal reactive group on the polypeptide. Alternatively, or in addition, the«eublastin
polypeptide may be linked via the side chain amino group of an internal lysine residue, e.g., a
lysine residue introduced into the amino acid sequence of a naturally occurring neublastin
polypeptide. Thus, conjugations can also be branched from the non terminal reactive groups.
The polymer with the reactive group(s) is designated herein as "activated polymer". The
reactive group selectively reacts with reactive groups on the protein, e.g., free amino.
Attachment may occur in the activated polymer at any available neublastin amino
group such as the alpha amino groups or the epsilon amino groups of a lysine residue or
residues introduced into the amino acid sequence of a neublastin polypeptide. Free
carboxylic groups, suitably activated carbonyl groups, hydroxyl, guanidyl, imidazole,
oxidized carbohydrate moieties and mercapto groups of the neublastin (if available) can also
be used as attachment sites.
Generally from about 1.0 to about 10 moles of activated polymer per mole of protein,
depending on protein concentration, is employed. The final amount is a balance between
maximizing the extent of the reaction while minimizing non-specific modifications of the
product and, at the same time, defining chemistries that will maintain optimum activity, while
at the same time optimizing, if possible, the half-life of the protein. Preferably, at least about
50% of the biological activity of the protein is retained, and most preferably near 100% is
retained.
The polymer can be coupled to the neublastin polypeptide using methods known in
the art. For example, in one embodiment, the polyalkylene glycol moiety is coupled to a
lysine group of the mutated neublastin polypeptide. Linkage to the lysine group can be
performed with an N-hydroxylsuccinimide (NHS) active ester such as PEG succinimidyl
succinate (SS-PEG) and succinimidyl propionate {SPA-PEG). Suitable polyalkylene glycol
moieties include, e.g.t carboxymethyl-NHS, norteucine-NHS, SC-PEG, ttesylate, aldehyde,
epoxide, carbonylimidazole, and PNP carbonate.
Additional amine reactive PEG linkers can be substituted for the succmimidyl moiety.
These include, e.g. isothiocyanates, nitrophenylcarbonates, epoxides, and benzotriazole
carbonates. Conditions are preferably chosen to maximize the selectivity and extent or
reaction.
If desired, polymer-conjugated neublastin polypeptides may contain a tag, e.g., a tag
that can subsequently be released by proteolysis. Thus, the lysine moiety can be selectively
modified by first reacting a His-tag modified with a low molecular weight linker-such as
Traut's reagent j(Pierce) which will react with both the lysine and amino-terminus, and then
releasing the his tag. The polypeptide will then contain a free SH group that^an be
selectively modified with a PEG containing a thiol reactive head group -such as a maleimMe
group, a vinylsulfone group, a haloacetate group, or a free or protected SH.
Traut's reagent can be replaced with any linker that will-set up a specific site for PEG
attachment. By way of example, Traut's reagent could be replaced with SPDP, SMPT,
SATA, or SATP (all available from Pierce). Similarly one + could react the protein with an
amine reactive linker that inserts a maleimide (for example SMCC, AMAS, BMPS, MBS,
EMCS, SMPB, SMPH, KMUS, or GMBS), a haloacetate group(SBAP, SIA, SIAB), or a
vinylsulfone group and react the resulting product with a PEG that contains a free SH. The
only limitation to the size of the linker that is employed is that it cannot block the subsequent
removal of the amino-terminal tag.
Thus, in other embodiments, the polyalkylene glycol moiety is coupled to a cysteine
group of the mutated neublastin polypeptide. Coupling can be effected using, e.g., a
maleimide group, a vinylsulfone group, a haloacetate group, and a thiol group.
In preferred embodiments, the polymer-conjugated neublastin polypeptide in the
composition has a longer serum half-life relative to the half-life of fee neublastin polypeptide
in the absence of the polymer. Alternatively, or in addition, the polymer-conjugated
neublastin polypeptide dimer in the composition binds 5FRcc, activates RET, normalizes
pathological-changes of a neuron, enhances survival of a neuron, or ameliorates neuropathic
pain, or performs a combination of these physiological functions. Assays for determining
whether a polypeptide enhances survival of a neuron, or normalizes pathological changes of a
neuron, are described in, e.g., WOOO/01815. Preferably, the neuron is a sensory neuron, an
autonomic neuron, or a dopaminergic neuron.
In preferred embodiments, the composition is provided as a stable, aqueous-soluble
conjugated neublastin polypeptide complex comprising a neublastin polypeptide or mutated
neublastin polypeptide coupled to a PEG moiety. If desired, the neublastin polypeptide or
mutated neublastin polypeptide may be coupled to the PEG moiety by a labile bond. The
labile bond can be cleaved in, e.g., biochemical hydrolysis, proteolysis, or sulfhydryl
cleavage. For example, the bond can be cleaved under in vivo (plrysiological) conditions.
Other reaction parameters, such as solvent, reaction times, temperatuites, etc., and
means of purification of products, can be determined'case by case-based on the published
information relating to derivatization of proteins with water soluble polymers.
If desired, a single polymer molecule forconjugation per neublastin polypeptides may
beemployed. Alternatively, more than one polymer molecule may be attached. Conjugated
neublastin compositions of the invention may find utility in both in vivo as well as non-in
29
vivo applications. Additionally, it will be recognized that the conjugating polymer may
utilize any other groups, moieties, or-other-conjugated -species, as appropriate to the end use
application. By way of example, it may be useful in some applications to covalently bond to
the polymer a functional moiety imparting UV-degradation resistance, or antioxidation, or
other properties or characteristics to the polymer. As a further example, it may be
advantageous in some applications to functionalize the polymer to render it reactive or erosslinkable
in character, to enhance various properties or characteristics of the overall
. conjugated material. Accordingly, the polymer may contain any functionality, repeating
groups, linkages, or other constituent structures that do not preclude the efficacy of the
conjugated neublastin mutein composition for its intended purpose.
Illustrative polymers that may usefully be employed to achieve these desirable
characteristics are described herein below in exemplary reaction schemes. In covalently
bonded peptide applications, the polymer may be functionaUzed and then coupled to &ee
amino acid(s) of the peptide(s) to form labile bonds.
The reactions may take place by any suitable method used for reacting biologically
active materials with inert polymers, preferably at about pH 5-8, e.g., pH 5, 6, 7, or 8, if the
reactive groups are on the alpha amino group at the ammo-terminus. Generally the process
involves preparing an activated polymer and thereafter reacting the protein with the activated
polymer to produce the soluble protein suitable for formulation. The aboVe modification
reaction can be performed by several methods, which may involve one or more steps.
Linear and branched forms of PEG can be used as well as other alkyl forms. The
length of the PEG can be varied. Most common forms vary in size from 2BC-100 kDa. While
the present examples report that targeted PEGylation at the amino-terminus does not affect
pharmokinetic properties, the fact that the material retained physiological function indicates
that modification at the site or sites disclosed herein is not deleterious. Consequently, in
generating mutant forms of neublastin that could provide additional sites of attachment
through insertion of lysine residues, the likely outcome that these forms would be PEGylated
both at the lysine and at the amino-terminus is encompassed by the invention.
One or more sites on a neublastin polypeptide can be coupled to a polymer. For
example, one two, three, four, or Eve PEG moieties can be attached to the polypeptide. In
some embodiments, a PEG moiety is attached at the amino terminus and/or amino acids 14,
39, 68, and 95 of a neublastin polypeptide numbered as shown in Table 1 and SEQ ID NO:1.
In advantageous embodiments, the polymer-conjugated neublastin polypeptide in the
composition has a longer serum half-life relative to the half-life of the neublastin wild-type or
mutated polypeptide in the absence of the polymer. Alternatively, or in addition, the
polymer-conjugated neublastin polypeptide in the composition binds GFRaS, activates RET,
normalizes pathological changes of a neuron, enhances survival of a neuron, or ameliorates
neuropathic pain, or performs a combination of these physiological functions.
In some embodiments, the mutated neublastin polypeptide or polymer conjugate in
the complex has a physiological activity selected from the group consisting of: GFRaS
binding, RET activation, normalization of pathological changes of a neuron, enhancing
neuron survival, or ameliorating neuropathic pain.
Also provided by the invention are multimeric polypeptides that include a polymerconjugated
neublastin polypeptide. The multimeric polypeptides are preferably provided as
purified multimeric polypeptides. Examples of multimeric-complexes include, e.g., dimeric
complexes. The multimeric complex can be provided as a heteromeric or homomeric
complex. Thus, the multimeric complex can be a heterodimeric polymer-conjugated
polypeptide complex including one mutated neublastin polypeptide and one non-mutated
neublastin or a heterodimeric polymer-conjugated polypeptide complex including two or
more mutated neublastin polypeptides.
In some embodiments, the polymer-conjugated neublastin polypeptide binds GFRaS.
Preferably, binding of the polymer-conjugated neublastin polypeptide stimulates
phosphorylation of a RET polypeptide. To determine whether a polypeptide binds GFRaS,
assays can be performed as described in WOOO/01815. For example, the presence of
neublastin in the media of CHO cell line supernatants-can be described using a modified form
of a ternary complex assay described by Sanicola et al. (Proc. Natl. Acad. Sci. USA, 1997,
94: 6238). In this assay, the ability of GDNF-like molecules can be evaluated for their ability
to mediate binding between the extracellular domain of RET and the various co-receptors,
GFRal, GFRa2, and GFRaS. Soluble forms of RET and the co-receptors are generated as
nision proteins. A rusion protein between the extracellular .domain .of rat RET and placenta!
alkaline phosphatase (RET-AP) and a fusion protein between the extracellular domain of tat
GFRa-1 (disclosed in published application WO9744356; November 27,1997) and the Fc
domain of human IgGl (rGFR(al-Jg) have been described {Sanicola et al., Pfoc. Natl. Acad.
Sci. USA 1997,94:6238).
The polymer of the invention is preferably a polyalkyleneglycol moiety, and more
preferably a PEG moiety. Income embodiments, a polymeric moiety has an average
molecular weight of about 100 Da to about 25,000 Da; of about 1000 Date about 20,000 Da;
or of about 5000 Da to about 20,000 Da. In some embodiments, at least one polymeric
moiety has an average molecular weight of about 5000 Da; an average molecular weight of
about 10,000 Da; or an average molecular weight of about 20,000 Da.
The functional group on the polyalkylene glyeol moiety can be, e.g.,
carboxymethyl-NHS, norleucine-NHS, SC-PEG, tresylate, aldehyde, epoxide,
carbonylimidazole, or PNP carbonate. Coupling can occur via an N-hydroxylsuccinimide
(MHS) active ester. The active ester can be, e.g., PEG succinimidyl succinate succinimidyl butyrate (SPB-PEG), or succinimidyl propionate-(SPA-PEG). In some
embodiments, the polyalkylene glyeol moiety is coupled to a cysteine group of the neublastin
polypeptide or mutated neublastin polypeptide. For example, coupling can occur via a
maleimide group, a vinylsulfone group, a haloacetate group, and a thiol group, fa various
embodiments, the neublastin polypeptide or mutated neublastin polypeptide comprises one,
two, three, or four PEG moieties.
In some embodiments, the polymer iscoupled to the polypeptide at a site on the
neublastin that is an N terminus. In some embodiments, the polymer is coupled to the
polypeptide at a site in a non-terminal amino acid of the neublastin polypeptide or mutated
neublastin polypeptide. hi some embodiments, the polymer is coupled to a solvent exposed
amino acid of the neublastin polypeptide or mutated neublastin polypeptide.
In some embodiments, the polymer is coupled to the neublastin polypeptide or
mutated neublastin polypeptide at a residue selected from the group consisting of the amino
terminal amino acid of the polymer-conjugated polypeptide, position 14 in the ammo acid
sequence of the neublastin polypeptide or mutated neublastin polypeptMe, position 39 in the
amino acid sequence of the neublastin polypeptide or mutated neublastin polypeptide,
position 68 in the amino acid sequence of the neublastin polypeptide or mutated neublastin
polypeptide, and position 95 in the amino acid sequence of the neublastin polypeptide or
mutated polypeptide.
Polymer-conjugated neublasiin fusion proteins
If desired, the polymer-conjugated neublastin polypeptide can be provided as a fusion
protein. Fusion polypeptide derivatives of proteins of the invention also include various
structural forms of the primary protein mat retain biological activity.
Polymer-conjugated neublastin-serum albumin fusions can beconstructed using
methods known in the art. Any of a number of cross-linkers that contain a corresponding
amino reactive group and thiol reactive group can be Used to link neublastin to serum
albumin. Examples of suitable linkers include amine reactive cross-linkers that insert a thiol
reactive-maleimide. These include, e.g., SMCC, AMAS, BMPS, MBS, EMCS,"SMPB,
SMPH, KMUS, or GMBS. Other suitable linkers insert a thiol reactive-haloacetate group.
These include, e.g., SBAP, SIA, SIAB and that provide a protected or non protected thiol for
reaction with sulfhydryl groups to product a reducible linkage are SPDP, SMPT, "SATA, or
SATP all of which are commercially available {e.g., Pierce Chemicals). One skilled in the
art'can similarly envision with alternative strategies that will link the amino-terminus of
neublastin with serum albumin.
It is also envisioned that one skilled in the art can generate conjugates to^erum
albumin that are not targeted at the amino-terminus of neublastin or at the thiol moiety on
serum albumin. If desired, neublastin-serum albumin fusions can be generated using genetic
engineering techniques, wherein neublastin is fused to the serum albumin gene at its aminoterminus
carboxy-terminus, or at both ends.
Any neublastin conjugate that results in a product with a prolonged half-life, for
example, in vivo or, specifically, in animals (including humans) can be generated using a
similar strategy. Another example of a neublastin conjugate that results in a product with a
prolonged half-life in vivo is a neublastin fusion protein where the fusion partner is an Ig.
Other derivatives of polymer-conjugated neublastins include covalent or aggregate
conjugates of mutated neublastin or its fragments with other proteins or polypeptides, such as
by synthesis in recombinant culture as additional ammo-termini, or carboxy-termini. For
example, the conjugated peptide may be a signal-(or leader) polypeptide •sequence at the
amino-terminal region of the protein which co-translationally or post-translationally directs
transfer of the protein from its site of synthesis to its site of function inside or outside of the
cell membrane or wall (e.g., the yeast alpha.-factor leader). Neublastin receptor proteins«an
comprise peptides added to facilitate purification or identification of neublastin fe,g.,
histidine/neublastin fusions). The amino acid sequence of neublastin can also be linked to the
peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:22)tHopp^«/.,
Biotechnology 6:1204 (1988)). The latter sequence is highly antigenic and provides an
epitope reversibly bound by a'Specific monoclonal antibody, enabling rapid assay and-facile
purification of expressed recombinant protein.
This sequence is also specifically cleaved by bovine mucosalenterokinase at the
residue immediately following the Asp-Lys pairing.
Bioactive Polypeptides
The polypeptides of the invention may be provided in any bioactive -form, including
the form of pre-pro-proteins, pro-proteins, mature proteins, glycosylated proteins, nonglycosylated
proteins, phosphorylated proteins, non-phosphorylated proteins, truncated
forms, or any other posttranslational modified protein. A bioactive neublastin polypeptide
includes a polypeptide that, for example, when dimerized, alone or in the presence of a
cofactor {such as GFRccS, or RET), binds to RET, induces dimerization of RET, and
autophosphorylation of RET.
The polypeptides of the invention may in particular be an N-glycosylated polypeptide,
which polypeptide preferably is glycosylated at the N-residues indicated in the sequence
listings.
In some embodiments, a polypeptide of the invention has the amino acid sequence
presented as SEQ ID NO:6, holding a glycosylated asparagine residue at position 122; or the
amino acid sequence presented as SEQ tt) NO: 14, holding a glycosylated asparagine residue
at position 95, or the analogous position in any mutated neublastin polypeptide when aligned
by, e.g., ClustalW computer software.
In some embodiments, the polypeptide of the invention has the amino acid sequence
presented as SEQ ID NO:23, referred to herein as NBN113-N95K, containing a lysine
residue substituted for the asparagine residue at position 95 of SEQ ID NO:2; or the amino
acid sequence presented as SEQ ID NO:24, referred to herein as NBN106-N95K; or the
analogous position in any mutated neublastin polypeptide when aligned by, e.g., ClustaiW
computer software.
This invention also includes mutated neublastin fusion proteins, such as Ig-fusions, as
described, e.g., in United States patent 5,434,131, or serum albumin fusions.
In some embodiments, the invention provides a polypeptide having the amino acid
sequence shown as SEQ ID NO: 1 with the exception of one substitution, or an amino acid
sequence that has at least about 85%, preferably at least about 9f)%, more preferably at least
about 95%, more preferably at least about 9S%, and most preferably at least about 99%
identity to the sequence presented as SEQ ID NO:1.
In other embodiments, the invention provides a polypeptide having the amino acid
sequence of SEQ ID NO:2 with the exception of one substitution, or an amino acid sequence
that has at least about 85%, preferably at least about 90%, more preferably at least about
95%, more preferably at least about 98%, and most preferably at least about 99% identity to
the sequence presented as SEQ ID NO:2.
In some embodiments, the invention provides a polypeptide having the amino acid
sequence of SEQ ID NO:3 witii the exception of one substitution, or an amino acid sequence
that has at least about 85%, preferably at least about 90%, more preferably at least about
95%, more preferably at least about 98%, and most preferably at least about 99% identity to
the-sequence presented as SEQ ID N0:3.
In some embodiments, the invention provides a polypeptides having the amino acid
sequence of SEQ ID NO:4 with the exception of one substitution, or an amino acid sequence
that has at least about 85%, preferably at least about 90%, more preferably at least about
95%, more preferably at least about 98%, and most preferably at least about 99% identity to
the sequence presented as SEQ ID NO:4.
In some embodiments, the invention provides a polypeptide having the amino acid
sequence of SEQ ID NO: 5 with the exception of one substitution, or an amino acid sequence
that has at least about 85%, preferably at least about 90%, more preferably at least about
95%, more preferably at least about 98%, and most preferably at least about 99% identity to
the sequence presented as SEQ ID NO:5.
In some -embodiments, the invention provides a polypeptides having the amino acid
sequence of SEQ ID NO:6 with the exception of one substitution, or an amino acid sequence
mat has at least about 85%, preferably at least about 90%, more preferably at least about
95%, more preferably at least about 98%, and most preferably at least about 99% identity to
the sequence presented as SEQ ID NO:6.
In some embodiments, the invention provides a polypeptide having the amino acid
sequence of SEQ ID NO:7 with the exception of one substitution, or an amino acid sequence
that has at least about 85%, preferably at least about 90%, more preferably at least about
95%, more preferably at least about 98%, and most preferably at least about 99% identity to
the-sequence presented as SEQ ID NO:7.
In some embodiments, the invention provides a polypeptide having the amino acid
sequence of any one of SEQ ID NQs:8-21 with Ihe exception of one substitution, or an amino
acid sequence that has at least about 85%, preferably at least about 90%, more preferably at
least about 95%, more preferably at least about 9S%, and most preferably at least about 99%
identity to the sequence presented as any one of SEQ ID NOs:8-21.
In -some embodiments, the invention provides a polypeptide having the amino acid
sequence of SEQ ID NO:36 with the exception of one substitution, or an amino acid sequence
that has at least about 85%, preferably at least about 90%, more preferably at least about
95%, more preferably at least about 98%, and most preferably at least about 99% identity to
the sequence presented as SEQ ID NO:36.
In further embodiments, the invention provides a polypeptide having the amino acid
sequence of any one of SEQ ID NOS:1-21 and 36 with the exception of one substitution, or
an amino acid sequence that has at least about 85%, preferably at least about 90%, more
preferably at least about 95%, more preferably at least about 98%, and most preferably at
least about 99% identity to any one of the sequences presented as SEQ ID NOS:1-21 and 36.
In other embodiments, the mutated polypeptide of the invention holds the GDNF
subfamily fingerprint, i.e, the conserved cysteine amino acid residues designated in Tables 3 and
4.
In some embodiments, the invention provides a mutated polypeptide encoded by a
polynucleotide sequence capable of hybridizing under high stringency conditions with the
polynucleotide sequence encoding the polypeptide of SEQ ID NO:1, its complementary
strand, or a sub-sequence thereof. In some^embodiments, the mutated polypeptide of the
invention is encoded by a polynucleotide sequence having at least 70% identity to the
polynucleotide sequence encoding the polypeptide of SEQ ID NO:1.
In some embodiments, the invention provides novel polypeptides encoded by a
polynucleotide sequence capable of hybridizing under high stringency-conditions with the
polynucleotide sequence encoding the poiypeptide of SEQ ID NO:2, its complementary
strand, or a sub-sequence thereof. In some embodiments, the mutated polypeptide of the
invention is encoded by a polynucleotide sequence having at least 70% identity to the
polynucleotide sequence encoding the polypeptide ofSEQ ID NO:2.
In some embodiments, the invention provides mutated polypeptides encoded by a
polynucleotide sequence capable of hybridizing under high •stringency conditions with the
polynucleotide sequence encoding the polypeptide of any one of SEQ ID N0s:"8-21, its
complementary strand, or a sub-sequence thereof. In other embodiments, the mutated
polypeptide of the invention is encoded by apnolynucteotide sequence having at least 70%
identity to the polynucleotide-sequence encoding flie polypeptide of any one of SEQ ID NO:
8-21.
In some embodiments, the invention provides novel polypeptides ^encoded by a
polynucleotide sequence capable of hybridizing under high stringency conditions wife the
polynucleotide sequence encoding the polypeptide of SEQ ID NO:36, ks complementary
•strand, or a sub-sequence thereof. In some embodiments, the mutated polypeptide-of the
invention is encoded by a polynucleotide sequence having at least 70% identity io the
polynucleotide sequence encoding thepolypeptide of SEQ ID NO:36.
Biological Origin
A non-conjugated neublastin polypeptide dimer can be isolated and then-conjugated
to one or more polymers to obtain a polymer conjugated neublastin polypeptide dimer of the
invention. The neublastin polypeptide dimer can be isolated from a mammalian cell,
preferably from a human cell or from a cell of murine origin or from a cell of Chinese
hamster ovary origin.
Neurotrophic Activity
Modified neublastin polypeptides, including truncated neublastin polypeptides, of the
invention are useful for moderating metabolism, growth, differentiation, or survival tf a nerve or
neuronal cell. In particular, modified neublastin polypeptides are used to treat or alleviate a
disorder or disease of a living animal, e&, a human, which disorder or disease is responsive
to the activity of a neurotrophic agent. Such treatments and methods are described in more
detail below.
Pharmaceutical compositions comprising neublastin-polymer conjugates
Also provided is a pharmaceutical composition comprising a modified neublastin
polypeptide dimer of the present invention.
The polymer-neublastin conjugates of the invention may be •administered per se as
well as in the form of pharmaceutically acceptable-esters, salts, and other physiologically
functional derivatives thereof. Jn such pharmaceutical and medicament formulations, the
polymer-conjugated neublastin conjugate preferably is utilized together with-one or more
pharmaceutically acceptable carrier(s) and optionally any other therapeutic ingredients.
The carrier(s) must be pharmaceutically acceptable in the«ense of being compatible
with the other ingredients of the formulation and not unduly deleterious to the tecipienl
thereof. The polymer-conjugated neublastin is provided in an amount effecliveto achieve a
desired pharmacological effect or medically beneficial effect, as described terein, and in a
quantity appropriate to achieve the desired bioavailable in vivo dose or concentration.
The formulations include those suitable for par-enteral as well as non parenteral
administration, and specific administration modalities include oral, rectal, buccal, topical,
nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal,
intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and ultra-uterine
administration. Formulations suitable for aerosol andparenteral administration, both locally
and systemically, are preferred.
When the polymer-conjugated neublastin is utilized in a formulation comprising a
liquid solution, the formulation advantageously may be administered orally, bronchially, or
parenterally. When the polymer-conjugated neublastin is employed in a liquid suspension
formulation or as a powder in a biocompatible carrier formulation, the formulation may be
advantageously administered orally, rectally, or bronchially. Alternatively, it may be
administered nasally or bronchially, via nebulization of the powder in a carrier gas, to form a
gaseous dispersion of the powder that is inspired by the patient from a breathing circuit
comprising a suitable nebulizer device.
The formulations comprising the proteins of the present invention may conveniently
be presented in unit dosage forms and may be prepared by any of the methods well known in
the art of pharmacy. Such methods generally include the step of bringing the active
ingredients) into association with a carrier that constitutes one or more accessory
ingredients.
Typically, the formulations are prepared by uniformly and intimately bringing the
active ingredient(s) into association with a liquid carrier, a finely divided solid carrier, or
both, and then, if necessary, shaping the product into dosage forms of the desired
formulation.
Formulations of the present invention suitable for oral administration may be
presented as discrete units such as capsules, cachets, tablets, or lozenges, each comprising a
predetermined amount of the active ingredient as a powder or granules; or a suspension in an
aqueous liquor or a non-aqueous liquid, such as a syrup, an elixir, an emulsion, or a-draught.
Formulations suitable for parenteral administration convenientlycomprise a-sterfle
aqueous preparation of the active conjugate, which preferably is isotonic with the blood of
the recipient {e.g., physiological saline solution). Such formulations may include suspending
agents and thickening agents or other microparticulate systems which are designed to target
the compound to blood components or one or more organs. The formulations ma)' be
presented in unit-dose or multi-dose form.
Nasal spray formulations comprise purified aqueous solutions of the active conjugate
with preservative agents and isotonic agents. Such formulations are preferably adjusted to a
pH and isotonic state compatible with the nasal mucus membranes.
Formulations for rectal administration may be presented as a suppository with a
suitable carrier such as cocoa butter, hydrogenated fats, or hydrogenated fatty carboxyiic
3S
acid. Ophthalmic formulations such as eye drops are prepared by a similar method to the
nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of
the eye.
Topical formulations comprise the conjugates of the invention dissolved or suspended
in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, or other bases
used for topical pharmaceutical formulations.
In addition to the aforementioned ingredients, the formulations of this invention may
further include one or more accessory ingredients) selected from diluents, buffers, flavoring
agents, disintegrants, surface active agents, thickeners, lubricants, preservatives (including
antioxidants), and the like. The foregoing considerations apply also to the neublastin fusion
proteins of the invention (e.g., neublastin-human serum albumin fusion proteins).
Accordingly, the present invention includes the provision of suitable fusion proteins
for in vitro "stabilization of a polymer-conjugated neublastin conjugate in-solution, as a
preferred illustrative application of the invention. The fusion proteins may be employed for
example to increase the resistance to enzymatic degradation of the polymer-conjugated
neublastin polypeptide and provides a means of improving shelf life, room temperature
stability, and the like. It is understood that the foregoing considerations apply also to the
neublastin-serum albumin fusion proteins (including the human neublastin-human serum
albumin fusion proteins) of the invention.
Methods of treatment
The compositions of the invention may be used for treating or alleviating a disorder or
disease in a mammal, e.g., a primate including a human, which disorder or disease is
responsive to the activity of neurotrophic agents.
The compositions of the invention may be used directly via, e.g., injected, implanted
or ingested pharmaceutical compositions to treat a pathological process responsive to the
neublastin polypeptides. The compositions may be used for alleviating a disorder or .disease
of a living animal body, including a human, which disorder or disease is responsive to the
activity of neurotrophic agents. The disorder or disease may in particu!arjbe .damage of the
nervous system caused by trauma, surgery, ischemia, infection, metabolic diseases,
nutritional deficiency, malignancy or-toxic agents, and genetic or idiopathic processes.
The damage may in particular have occurred to -sensory neurons or retinal ganglion
cells, including neurons in the dorsal root ganglia or in any of the following tissues: the
geniculate, petrosal and nodose ganglia; the vestibuloacoustrc complex of the eighth cranial
nerve; the ventrolateral pole of the maxillomandibular lobe of the trigeminal ganglion; and
the meseneephalic trigeminal nucleus.
In some embodiments of the method of the invention, the disease or disorder is a
neurodegenerative disease involving lesioned and traumatic neurons, such as traumatic
lesions of peripheral nerves, the medulla, and/or the spinal cord, cerebral ischemic neuronal
damage, neuropathy and especially peripheral neuropathy, peripheral nerve trauma or injury,
ischemic stroke, acute brain injury, acute spinal-cord injury, nervous system tumors, multiple
sclerosis, exposure to neurotoxins, metabolic diseases such as diabetes or renal dysfunctions
and damage caused by infectious agents, neurodegenerative disorders including Alzheimer's
disease, Huntington's disease, Parkinson's disease, Parkinson-Plus syndromes, progressive
Supranuclear Palsy (Steele-Richardson-Olszewski Syndrome), Olivopontocerebellar Atrophy
(OPCA), Shy-Drager Syndrome (multiple systems atrophy), Guamanianparkinsonism
dementia complex, amyotrophic lateral sclerosis, or any other congenital or
neurodegenerative disease, and memory impairment connected to dementia.
In some •embodiments, sensory and/or autonomic system neurons can be treated. In
particular, nociceptive and mechanoreceptive neurons can be treated, more particularly Adelta
fiber, C-fiber and A-beta fiber neurons. In addition, sympathetic and parasympathetic
neurons of the autonomic system can be treated.
In some embodiments, motor neuron diseases such as amyotrophic lateral sclerosis
("ALS") and spinal muscular atrophy can be treated. In other embodiments, the neublastin
molecules of this invention to can be used to enhance nerve recovery following traumatic
injury. Alternatively, or in addition, a nerve guidance channel with a matrix containing
polymer-conjugated neublastin polypeptides, or fusion or conjugates of mutated neublastin
polypeptides can be used in the herein described methods. Such nerve guidance channels are
disclosed, e.g., United "States Patent No. 5,834,029.
In some embodiments, the compositions disclosed herein {and pharmaceutical
compositions comprising same) are used in the treatment of peripheral neuropathies. Among
the peripheral neuropathies included for treatment with the molecules of this invention are
trauma-induced neuropathies, e.g., those caused by physical injury or disease state, physical
damage to the brain, physical damage to the spinal cord, stroke associated with brain damage,
and neurological disorders related to neurodegeneration. Also included herein are mose
neuropathies secondary to infection, toxin exposure, and drug exposure. Still further
included herein are those neuropathies secondary to systemic or metabolic disease. For
example, the herein disclosed compositions can also be usecUo treat chemotherapy-induced
neuropathies (such as those caused by delivery of chemotherapeutic agents, e.g., taxol or
cisplatin); toxin induced neuropathies, drug-induced neuropathies, vitamin-deSciencyinduced
neuropathies; idiopathic neuropathies; diabetic neuropathies; and post-herpetic
neuralgias. See, e.g., United States patents 5,496,804 and 5,91-6,555.
Additional conditions mat can be treated according to the invention are
mono-neuropathies, mono-multiplex neuropathies, and poly-neuropathies, including axonal
and demyelinating neuropathies.
In some embodiments, the-compositions of the invention (and pharmaceutical
compositions comprising •same) are used in the treatment of various disorders in the eye,
including photoreceptor loss in the retina in patients afflicted with macular degeneration,
retinitis pigmentosa, glaucoma, and similar diseases.
Methods and Pharmaceutical Compositions
This invention provides methods for treating neuropathic pain, for treating tactile
allodynia, and for reducing loss of pain sensitivity associated with neuropathy. The present
methods use polymer conjugated neublastin polypeptide dimers, including dimers comprising
bioactive full-length neublastin polypeptides or bioactive truncated neublastin polypeptides.
fa addition, the invention provides pharmaceutical-compositions comprising a polymer
conjugated neublastin polypeptide dimer suspended, dissolved, or dispersed hi a
pharmaceutically acceptable carrier.
1. Treatment of Neuropathic Pain
In one embodiment, the invention includes a method for teeating neuropathic pain in a
subject comprising administering to the subject an effective amount of a polymer conjugated
neublastin polypeptide dimer. In some embodiments, the invention includes a method for
treating neuropathic pain in a subject comprising administering to the subject a
pharmaceutically effective amount of a polymer conjugated neublastin polypeptide dimer
comprising, for example, wild-type, truncated or mutated neublastin polypeptides, including,
e.g., any one of SEQ ID NOS:1,2, 6-21 and 36 or a mutated form thereof, either alone, or by
also administering to the subject an effective amount of an analgesia-inducing compound
selected from the group consisting of opioids, anti-arrhythmics, topical analgesics, local
anaesthetics, anticonvulsants, antidepressants, corticosteroids and non-steroidal antiinflammatory
drugs ^(NS AIDS). In a preferred-embodiment, the analgesia-inducing
compound is an anticonvulsant. In another preferred embodiment, the analgesia-inducing
compound is gabapentin ((l-ammomethyl)cyclohexane acetic acid) orpregabalin (S-t+>4-
amino-3-(2-methylpropyl) butanoic acid).
The neublastin polypeptides and nucleic acids of this invention (andpharmaceutical
compositions comprising polymer conjugated neublastin polypeptide diaiers described
herein) are used in the treatment of pain associated with peripheral neuropathies. Among the
peripheral neuropathies which can be treated according to this invention are trauma-induced
neuropathies, e.g., those caused by physical injury or disease state, physical damage to the
brain, physical damage to the spinal cord, stroke associated with brain damage, and
neurological disorders related to neurodegeneration.
The invention also provides treatments of chemotherapy-induced neuropathies {such
as those caused by delivery of chemotherapeutic agents, e.g., taxol or cisplatin); toxininduced
neuropathies, drug-induoed neuropathies, pathogen-induced (e.g., virus induced)
neuropathies, vitamin-deficiency-induced neuropathies; idiopathic neuropathies; and diabetic
neuropathies. See, e.g., United States Patent Nos. 5,496,804 and 5,916,555, each herein
incorporated by reference. The invention still further can be used for treatment of mononeuropathies,
mono-multiplex neuropathies, and poly-neuropathies, including axonal and
demyelinating neuropathies, using the neublastin nucleotides and polypeptides of this
invention.
The neuropathic pain maybe associated with a number of peripheral neuropathies,
including: (a) trauma-induced neuropathies, (b) chemotherapy-induced neuropathies, (c)
toxin-induced neuropathies '(including but not limited to neuropathies induced by alcoholism,
vitamin B6 intoxication, hexacarbon intoxication, amiodarone, chloramphenicol, disulfjram,
isoniazide, gold, lithium, metronidazole, misonidazole, nitrofurantoin), neuropathies, including therapeutic drug-induced neuropathic pain {such as -caused by anticancer
agents, particularly anti-cancer agents selected from the group consisting of taxol,
taxotere, cisplatin, nocodazole, vincristine, vindesine and vinblasn'ne; and such as causedfty
anti-viral agents, particularly anti-viral agents selected from me group consisting of ddl,
DDC, d4T, foscarnet, dapsone, metronidazole, and isoniazid), (e) \dtamin-deficiencyinduced
neuropathies {including but not limited to vitamin B12 deficiency, vitamin B6
deficiency, and vitamin E deficiency), (f) idiopathic neuropathies, (g) diabetic neuropathies,
{h) pathogen-induced nerve damage, (i) inflammation-induced nerve damage, (j)
neurodegeneration, (k) hereditary neuropathy (including but not limited to friedreich ataxia,
Familial amyloid polyneuropathy, Tangier disease, Fabry disease), (1) metabolic disorders
{including but not limited to renal insufficiency and hypothyroidism), (m) infectious and
viral neuropathies (including but not limited to neuropathic pain associated with leprosy,
Lyme disease, neuropathic pain associated with infection by a virus, particularly a virus
selected from the group consisting of a herpes virus (e.g. herpes zoster which may lead to
post-herpetic neuralgia), a human immunodeficiency virus (HTV), and apapilloma virus),
auto-immune neuropathies (including but not limited to Guillain-Barre syndrome, chrome
inflammatory de-myelinating polyneuropathy, monoclonal gammopathy of undetermined
significance and polyneuropathy), (o) trigeminal neuralgia and entrapment syndromes
(including but not limited to Carpel tunnel), and (p) other neuropathic pain syndromes
including post-traumatic neuralgia, phantom limb pain, multiple sclerosis pain, complex
regional pain syndromes (including but not limited to reflex sympathetic dystrophy,
causalgia), neoplasia- associated pain, vasculitic/angiopathic neuropathy, and sciatica.
Neuropathic pain may be manifested as allodynia, hyperalgesia, spontaneous pain or phantom
pain.
2. Treatment of Tactile Allodynia
The term "tactile allodynia" typically refers to theicondition in a subject where pain is
evoked by stimulation of the skin (e.g. touch) that is normally innocuous. This invention
includes a method for treating tactile allodynia in a subject.
In some-embodiments, tactile allodynia is treated by administering to the subject a
pharmaceutically effective amount of a polymer conjugated mutated neublastin poiypeptide
dimer alone.
hi a related embodiment, the invention includes a method for treating tactile allodynia
in a subject, either by administering to the subject an effective amount of a polymer
conjugated neublastin polypeptide dimer containing truncated wild-type or mutated
neublastin polypeptides, including, e.g., at least one of SEQ ID NOS:1,2,6-21 and 3t> or a
mutated form thereof, either alone, or by administering to the-subject an effective amount of a
neublastin polypeptide with an effective amount of an analgesia-inducing compound selected
from the group consisting of opioids, aiiti-arrhythmics, topical analgesics, local anaesthetics,
anticonvulsants, antidepressants, corticosteroids and NSAJDS. M a preferred embodiment,
the analgesia-inducing compound is an anticonvulsant. hi another preferred -embodiment, .the
analgesia-inducing compound isgabapentin((l-aminomethyl)cyclohexane acetic acid) or
pregabalin (S-(+)-4-amino-3-(2-methylpropyl)butanoic acid).
m some embodiments, a polymer conjugated mutated neublastin polypeptide dimer is
administered in association with a therapeutic agent, including but not limited to an anti-
cancer agent or an anti-viral agent. Anti-cancer agents include, but are not limited to, taxol,
taxotere, cisplatin, nocodazole, vincristke, vindesine and vinbf astine. Anti-viral agents
include, but are not limited to, ddl, DDC, d4T, foscarnet, dapsone, metronidazole, and
isoniazid.
3. Treatment for Reduction of Loss of Pain Sensitivity
In another embodiment, the invention includes a method for reducing the loss of pain
sensitivity in a subject afflicted with a neuropathy. In a preferred embodiment, the
neuropathy is diabetic neuropathy. In a preferred-embodiment, the loss of pain sensitivity is a
loss in thermal pain sensitivity. This invention-contemplates both prophylactic and
therapeutic treatment.
In prophylactic treatment, a polymer conjugated mutated neubiastin polypeptide
dimer is administered to a subject at risk of developing loss of pain sensitivity; such subjects
would be expected to be subjects with an-early-stage neuropathy. The treatment with
neublastin under such circumstances would serve to treat at-risk patients preventively.
hi therapeutic treatment, a polymer conjugated mutated neublastin polypeptide dimer
is administered to a subject who has experienced loss of pain-sensitivity as a result of
affliction with a neuropathy; 'such subjects would be expected to be -subjects with a late stage
neuropathy. The treatment with a polymer conjugated mutated neublastin polypeptide dimer
under such circumstances would serve to rescue appropriate pain sensitivity in the subject.
4. Treatment of Viral Infections and Viral-Associated Neuropathies
Prophylactic treatment of infectious and viral neuropathies is contemplated.
Prophylactic treatment is indicated after determination of viral infection and before onset of
neuropathic pain. During treatment, a polymer conjugated mutated neublastin polypeptide
dimer is administered to prevent appearance of neuropathic pain including but not limited to
neuropathic pain associated with leprosy, Lyme disease, neuropathic pain associated with
infection by a virus, particularly a. virus selected from the group consisting of a herpes virus
(and more particularly by a herpes zoster virus, which may lead to post-herpetic neuralgia), a
human immunodeficiency virus {HIV), and a papilloma virus). In an alternative
embodiment, a polymer conjugated mutated neublastin polypeptide dimer is administered to
reduce the severity of neuropathic pain, should it appear.
"Symptoms of acute viral infection often include the appearance of a rash. Other
symptoms include, for example, the development of persistent pain in the affected area of tiie
body, which is a common-complication of a herpes zoster infection (shingles). Post-herpetic
neuralgia can last For a month or more, and may appear -several months after any rash-like
-symptoms have disappeared.
5. Treatment of Painful Diabetic Neuropathy
Prophylactic treatment of painful diabetic neuropathy is contemplated. Prophylactic
treatment of diabetic neuropathies would commence after determination of the initial
diagnosis of diabetes or diabetes-associated symptoms and before onset of neuropathic pain.
Prophylactic treatment of painful diabetic neuropathy may also commence upon determining
that a subject is at risk for developing diabetes or diabetes-associated symptoms. During
treatment, a polymer-conjugated mutated neublastin polypeptide dimer is administered to
prevent appearance of neuropathic pain. In an alternative embodiment, a polymer conjugated
mutated neublastin polypeptide dimer is administered to reduce theseverity of neuropathic
pain that has already appeared.
6. Nervous System Disorders
Jn a further aspect, the invention provides a method of treating or preventing a
nervous system disorder in a subject (such as a human), by administering to a subject in need
thereof a therapeutically effective amount of a polymer-conjugated neublastin polypeptide, a
composition containing a neublastin polypeptide or mutated neublastin polypeptide-coupled
to a polymer, or a complex that includes a stable, aqueous soluble conjugated neublastin
polypeptide or mutated neublastin polypeptide complex comprising a neublastin polypeptide
or mutated neublastin polypeptide coupled to a poiyalkylene moiety such as, e.g., PEG.
The nervous system disorder can be a peripheral nervous system disorder, such as a
peripheral neuropathy or a neuropathic pain syndrome. Humans are preferred subjects for
treatment.
A polymer-conjugated neublastin polypeptide dimer t>f the invention is useful for
Seating a defect in a neuron, including without limitation lesioned neurons and traumatized
neurons. Peripheral nerves that experience trauma include, but are not limited to, nerves of
the medulla or of the spinal .cord. Inventive polymer-conjugated neublastin polypeptide
dimers are useful in the treatment of neurodegenerative disease, e.g., cerebral ischemic
neuronal damage; neuropathy, e.g., peripheral neuropathy, Alzheimer's disease, Ftuntington's
disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS). Such neublastin
polypeptide dimers can be used in the treatment of impaired memory, e.g., memory
impairment associated withtternentia.
Additional-examples of-conditions or diseases are disorders of the peripheral nervous
system, the medulla, or the spinal cord, as well as trauma-induced neuropathies,
chemotherapy-induced neuropathies, toxin-induced neuropathies, vitamin-deficiency-induced neuropathies; idiopathic neuropathies; and diabetic neuropathies,
neuropathic pain associated with toxin-induced nerve damage, pathogen-induced nerve
damage, inflammation-induced nerve damage, or neurodegeneration. An inventive
neublastin polypeptide dimer is additionally useful for treating neuropathic pain, for teeating
tactile allodynia and for reducing loss of pain sensitivity associated with neuropathy.
7. Dosage
The foregoing methods contemplate administering a to the subject, preferably
systemically, a formulation comprising a polymer conjugated mutated neublastin polypeptide
dimer that may or may not be truncated at a dosage from 0.0lug/kg to 1000 jig/leg body
weight of the subject, per dose. Preferably the dosage is from 1 ug/kg to 100 ug/kg body
weight of the subject, per dose. More preferably the dosage is from 1 ug/kg to 30 ug/kg body
weight of the subject, per dose, e.g., from 3 ng/kg to 10 (ig/kg body weight of fee subject, per
dose. Therapeutically'effective amounts of the formulation of the invention may be
administer-ed to a subject in need thereof in a dosage regimen ascertainable by one of skill in
the art, without undue experimentation..
. 8. Delivery
The polypeptide dimer used in the foregoing methods can be administered via any
suitable delivery system, and preferably from the group consisting of intravenous delivery,
intramuscular delivery, intrapulmonary delivery, subcutaneous delivery, and interaperitoneal
delivery, most preferably via intramuscular delivery, intravenous delivery, or subcutaneous
delivery. The neublastin polypeptide used in the foregoing methods can also be administered
via intrathecal delivery.
Administration of a polymer-conjugated neublastin polypeptide dimer can be, e.g.,
-systemic or local. The formulations include those suitable for parenteral as well asnon
parenteral administration, and specific administration modalities include oral, rectal, buccal,
topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intralhecal,
intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine
administration. Formulations-suitable for aerosol and patenteral administration, both locally
and systemically, are included. Preferred formulations are suitable for subcutaneous,
intramuscular, or intravenous administration.
8. Regimes
In some embodiments, the frequency of dosing for the polypeptide dimer of the
invention provides for administering to the subject a formulation three times a week for two
weeks. In order to optimize therapeutic efficacy, a polymer conjugated mutated neublastin
polypeptide dimer is first administered at different dosing regimens. The unit dose and
regimen depend on factors that include, &g., the species of mammal immunized, its immune
status, the body weight of the mammal. Typically, protein levels in tissue are monitored
using appropriate screening assays as part of a clinical testing procedure, e.g., to determine
the efficacy of a given treatment regimen.
The frequency of dosing for a polymer conjugated mutated neublastin polypeptide
dimer of this invention is within the skills and clinical judgement of physicians. Typically,
the administration regime is established by clinical trials which may-establish optimal
administration parameters. However, the practitioner may vary such administration regimes
according to the subject's age, health, weight, sex and medical status. The frequency of
dosing may also vary between acute and-chronic treatments for neuropathy, hi addition, the
frequency of dosing may be varied depending on whether the treatment is prophylactic or
therapeutic.
The invention is further illustrated in the following non-limiting examples.
EXAMPLES
Example 1: Unavailability of ammo-terminal PEGylated Neublastin
GHO cell - and E. coli - derived recombinant neublastins were observed to be rapidly
cleared from circulation if administered intravenously in rats. The proteins were below the
threshold of .detection in the serum following subcutaneous administration. To increase
bioavailability of neublastin, PEGylated forms of mutated neublastin were constructed.
Because no lysines occur in the neubiastin sequence, amine-specific PEGylation
chemistries will result in PEGylation of a wild-type neublastin polypeptide at its amino
terminus. Thus, for each neublastin dimer, two PEG moieties should be attached.
Accordingly, PEG forms were first directly targeted to the ammo-terminus through amine
specific chemistries. Surprisingly, PEGylation of E. coli expressed wild-type neublastin, -even
with two 20 kDa PEGs attached, had little benefit on half life, indicating that a mechanism
based clearance pathway was overriding the enhancement in half life that was expected to be
achieved by PEGylation.
Example 2: Construction of a PEGylated Mutated Neublastin (N95K)
The bioavailability of mutated neublastin forms PEGylated at internal amino acid
residues was next examined. A series of four mutants replacing naturally occurring residues
at positions 14,39,68, and 95, when numbered as shown in SEQ ID NO:1, were designed to
insert lysines at selected sites in the sequence. These lysines would provide alternative sites
for PEG attachment These sites were selected using the crystal structure of GDNF (Nat.
Struct Biol. 4: 435-8, 1997) as a framework to identify surface residues. The persephin/
neublastin chimera mutagenesis study (J. Biol. Chem. 275: 3412-20,2000) was used to
identify functionally important regions of the structure that should be avoided.
In order to express the wild-type neuWastin-gene in E. cdi, syngenes were
constructed with lower GC content and preferred E. coli codons. The syngene was cloned
into two vectors, pET19b and pMJB164, a derivative of pET19b. In pET19b, the sequence
encoding the mature domain of neublastin (NBN113) is directly fused to an initiating
methionine. hi pMJB164, the mature domain of neublastin is fused to a histidine tag {ill. 10
histidines) and separated from the histidine tag by an enterokinase cleavage site (SEQ ID
NOs: 35 and 36). The initiating methionine precedes the histidine tag.
(Table Removed)
Two of the mutations distribution of positive-charges on the surface, might represent a heparin binding site. This
site likely contributes to the -rapid clearance of the protein. A third site was targeted at HP'S,
the natural glycosylation site in wild-type neublastin. This site is naturally modified with a
complex carbohydrate structure. Therefore, modification with PEG at this-site was not
expected to impact function. The fourth site {R14) was selected in a region that was not
covered by any other of the modifications. A mutant in which the asparagine residue at
position 95 was replaced with a lysine (the "N95K mutant") was chosen for the studies
disclosed herein.
Four different mutated rat neublastins comprising one or more alterations in the
wild-type sequence of rat neublastin polypeptide were constructed. These mutated
neublastins contained single amino acid substitutions: R14K; R68K; R39K; or N95K. Table
1A identifies these exemplary point mutations in bold. In the "XiNiX2" nomenclature, Xi
refers to an amino acid of a wild-type neublastin polypeptide, NI refers to the numerical
position of the Xi amino acids in the sequence, as numbered according to SEQ ID NO:1, and
Xa refers to an amino acid substituted for the wild-type amino acid at the indicated numerical
position NI.
To construct the rat N95K neublastin mutation, site-directed mutagenesis was
performed on pCMB020, a plasmid encoding wild-type rat neublastin. The wild-type rat
neublastin nucleic and the amino acid sequence of the polypeptide encoded thereby are
presented below:
(Table Removed)
Mutagenesis of pCM020 using oligonucleotides KD3-210 andfCD3-2i 1 resulted in
formation of the plasmid pCMB027:
•KD3-210 5' - GTATCTTTCATGGAOGTTATGTTCTACATGGAGAACC - 3 '
KSEQ ID NO:27)
KD3-211
5 ' -GGTTCTCCATGTAGAACATA In pCMB027, the codon encoding asparagine at position 95 was replaced with a
codon encoding lysine.
A R14K mutated neublastin was formed by replacement of a at position 14 with a codon encoding lysine in the neublastin coding-sequence of pGMB020.
Site-directed mutagenesis was perfonned on pCMB020 using oligonucleotides KD3 -254 and
KD3-255:
KD3 -254 5 ' -3CT-GGTGCAAGGGATGCAAAAGGCK3Ta3TCTGCG-3 '
{SEQ ID NO: 29}
KD3-255 5'
The resulting construct was named pCMB029.
An R68K mutated neublastin was formed by replacement of a codon encoding
arginine at position 68 with a codon encoding lysine in the neublastin -coding sequence of
pCMB020. Site-directed mutagenesis was performed on pCMB020 using oligonucleotides
KD3-258andKD3-259:
KD3-258 S'-GGA KD3-259 5'-GGTCTA The resulting construct was named pCMBQ30.
A R3.9K mutated neublastin was formed by replacement of arginine at amino acid 39
with lysine in the neublastin .coding sequence of pCMB020. Site-directed mutagenesis of
pCMB020 was performed using oligonucleotides KD3-256 and KD3-257:
KD3-256 S'-GAGGAATTAATTAAGTTTGGTTTTTGTTCAGG-3' KD3-2S7 S ' -'CCT Expression and Characterization of Mutated Neublastin in E. coli
For expression and purification, a plasmid encoding the rat neublastin N95K
polypeptide was expressed in E. coli as a Hjs-tagged fusion protein wifli an-entelokinase
-cleavage site immediately adjacent to the start of the mature 113 amino acid neublastin
sequence. The E. coti was grown in a 500 L fermentor and frozen cell paste was provided.
The E. coli cells were lysed in a APV Gaulin Press and the rat neublastin N95K recovered
from the insoluble washed pellet fraction.
The N95K mutated neublastin was extracted from the pellet with guanidine
hydrochloride, refolded, and the His-tag removed with enterdkinase (see Example 5). The
product was then subjected to chromatography on Ni NTA agarose (Qiagen) and on
Bakerbond WP CBX cation exchange resin.
Bnterokinase treatment of the His tagged product resulted in an aberrant cleavage of
the protein at arginine 7 in the mature sequence. The resulting des 1-7 neublastin product
(NBN106-N95K) was fully active in the KCRA ELISA and structurally indistinguishable
from the mature form in its susceptibility to guanidine-induced denaturation and therefore
was used for subsequent work.
Rat mutated neublastin NBN106-N95K was PEGylated at an average of 3.3 PEG
moieties per neublastin molecule using methoxylpoly(ethylene glycol)-succinimidyl
propionate (SPA-PEG) with a molecular mass of 10,000 Da as the reactant. The resulting
PEGylated product was subjected to extensive characterization including analysis by
SDS-PAtjE, size exclusion chromatography (SEC), reverse phase HPLC, matrix assisted
laser desorption/ionization mass spectrometry (MALD/IMS), peptide mapping, assessment of
activity in the KIRA ELISA, and determination of endotoxin content. The purity of the
neublastin N95K product prior to PEGylation as measured by SDS-PAGE and SEC was
greater than 95%. The neublastin N95K product migrated under nonreducing-conditions as a
dimer, consistent with its predicted structure. After PEGylation, the resulting product
consisted of a series of modified adducts comprising 2 PEGs per molecule, which was 5% of
the product, 3 PEGs per molecule, which was 60% of the product, 4 PEGs per molecule,
which was 30% of the product, and several minor forms of higher mass. In the PEGylated
sample there was no evidence of aggregates. Residual levels of unmodified neublastin in the
product were below the limits of quantitation. The endotoxin content of the material is
routinely less than 1 EU/mg. The specific activity of the PEGylated neublastin in the KIRA
ELISA is 10 nM. The PEGylated product was formulated at 1.1 mg/mL in PBS pH 6.5. The
material, which is similar in potency to wild-type neublastin (NBN113),-can he-supplied as a
frozen liquid, which is stored at -70°C.
51
The R14K, R39K, and &6BK mutated neublastin polypeptides were expressed in E.
i and-can be subjected to the same methods for purification, PEGylation and assessment of
function as described above for the NBN106-N95K neublastin.
Preparation of PEGylated Mutated Neublastin NBN106-N9SK
230 mL of the refolded rat N95K mutated neublastin (2.6 mg/mL) that had been
produced in E. coli and stored at 4°C in 5 mM sodium phosphate pH 6.5,100 mM NaCl was
diluted with 77 mL of water, 14.4 mL of 1M HEPES pH7.5, and 2.8 g (10 mg/mL final) of
PEG SPA 10,000 Da (Shearwater Polymers, Inc.). The sample was incubated at room
temperature for 4 hours in the dark, then treated with 5 mM imidazole {final), filtered, and
stored overnight at 4°C. The product was generated in two batches one containing 130 mL of
the N95K bulk and the other containing 100 mL of the bulk. The PEGylated neublastin was
purified from the reaction mixture on Fractogel EMD SOa" (M) (EM Industries). The column
was run at room temperature. All buffers were prepared pyrogen free. Sodium chloride was
added to the reaction mixture to a final concentration of 87 mM and the sample was loaded
onto a 45 mL Fractogel column (5 cm internal diameter).
The column was washed with one column volume of 5 mM •sodium phosphate pHtf.5,
80 mM NaCl, then with three one column volume aliquots of 5 mM sodium phosphate
containing 50 mM NaCl. The resin was transferred into a 2.5 cm diameter column and the
PEGylated neublastin was eluted from the column with six ten mL steps containing 5 mM
sodium phosphate pH.6.5,400 mM NaCl, three steps containing 500 mL NaCl, and six steps
containing 600 mM NaCl. Elution fractions were analyzed for protein content by absorbance
at 280 ran and then for extent of modification by SDS-PAGE. Selected fractions were
pooled, filtered through a 0.2 |J,m filter, and diluted with water to 1.1 mg PEGylated rat
neublastin/mL. After assessing endotoxin levels in the individual batches, they were pooled
and refiltered through a 0.2 fim membrane. The final material was aliquoted and stored at -
70°C.
DV Spectrum of Purified PEGylated Mutated Neublastin NBW106-N95K
The UV spectrum (240-340 nm) of PEGylated NBN N95K was taken on the neat
sample. The sample was analyzed in triplicate. The PEGylated sample exhibited an
absorbance maximum at 275-217 nm and an absorbance minimum at 247-249. This result is
consistent with what is observed on thetmlk intermediate. The protein concentration of tiie
PEGylated product was -estimated from the spectrum using an extinction coefficient of
Z28o°'1%=0.50. The protein concentration of the PEGylated neublastin bulk is Llmg/mL. No
turbidity was present in the sample as evident by the lack of absorbanee at 320 nm.
Characterization of PEGylated Mutated Neublastin NBN106-N95K by
SDS-PAGE
Aliquots of PEGylated neublastin containing 3,1.5,0.75, and 0.3 .fig of the product
were subjected to SDS-PAGE on a 4-20% gradient gel (Owl). The gel was stained with
Goomassie brilliant blue R-250. Molecular weight markers (GIBCO-BRL) were run in
parallel.
SDS-PAGE analysis of PEGylated mutated neublastin NBN106-N95K under non
reducing conditions revealed a series of bands corresponding to modifications with 2,3,4,
and more than 4 PEGs per molecule. The major band with apparent mass of 98 kDa contains
3 PEGs per molecules. In the purified PEGylated product, non-PEGylated neublastin was not
detected. The presence of a mixture of products with 2,3 and 4 PEGS attached was verified
by MALDI mass spectrometric analysis. The ratio of product containing 2,3, and 4 PEGs
was determined by densitometry and determined to be 7,62, and 30 percent of the total,
respectively.
Characterization of PEGylated Mutated Neublastin NBN106-N95KBy Size
Exclusion Chromatography
PEGylated mutated neublastin NBN106-N95K was subjected to -size exclusion
chromatography on an analytical Superose 6 HR1O/30 FPLC column using 5 mM MES pH
6.5, 300 mM NaCl as the mobile phase. The column was run at 20 mL/h. Elution fractions
were monitored for absorbanee at 280 nm. The PEGylated mutated neublastin eluted as a
single peak with an apparent molecular weight of about 200 kDa consistent with the large
hydrodynamic volume of the PEG. :Nb evidence of aggregates was observed. Free
neublastin, which elutes with an apparent molecular mass of about SOkDa, was not detected
in the preparation.
Analysis of PEGj'lated Mutated Neublastin NENlftS-NPSKby Reverse Phase
HPLC
PEGylated mutated neublastin NBN106-N95K was subjected to reverse phase &PLC
on a Vydac €4 (5 urn, 1 x 250 mm)-column. The column was developed using a€0 mm
gradient from 40 to 60% B (Buffer A: 0.1% TFA, Buffer B: 75% acetonkrile/0.085% TFA).
The column efSuent was monitored for absorbanee at 214 nm and fractions collected for
subsequent analysis. PEGylated NBN106-N95K was fractionated into its various di (60.5
nan), tri {63.3 mm), and tetra (67.8 mm) PEGylated components by r-everse phase fiPLC on a
C4 column. The relative intensities of the peaks suggest that the ratios of fee components are
5.4, 60.5, and 30.1 %, respectively. Peak identities were verified by MALDI-MS. There was
no evidence of non-PEGylated NBN106-N95K (elutes at 5-15 mm) in the product.
Analysis of PEGylated Mutated Neublastin NBN106-N95K by Mass
Spectrometry
PEGylated mutated neublastin NBN106-N95K was desalted on a C4 Zip Tip and
analyzed by mass spectrometry on a Voyager-DE™ STR (PerSeptive Biosystems)
matrix-assisted laser desorption/ ionization time-of- fh'ght{MALDI-TOF) mass spectrometer
using sinapinic acid as a matrix. 0.5 \\L of the purified protein was mixed with 0.5 \\L of
matrix on the target plate. Mass spectrometry of PEGylated mutated neublastin NBN106-
N95K revealed singly and doubly charged forms of three adducts. The observed masses of
43803 Da, 54046 Da, and 64438 Da are consistent with modifications of 2,3, and 4 PEGs per
molecule.
Analysis of PEGylated Mutated Neublastin NBN106-N95K by Peptide Mapping
The specificity of the PEGylation reaction was evaluated by peptide mapping.
PEGylated neublastin was separated into di, tri, and tetra PEGylated components, which were
then reduced, alkylated, and further separated into their single chain components by HPLC on
a €4 column. These components and reduced and alkylated non-PEGylated NBN106-N95K
as a-control were digested with Asp-N proteinase and the resulting cleavage products were
fractionated by reversed phase HPLC on a Vydac Cig (5 ^m, 1 x 25t) mm) column using a 60
mm gradient from 0 to 60% B (Buffer A: 0.1% TFA, Buffer B: 75% acetorutrik/0.085%
TFA). Thecolumn effluent was monitored for absorbance at 214 mm.
The rat neublastin sequence contains five internal aspartic acids and therefore was
expected to yield a simple cleavage profile when digested with endoproteinase Asp-N. All^f
the peaks from the Asp-N digest of rat K95K have been, identified by mass spectrometry
and/or Edman amino-terminal sequencing and thus the peptide map -can be used as asimple
tool to probe for the sites of modification by the presence or absence of a peak. The identity
of the various peaks are summarized below in Table 7.
(Table Removed)
due to oxidation of methionine containing peptide on MALDI.
Since neublastin naturally exists as a homodimer, the rat mutated neublastin NBN106-
N95K product contains four potential sites for PEGylation, the two amino-tenninal amines
from each of the chains and the two N95K sites that were engineered into the construct. In
the peptide map of the di-PEGylated chain, only the peak that contains the peptide with the
N95K mutation was altered by the PEG modification. None of the other peaks were afifected
by the PEG modification. The mapping data therefore indicate that the PEG moiety is
specifically attached to this peptide and not to any of the other peptides mat were-screened.
The second potential site of attachment, the ammo-terminus is on a peptide that is only three
amino acids long and is not detected in the peptide map. It is inferred that additional PEG
attachments are at this site. Consistent with this notion, a small percentage of the rat mutated
neublastin N95K is not truncated and contains the mature Ala-1 sequence. This peptide
elutes at 30 jam and is visible in the peptide map from the non-PEGylated digest, but is absent
from the PEGylated mutated neublastin NBN106-N95K digests.
Example 3: Assessing the Potency of Internally PEGylated Mutated Neublastin
NBN106-N95K in a Kinase Receptor Activation (tORA) ELISA
The potency of PEGylated mutated rat neublastin was measured using neublastin
dependent activation/ phosphorylation of c-Ret as a reporter for neublastin activity in an
ELISA that was specific for the presence of phosphorylated RET. NB41 A3-mRL3 cells, an
adherent murine neuroblastoma cell line which expresses Ret and GFRocS, were plated at 2 x
10s cells per well in 24-well plates in Dulbecco's modified eagle medium (DMEM),
supplemented with 10 % fetal bovine serum, and .cultured for 18 h at 37 °C and 5 % CO2.
The cells were washed with PBS, and treated with serial dilutions of neublastin in
0.25 mL of DMEM for 10 min at 37 °C and 5 % C02. Each sample was analyzed hi
duplicate. The cells were washed with 1 mL of PBS, and lysedfor Ih at 4 °C with 0.30 mL
of 10 mM Tris HC1, pH 8.0,0.5 % Nonidet P40,0.2 %-sodium deoxycholate, 50 mM NaF,
0.1 mM Na3 VC4,1 mM phenylnaethylsulfonyl fluoride with gently rocking the plates. The
lysates were further agitated by repeated pipetting and 0.25 mL of sample was transferred to a


96-well ELISA plate that had been coated with 5 µg/mL of anti-Ret mAb (AA.GE7.3) in 50 mM carbonate buffer, pH 9.6 at 4°C for 18 h, and blocked at room temperature for one hour with block buffer (20 mM Tris HC1 pH 7.5,150 mM NaCl, 0.1% Tween-20 (TEST)
containing 1 % normal mouse serum and 3 % bovine serum albumin).
After a 2 h incubation at room temperature, the wells were washed 6-times
with TBST. Phosphorylated Ret was detected by incubating the wells at room
temperature for 2 h with 2µg/mL of horseradish peroxidase (HRP)-conjugated anti-
phosphotyrosine 4G10 antibody in block buffer, washing 6-times with TBST, and
measuring HRP activity at 450 nm with a colorometric detection reagent. The
absorbance values from wells treated with lysate or with lysis buffer were measured and
the background corrected signal was plotted as a function of the concentration of
neublastin present in the activation mixture. The potency of PEGylated mutated
neublastin (3(,4)X10 kDa PEG NBN106-N95K) in the KIRA ELISA was
indistinguishable from that of the wild-type NBN113 material (Table 8).
There was no effect of two freeze-thaw cycles bn potency and following this treatment
there was no significant increase in the turbidity of the sample, indicating that the
samples can be safely thawed for the study. In independent studies accessing the activity
of product with three and four 10 kDa PEGs per molecule separately, it was determined
that the adduct with three PEGs was fully active, while the four PEG product had reduced
potency (Table 8). These data demonstrate that 3,(4) X 10 kDa PEG NBN106-
N95K - and 3 X 10 kDa PEG NBN106-N95K -activate Ret to a similar
extent and with the same dose-dependence as non-mutated (wild-type) neublastin,
NBN113. However, although 4X10 kDa PEG NBN106-N95K activates Ret to
a similar extent as non-mutated (wild-type) NBN113, 4X10 kDa PEG NBN106-N95K is
approximately 10-fold less potent than non-mutated (wild-type) NBN113 in activating Ret. Estimated EC50's are provided in Table 8.
Example 4: Pharmokinetic Studies of Internally PEGylated Mutated Rat Neublastin
NBN106-N95K in Rats and Mice
The pharmokinetic properties of various PEGylated and non-PEGylated mutated neublastin products in rat and mouse models were examined (see Table 8 for summary of results).
The data revealed that PEGylation of rat mutated neublastin NBN1-06-N95K with 3.3, 10000 Da PEGs resulted in a-significant effect on the half life and bioavailability of the neublastin. Following a 1 mg/kg IV administration in sprague Dawley rats, peak levels of

PEGylated mutated neublastin of 3000 ng/mL were detected after 7 minutes, and levels of
700 ng/mL were detected after 24 h, 200 ng/mL after 4? h, and 100 ng/mL after 72 h. In
contrast for non-PEGylated mutated neublastin N95K following a 1 mg/kg IV administration,
levels of 1500 ng/mL were detected after 7 minutes, but then the levels quickly dropped to 70
ng/mL after 3 h and were not detectable after 7 h. The effects of PEGylation wereeven more
pronounoed in animals treated with PEGylated neublastin by subcutaneous administration.
Following a 1 rag/kg s.c. administration, circulating levels of PEGylated neublastin
reached a maximum of 200 ng/mL after 24 h and remained at this level for the duration of the
three day study. In contrast, no detectable neublastin was observed at any time point
following administration of non-PEGylated mutated neublastin.
The analysis of the PEGylated N95K samples are complicated by the presence of
adducts comprising 2, 3 and 4 PEGs per molecule, which each will display a different PK
profile. In early PK studies, mice were used to facilitate screening through a variety of
candidates and routes of administration. The mouse studies revealed dramatic difierences in
the bioavailability of the candidates. However, when the 3.3 IDkDaPEGadductwas
evaluated in rats, it was found to be less bioavailable in rats than it was in mice. This
difference in bioavailability was particularly pronounced following i.p. administration.
Levels in mice reached 1600 ng/mL after 7 hr and remained at 400 ng/mL after 24 hr. In
contrast, rat levels were constant at 100 ng/mL for 4-48 hr.
Two surprising and unexpected results emerged from the PfC studies summarized in
Table 8: 1) PEGylation of the amino-terminal ammo acids of non-glycosylated neublastin
was not sufficient to increase serum exposure substantially; and 2) PEGylation of the aminoterminal
amino acid(s) of neublastin together with modification *(e.g. PEGylation or
glycosylation) of amino acid 95 was sufficient to increase serum exposure substantially. For
example, glycosylated NBN104*(CHO) gave no detectable exposure after s.c. administration;
hov/ever, glycosylated 1X20 kDaPEGNBN104 (CHO)gave high serum exposure after s.c.
administration. Similarly, 2X20 kDa PEG MBN113 gave low-to-moderate serum exposure
after s.c. administration; however, glycosylated 2X20 kDa PEG NBN104 (CHO) gave high
serum exposure after s.c. administration. These results indicated that polymer conjugation of
the amino-terminal amino acid(s) of neublastin together with either polymer conjuation of an
internal amino acid (e.g. at position 95) or glycosylation at an internal amino acid (e.g. at
position 95) results in substantially increased serum exposure after-systemic administration.
Both wild-type rat neublastin and mutated neublastin N95K were refolded and
purified to >95% for efficacy tests in the STZ diabetic rat neuropathy model. Wild-type
neublastin was formulated to go directly into animal testing while N95K was prepared for
PEGylation with 10 kDa PEG-SPA. To accomplish the refolding and purification goal, a
refolding method utilizing size exclusion chromatography (SEC) was developed that
permitted the renaturation of neublastin from E. coli inclusion bodies in large quantities and
at high concentrations. In addition to SEC, both Ni-NTA and -CM silica column
chromatography steps were employed to increase the final protein purity. The proteins were
subjected to extensive characterization including analysis bySDS-PAGE, size exclusion
chromatography, ESMS, assessment of activity by KIRA ELIS A, and determination of
endotoxin content SDS-PAGE and SEC of the final protein products indicated a purity of
greater than 95%. The endotoxin level of each product was activity of both proteins in the KIRA ELIS A is approximately 10 nM. Wild-type neublastin
was formulated at 1.0 mg/ml and N95K was formulated at 2.6 mg/ml in phosphate-buffered
saline (PBS) pH6.5. Wild-type neublastin was aliquoted into 15 ml tubes and stored frozen at
70°C while N95K was subjected to PEGylation prior to aliquoting and freezing.
Examples: Refolding and Purification of a Wild-Type Neublastin and the Mutated
NPSKNeublastin
Both neublastin forms were expressed in E. coli as Histidine (His)-tagged fusion
proteins with an enterokinase cleavage site immediately adjacent to the-start of the mature
113 amino acid sequence. Bacteria expressing either wild-type (1.8 kg pellet) or N95K (2.5
kg pellet) neublastin were subjected to lysis in 2 liters of PBS using a APV Gaulin Pcess.
Following •centrifugation (10,000 rpm) to pellet the inclusion bodies, the supernatants from
-each preparation were discarded. The inclusion body pellets were washed two times with
wash buSert0.02M Tris-HCl pH 8.5,0.5 mM EDTA) then washed two times with the same
buffer containing Triton X-l 00(2%, v/v) followed by two additional buffer washes without
detergent. Both pellets were solubilized using 6M guanidine hydrochioride, 0.1M Tris pH
8.5,0.1M DTT, and 1 mM EDTA. To aid in the solubilizaiion proxsess, each pellet was
subjected to homogenisation using apolytron homogenizer followed by overnight stirring at
room temperature. The solubilized proteins were clarified by centrifugation prior to
denaturing chromatography through Superdex 200 (5.5 liter column-equilibrated with 0.05M
£lycine/H3P04 pH 3.0 with 2M Guanidine-HCl) at 20 ml per minute.
Denatured neublastin was identified by SDS-PAGE. Fractions containing either wild
type-neublastint>r N95KL were pooled and concentrated to approximately 250 mL using an
Amicon 2.5-liter stirred cell •concentrator. After filtration to remove any precipitate, the
concentrated protein was subjected to renaturing sizing chromatography through Superdex
200 equilibrated with 0.1 M Tris-HCl pH 7.8,0.5M guanidine-HCl, O mM reduced
glutathione and 0.22 mM oxidized glutathione. The column was developed using 0.5M
guanidine-HCl at 20 mL per minute. Fractions containing renatured wild-type neublastin or
N95K neublastin were identified by SDS-PAGE, pooled, and stored at 4°C until needed for
His tag removal.
Alternative Method of Refolding Wild-type Neublastin and Mutated N95K
Neublastin by Dilution.
To refold neublastin by dilution, solubilzed protein was rapidly diluted in refolding
buffer (0.5 M guanidine-HCl, 0.35 M L-Arginine, 50 mM potassium phosphate (pH 7.8), 0.2
mM glutathione reduced, 1 mM glutathione oxidized, and 0.1% Tween-BO) at a final
concentration of 0.1 rag/ml and incubated at room temperature for 48 hours without stirring.
Refolded neublastin was then concentrated 25 fold, brought to 40 mM imidazole, and applied
to a chromatography column containing Ni-NTA agarose to further concentrate the product
and eliminate host cell proteins. The column was washed with 10 times column volume with
wash buffer (40 mM imidazole, 0.5 M guanidine-HCl). Neubiastin was then eluted from the
resin with 0.2 M Imidazole and 0.5 M guanidine-HCl.
Concentration of Column-Refolded Neublastin by Ni-NTA Chromatography.
Column-renatured neublastin was stored at 4°C for at least 24 hours before
proceeding with the purification to promote disulfide formation between the neublastin
monomers. During this time, a precipitate formed and was removed by filtration through a
0.2 n polyether sulfone (PES) filter unit. To decrease non-specific binding, the protein
solution was brought to 20 mM imidazole prior to loading on a 100 ml Ni-NTA tQiagen)
column equilibrated with column buffer (0.5 M guanidine and 20 mM imMazoIfi) at 50 ml per
minute. Following the protein application, the column was washed to baseline using 4he
same buffer. Neublastin was eluted from the resin using approximately 300 mL of elution
buffer containing 0.5 M guanidine-HCl and 0.4 M imidazole. After elution, neublastin was
dialyzed overnight (using 10 kDa dialysis tubing) at room temperature against ten volumes of
5 mM HC1. Dialysis promotes the hydrolysis of contaminating substances andxtecr-eases the
guanidine-HCl and imidazole concentrations to 0.05M and 0.04M, respectively.
Cleavage of the His Tag by Lysyl Endopeptidase or Enterokinase,
The next day, any precipitate that formed during dialysis was removed by filiation.
The following purification steps apply •to both the column- and dilution-refolded neublastin
products. The protein sample was made to 0.1 M NaCl by the addition of NaCl from a 5M
stock for a final salt concentration including the remaining guanidine-HCI of approximately
150mM. This concentration was confirmed using a conductivity meter. Additionally, 1M
HEPES pH 7.8 was added for a final concentration of 25mM. To cleave the His -tag, lysyl
endopeptidase was added to the wild-type neuhlastin and enterokinase was added to the
N9SK mutated neublastin, bom at an approximately 1:300 ratio of protease to neublastin.
Enterokinase was used in place of lysyl endopeptidase for the N95K mutated neublastin due
to an additional protease cleavage site in the mutated protein at Lys95. The samples were
stirred at room temperature for 2 hours and the digestions monitored by SDS-PAGE.
His Tag Removal by Ni-NTA Chromatography.
Protease-treated neublastin was applied to a 100 mL Ni-NTA column-equilibrated
with 0.5M guanidine-HCI and 20 mM imidazole at 50 mL per minute. The column was
washed to baseline with the same buffer. Any protein washing off the column was pooled
with the flow-through protein containing neublastin without the His tag.
CM Silica Chromatography.
Following Ni-NTA Chromatography, the protein was immediately subjected to further
purification through CM silica resin. A 20 mL CM silica column equilibrated with loading
buffer (5 mM phosphate pH 6.5,150 mM NaCl) was loaded with neublastin at 20 mL per
minute. The column was washed with twenty column volumes of wash buffer (5 mM
phosphate pH 6.5,400 mM NaCl) and the protein step eluted with elution buffer containing 5
mM phosphate pH 6.5 but with 1 M NaCl. The eluted protein was dialyzed overnight against
the phosphate alone to bring the salt concentration down to 100 mM for N95K and 150 mM
for wild type neublastin. Both samples were filtered through a 0.2 |i PES filter unit, analyzed
by SDS-PAGE, and stored at 4°C until needed for further characterizations and/or
PEGylation.
Wild-type and N95K mutated neublastin protein preparations were subjected to UV
spectrum anatysis to assess their absorbance at 280. Using a micro quarts cuvette and
blanking against buffer alone, 100 \il of either wild-type or N95K mutated neublastin was
continuously scanned from 230 to 330 nm using a Beckmanspectrophotometer. Based on
this analysis, wild-type neublastin was determined to be at a concentration of 1.1 rag/ml and
N95K mutated neublastin at 2.6 mg/ml (A2SO nmE°'1%=0.5 used for-each protein). Less
than 1% precipitated material was identified based on absortiance at 330 ran.
To assess the purity of both protein preparations, each •sample -(0.5 mg) was-subjected
to size exclusion chromatography through a 16/30*Superdex 75 column. The column was
equilibrated with 5mM phosphate pH 6.5 containing 400 mM NaCl and developed with a 1.5
mL per minute flow rate. Based on the absorbance at 280 nm, both wild-type and N95K
mutated neublastin preparations migrated as a single peak with an expected molecular weight
(23-24 kDa), and they did not contain any significant protein contamination.
Both wild-type and N95K mutated neublastin proteins were reduced in 2.5 M
guanidine-HCl, 60 mM Tris pH 8.0 and 16 mM DTT. The reduced samples were desalted
over a short C4 column and analyzed on-line by ESMS using a triple quadrupole instrument.
The ESMS raw data were deconvolved by the MaxEnt program to generate mass spectra.
This procedure allows multiple charged signals to collapse into one peak that directly
corresponds to the molecular mass inkilodaltons (kDa). The4econvoluted mass spectrum
for wild-type showed the predominant species is 12046 Da, which is in agreement with the
predicted molecular weight of 12046.7 Da for the 113 amino acid form of the protein. A
minor component was also observed (12063 Da) suggesting the presence of an oxidation
product. Three peaks were identified in the N95K mutated neublastin protein sample. The
major component demonstrated an apparent molecular mass of 11345 Da in agreement with
the predicted mass for the 106 amino acid form of the protein. The other two peaks had
masses of 11362 and 12061 Da, suggesting N95K oxidation and the presence of the 113
amino acid form, respectively.
The presence of the 106 and 113 amino acid forms in the N95K mutated neublastin
protein preparation is attributable to digestion with enterokinase. This protease from
Biozyme is a natural enzyme preparation purified from calf intestinal mucosa and is imported
to contain a slight trypsin contamination (0.25 ng trypsin per jig enterokinase). Therefore,
trypsin may be acting on the N95K. mutated neublastin protein on the carboxy terminal side
of Arg7 to produce the predominant 106 amino acid form. On the other hand, lysyl
endopeptidase used to cleave wild-type neublastin is a single protease activity acting on 4he
carboxy terminal side of the lysine residue contained within the His .tag to produce the mature
113 amino acid neublastin form. Both the 106 and 113 amino acid forms of neublastin are
equally active in all assays tested and behave similarly in guanidine-HCl stability tests.
Neublastin activity was determined by its ability to stimulate c-Ret phosphorylation in
NB41A3-mRL3 cells using the KfilA ELfSA described in Example 3. Phosphoryiated Ret
was detected by incubating (2 hours) the captured receptor with HRP-conjugated
phosphotyrosine antibody (4G10; 0.2 ^g per well). Following the incubation, the wells were
washed six times with TBST, and the HRP activity detected at 450 nm with a eolorimetric
assay. The absorbance values from wells treated with lysate or with lysis buffer alone were
measured, background corrected, and the data plotted as a function of the concentration of
neublastin present in the activation mixture. The data demonstrate that the purified
neublastin polypeptides resulted in the appearance of phosphorylated RET, indicating that the
purified neublastin was active in this assay.
Example 6: Preparation of a Serum Albumin-Neublastin Conjugate
Wild-type rat neublastin at a concentration of 1 mg/ml in PBS was treated with 1 mM
sulfo-SMCC (Pierce) and desalted to remove excess cross-linker. Since the wild-type
neublastin protein contains only a single amihe at its ammo-terminus and no free suUhydryl
groups, reaction with SMCC was expected to result in site-specific modification of the
neublastin with SMCC attached at its amino-terminus.
Next, 60 fj,g of the neublastin-SMCC conjugate was incubated with 120 jig of bovine
serum albumin and analyzed for extent of cross-linking by SDS-PAGE. BSA contains a
single free SH group and consequently reaction with the neublastin-SMCC conjugate is
expected to result in modification at this site through the maleimide on the SMCC. Under
these conditions, two additional bands of higher molecular weight were observed, which are
consistent in mass with modification of the neublastin with a single BSA moiety and with
two BSA molecules since each neublastin molecule contains two amino-termini that can
undergo reaction, and consequently are in agreement with this notion. Concurrent with the
formation of these bands, was a decrease in the intensity of the neublastin-SMCC and BSA
bands. Based on the intensity of the remaining neublastin band, the reaction appeared to have
gone to 70-80% completion.
The monosubstituted product was purified from the reaction mixture by subjecting the
material to cation exchange chromatography and size exclusion chromatography on a
Superdex 200 column .(Pharmacia) essentially as described for PEGj'lation studies discussed
above. Column fractions from the gel filtration run were ana^fzed by SDS-PAGE and those
t
containing the monosubstituted product were analyzed for protein content by absorbance at
280 nm. Since the mass of BSA is approximately twice that of neublastin, the apparent
concentration was divided by a factor of 3 to give the neublastin equivalent. This fraction
was subjected this to analysis for fuactixm in the K1RA ELISA. IC50 values for both the wtand
BSA-conjugated neublastin were 3-6 nM, indicating that conjugation to the BSA had not
compromised function.
While these preliminary studies were generated with BSA, the corresponding serum
albumin proteins from rats and humans also contain a free SH. Consequently a similar
approach can be applied to generate a rat serum albumin-rat neublastin conjugate for
performing PK and efficacy studies in rats and human serum albumin-human neublastin for
performing clinical trials. Similarly SMCC can be substituted with any of a number of crosslinkers
that contain an ammo reactive group on one side and a thiol reactive group on the
other side. Examples of amine reactive •cross-linkers that insert a thiol reactive-maleimide are
AMAS, BMPS, MBS, EMCS, SMPB, SMPH, KMUS, or GMBS, that insert a thiol reactivehaloacetate
group are SBAP, SIA, SIAB and that provide a protected or non protected thiol
for reaction with sulfhydryl groups to product a reducible linkage are SPDP, SMPT, S ATA,
or SATP all of which are available from Pierce. Such cross linkers are merely exemplary and
many alternative strategies are anticipated for linking the amino-terminus of neublastin with
serum albumin. A skilled artisan also could generate conjugates to serum albumin that are
not targeted at the amino-terminus of neublastin or at the thiol moiety on serum albumin.
Neublastm-serum albumin fusions created using genetic engineering where neublastin is
fused to the serum albumin gene at its amino-terminus, carboxy-terminus, or at both ends, are
also expected to be functional.
This method can be extended through routine adaptations to any neublastin-serum
albumin conjugate that would result in a product with a prolonged half-life in animals and
consequently in humans.
Example 7: Crystallization and Structure Determination of Human Neublastin
Selenomethionine labeled neublastin was expressed using a standard procedure to
inhibit methionine biosynthesis (Van Duyne, et al., 1991, Science 252, 839-842). Both
wildtype neublastin and selenomethionine-incorporated neublastin wastjoncentrated to 17
nig/ml in 0.8 M arginine. The protein stock was concentrated to 17 mg/ml in 0.8 M arginine.
Crystals were grown by the hanging-drop vapor diffusion method (Jancarik, J. •& -Kirn, S.H.
,1991, J. Appl. Crystdlogr. 24,409-411) out of 1.25 M magnesium sulfate, 0.1 M MES
6.5 at 20°C. The most reproducible crystals were obtained by microseeding. The crystals
were cryoprotected by the addition of 5% ethylene glycol every 60 seconds to a-final
concentration of 1.25 M magnesium sulfate, 0.1 M MES pH 6.5, and 30% {v/v) ethylene
glycol and then frozen by quick transfer into liquid nitrogen.
Crystals approximately 100 microns on each side diffracted to 1.6A at beamline X4A
at the National Synchrotron Light Source (Upton, NY). Data processing with the HKL
program package (Otwinowski (1993) & Proceeding of thetlOM Study Weekend: Data
63
Collection and Processing {Sawer et al., Eds.) pp. 56-62, Daresbury Laboratory, Warrington)
revealed the-crystals to belong to a C2 space group with 1 covalent dimer per asymmetric unit
and approximate-cell dimensions a=l 15 A, b=33 A , c=55 A and cc= y =90°,*J3=99°.
The crystal structure was solved by multiple isomorphous replacements. Native
neublastin crystals were soaked in 1 mM PtCLj for 4 hours, 10 mM IrCla for 72 hours, and 10
mM IrClg for 18 hours and data collected and processed by the HKL suite (Otwinowski et al.,
supra). The two selenomethionine sites were located by inspection of isomorphous and
anomalous difference pattersons. The remaining sites were located using SOLVE {4). The
phases were improved by RESOLVE (Terwilliger et al., 1999,. Ada Ciystallogr. D. 55, 849-
861) to a figure of merit of 0.56 and resulting maps were of sufficient quality to trace the
neublastin model. Alternating cycles of model building with O2D (5 G J Kleywegt & T A
Jones, "O2D - the manual", software manual, Uppsala, 1994) and refinement with CNX using
a mlhl target and refined against the selenomethionine data resulted in a complete model of
the neublastin protein, excluding the first amino-tenninal 13 amino acids, as well as 89 water
molecules and 6 sulfate anions. The final R^e is 28.5% and R-factor is 24.7% to 2.0A with
good stereochemistry.
Example S: Sulfate Binding Sites and Modeling of Heparin Sulfate
A cluster of three sulfates at the vertices of an approximate equilateral triangle are
located on at the pre-helix region of the neublastin surface and could represent a binding site
for heparin sulfate. There are three arginine residues that appear to have key interactions
with these sulfates. The arginine residue R48 links together all three sulfates (#2, #6, and
#3). Its backbone amide interacts with sulfate #2, while its side chain guanidinium group
forms a bifurcated hydrogen bond with sulfates #6 and #3. A second arginine residue (R49)
forms a hydrogen bond with sujfate #2 and a third arginine residue (RSI) forms a long
hydrogen bond to sulfate #6.
These sulfates may represent binding pockets for heparin sulfate, which if mutated to
non-positively charged residues could reduce heparin sulfate binding and possibly decrease
and/or delay clearance of the neublastin molecule upon in vivo administration. A model of
heparin sulfate bound to neublastin may be constructed by superimposing the sulfates of the
glycosaminoglycan with the existing sulfates of the neublastin crystal structure. The n and
n+2 saccharide-linked sulfates in heparin sulfate from the crystal structure ofFGF-1
complexed to heparin sulfate are separated by approximately 8.5A (Pellegrini et al., Nature 407,1029-1034). This measurement closely matches &e distance between sulfates of
3-Sulfate cluster in the neublastin structure; sulfates #3 and #6 are separated by 8.3 A and
sulfates #3 and #2 are separated by $. 1 A. This distance measurement correspondence could
indicate that this R48/R49/R51 motif is part of a heparin binding site. This suggests that
single site mutations of R48, R49 or R51 to -glutamate or aspartate, or any other non-positive
amino acid, might reduce heparin sulfate binding affinity without reducing the receptorbinding
activity-of neublastin, thereby resulting in a biologically active product that has a
prolonged half-life in animals and consequently hi humans. To test this possibility, we are
generating a series of iconstructs that contain one or more arginines mutated to glutamic acids.
The mutated neublastin products will be expressed in E. coli, purified and refolded, and
tested for function. Finally, the products will be tested for ability to bind heparin and for
pharmacokmetics and pharmacodynamics hi animals.
One or more of these sites might also provide other sites for PEGylation which can be
accomplished by replacing R with K orC and applying the methods described above.
Example 9: Dosage of Begylated Mutated Neublastin NBN106-N95K on
Reversal of Tactile and Thermal Hyperalgesia in Nerve Ligation Animal Model of
Neuropathic Pain
We had previously demonstrated that 1 mg/kg wild-type neubiastin, administered s.c.
3 times per week, results hi nearly complete reversal and normalization of neuropathic pain
behaviors (tactile allodynia and thermal hyperalgesia) induced by spinal nerve ligation hi the
rat, whereas 0.03 mg/kg wild-type neublastin, administered s.c. 3 times per week had no
effect and 0.1 mg/kg and 0.6 mg/kg wild-type neublastin, administered s.c. 3 times per week
had intermediate effects in this model.
Here, we describe studies to address the reversal effect of pegylated N95K neublastin
on tactile allodynia and thermal hyperalgesia in theChung L5/L6 spinal nerve ligation
("SNL") model. Sprague-Dawley male rats (230 - SSOg) were divided into two groups. All
rats received the spinal nerve ligation. One group of rats (n=6) was administered vehicle by
subcutaneous injection. A second group of rats (n=6 per group) wer-e administered pegylated
N95K. neublastin (3 ,(4) X 10 M)a PEG NEN106-N95K) by subcutaneous injection at 10
fig/kg. 3(,4) X 10 kDa PEG NBN106-N95K, comprised neublastin protein mat was E. coliderived
and contained an Asn-to-Lys amino acid substitution at position 95, then truncated
{amino-termhius truncation of 7 amino acids; NBN106), and-finally pegylated with an
average of 3.3 PEG moieties per dimer of NBN, using methoxylpoly(ethylene glycol)-
succinimidyl propionate (SPA-PEG) with a molecular massif 10,000 Da as theieactant. The
vehicle consisted of 5 mM phosphate and 150 mM sodium chloride at pH-6.5. Subcutaneous
injections were administered on days 3,5, 7, 10,12 and 14 following the operation (post-
SNL). The Von Frey (Chaplan et al. (1994), J. Neurosci. Meth. 53:55-63) and Hargreave's (Hargreaves et al. (1988), Pain.32: 77-88) behavioral tests were used to monitor tactile and thermal responses, respectively. These pain responses were monitored prior to the spinal nerve ligation to establish baseline responses, on day 2 post-SNL to verify the presence of tactile and thermal hyperalgesia, and then on days 3,5,7,10,12,14 and 15 post-SNL. To assess statistical significance of drug treatment relative to vehicle treatment, a 1-way analysis of variance (1-way ANOVA) was carried out followed by a post-hoc "Student Neuman KeuIs (SNK) test.
Both types of neuropathic pain behavior (tactile allodynia and thermal hyperalgesia developed folly by day 2 post-SNL, as expected. Subcutaneous administration of 10 µg/kg 3(,4) X 10 kDa PEG N95K-NBN106 led to substantial and statistically significant reversal of both types of neuropathic pain in rats with spinal nerve ligation. In rats with spinal nerve ligation, the effect of 10 µg/kg 3(,4) X 10 kDa PEG NBN106-N95K on thermal sensitivity and tactile allodynia first became statistically significant 4 days after the initiation of administration of pegylated N95K neublastin. The effect of 10 µg/kg 3(,4) X 10 kDa PEG NBN106-N95K on thermal sensitivity and tactile allodynia reached a plateau approximately 4 days after the initiation of administration of pegylated N95K neublastin. The effects of 3(,4) X 10 kDa PEG NBN106-N95K did not diminish during the 2 to 3 day interval between administrations. In fact, there was substantial normalization of pain behaviors between the administrations of pegylated N95K neublastin on days 5 and 7. In another experiment (data not shown), subcutaneous administration of 3µg/kg 3C4) X 10 kDa PEG NBN106-N95K on days 3, J, 7, 10, 12 and 14 led to a significant normalization of pain behaviors (tactile and thermal hyperalgesia) in the SNL model, though the onset of the effect was somewhat slower.
These results demonstrated that^of 3(,4) X 10 kDa PEG NBN1D6-N95K has an increased potency of at least 100 to 333-fold over non-mutated non-glycosylafed neublastin on tactile allodynia and thermal hyperalgesia pain behaviors in the SNL model. The enhanced pharmacokinetic properties of 3(,4) X 10 kDa PEG N95K NBN106 compared to non-mutated non-glycosylated neublastin indicated that efficacy of systemically adrninretered neublastin correlates with serum levels of neublastin. These results demonstrated that polymer conjugates of mutated neublastin can be used to treat neuropathic pain in patients with greatly

reduced doses, and potentially reduced dosing frequency, due to their-enhanced
bioavailability compared to unconjugated neublastin.
(Table Removed)
Other Embodiments
Although particular embodiments have been disclosed herein in detail, this has been
done by way of example for purposes of illustration only, and is not intended to be limiting
with respect to the scope of the appended claims, which follow. In particular, it is
contemplated by the inventors that various substitutions, alterations, and modifications may
be made to the invention without departing from the spirit and scope of the invention as
defined by the claims.





WE CLAIM:
1. A neublastin polypeptide containing a substitution of the arginine at position 48, the arginine at position 49, or the arginine at position 51 with a non-positive amino acid residue.
2. The polypeptide as claimed in claim 1, wherein the non-positive amino acid residue is glutamic acid.
3. The polypeptide as claimed in claim 1, wherein the arginine residue at position 48 is substituted with glutamic acid.
4. The polypeptide as claimed in claim 1, wherein the arginine residue at position 49 is substituted with glutamic acid.
5. The polypeptide as claimed in claim 1, wherein the arginine residue at position 51 is substituted with glutamic acid.

Documents:

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3372-DELNP-2005-Claims-(16-02-2009).pdf

3372-delnp-2005-claims.pdf

3372-DELNP-2005-Correspondence-Others-(02-09-2008).pdf

3372-DELNP-2005-Correspondence-Others-(16-02-2009).pdf

3372-DELNP-2005-Correspondence-Others-(30-04-2009).pdf

3372-delnp-2005-correspondence-others.pdf

3372-delnp-2005-description (complete)-02-09-2008.pdf

3372-delnp-2005-description (complete).pdf

3372-DELNP-2005-Form-1-(02-09-2008).pdf

3372-DELNP-2005-Form-1-(30-04-2009).pdf

3372-delnp-2005-form-1.pdf

3372-delnp-2005-form-18.pdf

3372-DELNP-2005-Form-2-(02-09-2008).pdf

3372-delnp-2005-form-2.pdf

3372-DELNP-2005-Form-3-(02-09-2008).pdf

3372-delnp-2005-form-3.pdf

3372-delnp-2005-form-5.pdf

3372-DELNP-2005-GPA-(02-09-2008).pdf

3372-delnp-2005-gpa.pdf

3372-DELNP-2005-Others-Document-(02-09-2008).pdf

3372-DELNP-2005-Others-Document-(16-02-2009).pdf

3372-delnp-2005-pct-101.pdf

3372-delnp-2005-pct-304.pdf

3372-delnp-2005-pct-306.pdf

3372-DELNP-2005-Petition-137-(02-09-2008).pdf

3372-DELNP-2005-Petition-138-(02-09-2008).pdf


Patent Number 233338
Indian Patent Application Number 3372/DELNP/2005
PG Journal Number 13/2009
Publication Date 27-Mar-2009
Grant Date 28-Mar-2009
Date of Filing 28-Jul-2005
Name of Patentee BIOGEN IDEC MA INC.
Applicant Address 14 CAMBRIDGE CENTER, CAMBRIDGE, MASSACHUSETTS 02142, USA
Inventors:
# Inventor's Name Inventor's Address
1 DINAH WEN-YEE SAH 4 LONGFELLOW PLACE, APT. 2608. BOSTON, MASSACHUSETTS 02114, USA
2 R. BLAKE PEPINKSY 30 FALMOUTH ROAD, ARLINGTON, MASSACHUSETTS 02474, USA
3 PAULA ANN BORIACK-SJODIN 46 FLORENCE ROAD, WALTHAM, MASSACHUSETTS 02453, USA
4 STEPHAN S. MILLER 6 WOODSIDE LANE, ARLINGTON, MASSACHUSETTS 02474, USA
5 ANTHONY ROSSOMANDO 50 ASTI AVENUE, REVERE, MASSACHUSETTS 02151, USA
PCT International Classification Number A61K
PCT International Application Number PCT/US2004/002763
PCT International Filing date 2004-02-02
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10/356,264 2003-01-31 U.S.A.