Title of Invention

"POLYPEPTIDE COMPRISING AN AMINO ACID SEQUENCE AND CONJUGATE COMPRISING SAID POLYPEPTIDE"

Abstract Variant Neublastin polypeptides having substitutions at selected amino acid residues are disclosed. Substitution at one or more selected amino acid residues decreases heparin binding and increases serum exposure of variant Neublastin polypeptides. Also disclosed are methods of using variant Neublastin polypeptides to treat disorders and activate the RET receptor in a mammal.
Full Text NEUBLASTIN VARIANTS
Cross Reference To Related Applications
This application claims priority from provisional application number 60/602,825,
filed August 19, 2004 and provisional application number 60/694,067, filed
June 24, 2005. The entire content of each of these prior applications is incorporated
herein by reference in its entirety.
Technical Field
The invention relates to protein chemistry, molecular biology, and neurobiology.
Background
Neublastin, also known as Artemin and Enovin, is a 24-kDa homodimeric
secreted protein that promotes the survival of neurons of the peripheral and central
nervous system such as dopaminergic neurons (Baudet et al., 2000, Development,
127:4335; Rosenblad et al., 2000, Mol. Cell Neurosci., 15(2):199; GenBank AF120274).
The gene encoding neublastin has been cloned and sequenced (Roseblad et al., 2000,
Mol. Cell Neurosci., 15(2):199;Baloh et al.,Neuron, 21:1291).
Neublastin is a member of the glial cell line-derived neurotrophic factor (GDNF)
ligand family. At the cellular level, GDNF members activate the receptor tyrosine
kinase, RET. RET associates with a co-receptor, GDNF family receptor alpha
(GFRalpha), a glycosylphosphatidyl inositol (GPI) linked membrane protein that
provides ligand specificity for RET. Four GFRalphas are known (GFRalpha 1-4).
Neublastin binds to GFRalpha3 together with RET forming a ternary signaling complex(Baudet et al. 2000, Development, 127:4335; Balohetal., 1998, Neuron, 21:1291),which is localized predominantly on nociceptive sensory neurons (Orozco et al., 2001,Eur. J. Neurosci., 13(11):2177). These neurons detect pain and injury. Thus, neublastinhas clinical application in the general treatment of neuropathy and more specifically inthe treatment of neuropathic pain.
Neublastin and the other GDNF family members are members of the transforming
growth factor beta (TGF beta) superfamily and thus, are characterized by the presence ofseven conserved cysteine residues with similar spacing which form the structure of acysteine knot (Saarma, l999,Microsc. Res. Tech., 45:292). Each monomer contains twodisulfide bonds that form a closed loop structure encircling the third disulfide to form atight knot structure. The seventh cysteine contained within each monomer forms anintermolecular disulfide bond, covalently linking the monomers to form the final dimerproduct (Rattenholl et al 2000, J. Mol. Biol, 305:523).
TGF beta family members are synthesized as pre pro proteins that eventually are
secreted as a mature homodimer after cleavage of the signal peptide and pro-domain (seee.g. Rattenholl, et al., 2000, J. Mol. Biol., 305:523; Fairlie et al., 2001, J. Biol. Chem.,
276(20): 16911). Both the signal peptide and pro-domain mediate proper secretion for
TGF beta family members (Rattenholl et al., 2000, J. Mol. Biol., 305:523; Rattenholl et
al., 2001, Eur. J. Biochem., 268:3296).
Summary
The invention is based, at least in part, on the discovery that Neublastin binds to
heparin sulfate and that particular amino acid residues in the Neublastin polypeptide
contribute to this binding event. Substitution of selected amino acid residues was found
to decrease heparin binding by variant Neublastin polypeptides and increase bioactivity
and bioavailabiliry of the variants.
In one aspect, the invention features a polypeptide containing an amino acid
sequence that is at least 80% identical to amino acids 15-113 of SEQ ID NO: 1, wherein
the amino acid sequence contains at least one amino acid substitution, relative to SEQ
NO: 1, selected from the group consisting of: (i) an amino acid other than arginine at the
position corresponding to position 48 of SEQ ID NO:1 (e.g., the arginine is substituted
with a non-conservative amino acid residue such as glutamic acid); (ii) an amino acid
other than arginine at the position corresponding to position 49 of SEQ ID NO:1 (e.g., the
arginine is substituted with a non-conservative amino acid residue such as glutamic acid);
and (iii) an amino acid other than arginine at the position corresponding to position 51 of
SEQ ID NO:1 (e.g., the arginine is substituted with a non-conservative amino acid
residue such as glutamic acid). The polypeptide, when dimerized, binds to a complex
containing GFRalphaS and RET.
In some embodiments, the amino acid sequence contains amino acids other than
arginine at the positions corresponding to position 48 and position 49 of SEQ ID NO: 1.
For example, the arginine residue at position 48 and the arginine reside at position 49 of
SEQ ED NO: 1 can be substituted with non-conservative amino acid residues (e.g.,
glutamic acid).
In some embodiments, the amino acid sequence is at least 90%, at least 95%, or at
least 98% identical to amino acids 15-113 of SEQ ID NO:1.
Also disclosed is a polypeptide containing amino acids 15-113 of SEQ ID NO:2,
amino acids 15-113 of SEQ IDNO:3, amino acids 15-113 of SEQ IDNO:4, amino acids
15-113 of SEQ IDNO:5, amino acids 15-113 of SEQ IDNO:8, or amino acids 15-113 of
SEQ ED NO:9. In some embodiments, the polypeptide contains amino acids 10-113 of
SEQ ED NO:2, amino acids 10-113 of SEQ ID NO:3, amino acids 10-113 of SEQ ID
NO:4, amino acids 10-113 of SEQ ID NO:5, amino acids 10-113 of SEQ ED NO: 8, or
amino acids 10-113 of SEQ ID NO:9. In some embodiments, the polypeptide contains
the amino acid sequence of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:3, the
amino acid sequence of SEQ ED N0:4, the amino acid sequence of SEQ ID NO:5, the
amino acid sequence of SEQ ED N0:8, or the amino acid sequence of SEQ ID NO:9.
Also disclosed is a polypeptide containing an amino acid sequence at least 80%,
identical to amino acids 15-113 of SEQ ED NO:1, wherein the amino acid sequence
comprises at least one amino acid substitution, relative to SEQ ED NO:1, selected from
the group consisting of: (i) an amino acid other than serine at the position corresponding
to position 20 of SEQ ED NO: 1 (e.g., the serine is substituted with a non-conservative
amino acid residue); (ii) an amino acid other than glutamine at the position corresponding
to position 21 of SEQ ED NO: 1 (e.g., the glutamine is substituted with a non-conservative
amino acid residue); (iii) an amino acid other than histidine at the position corresponding
to position 32 of SEQ ED NO: 1 (e.g., the histidine is substituted with a non-conservative
amino acid residue); (iv) an amino acid other than arginine at the position corresponding
to position 33 of SEQ ED NO:1 (e.g., the arginine is substituted with a non-conservative
amino acid residue); (v) an amino acid other than arginine at the position corresponding
to position 39 of SEQ ID NO:1 (e.g., the arginine is substituted with a non-conservative
amino acid residue); (vi) an amino acid other than serine at the position corresponding to
position 46 of SEQ ID NO: 1 (e.g., the serine is substituted with a non-conservative amino
acid residue); (vii) an amino acid other than arginine at the position corresponding to
position 68 of SEQ ID NO:1 (e.g., the arginine is substituted with a non-conservative
amino acid residue); (viii) an amino acid other than glycine at the position corresponding
to position 72 of SEQ ID NO:1 (e.g., the glycine is substituted with a non-conservative
amino acid residue); (ix) an amino acid other than serine at the position corresponding to
position 73 of SEQ ID NO: 1 (e.g., the serine is substituted with a non-conservative amino
acid residue); and (x) an amino acid other than valine at the position corresponding to
position 94 of SEQ ID NO:1 (e.g., the valine is substituted with a non-conservative
amino acid residue). The polypeptide, when dimerized, binds to a complex containing
GFRalphaS and RET. In some embodiments, the amino acid sequence is at least 90%, atleast 95%, or at least 98% identical to amino acids 15-113 of SEQ ID NO:1.
Also disclosed is a polypeptide containing an amino acid sequence at least 80%
identical to SEQ ID NO:1, wherein the amino acid sequence comprises at least one aminoacid substitution, relative to SEQ ID N0:l, selected from the group consisting of: (i) anamino acid other than arginine at the position corresponding to position 7 of SEQ IDN0:l (e.g., the arginine is substituted with a non-conservative amino acid residue such as
glutamic acid); (ii) an amino acid other than arginine at the position corresponding to
position 9 of SEQ ID NO: 1 (e.g., the arginine is substituted with a non-conservative
amino acid residue such as glutamic acid); and (iii) an amino acid other than arginine at
the position corresponding to position 14 of SEQ ID NO: 1 (e.g., the arginine is
substituted with a non-conservative amino acid residue such as glutamic acid). The
polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET. In
some embodiments, the amino acid sequence is at least 90%, at least 95%, or at least 98%
identical to SEQ ID NO: 1.
The invention also features conjugates containing a polypeptide described herein
conjugated to a non-naturally occurring polymer. An exemplary polymer is a watersoluble
synthetic polymer such as a polyalkylene glycol (e.g., polyethylene glycol).
The invention also features a fusion protein containing a polypeptide described
herein and a heterologous ammo acid sequence.
The invention also features a dimer containing two of the polypeptides,
conjugates, or fusion proteins described herein.
The invention also features a pharmaceutical composition containing a
polypeptide, dimer, conjugate, or fusion protein described herein and a pharmaceutically
acceptable carrier or excipient.
Also disclosed is a nucleic acid containing a sequence that encodes a polypeptide
described herein, an expression vector containing the nucleic acid, and a cell containing the
expression vector.
Also disclosed is a method of making a polypeptide, the method including the
following steps: (i) providing a cell containing an expression vector containing a nucleic
acid encoding a polypeptide described herein, and (ii) culturing the cell under conditions that
permit expression of the nucleic acid.
The invention also features a method of treating or preventing a nervous system
disorder in a mammal by administering to the mammal a therapeutically effective amount of
a polypeptide, dimer, conjugate, fusion protein, or pharmaceutical composition described
herein.
The invention also features a method of treating neuropathic pain in a mammal by
administering to the mammal a therapeutically effective amount of a polypeptide, dimer,
conjugate, fusion protein, or pharmaceutical composition described herein.
The invention also features a method of activating the RET receptor in a mammal by
administering to the mammal an effective amount of a polypeptide, dimer, conjugate, fusion
protein, or pharmaceutical composition described herein.
An advantage of selected variant Neublastin polypeptides described herein is that
they have decreased heparin binding ability as compared to wild type Neublastin.
Decreased heparin binding results in a decreased clearance of the variant polypeptide
in vivo.
A variant Neublastin polypeptide having substitutions at amino acid positions 48
and 49 was unexpectedly found to have greatly deceased heparin binding ability and
greatly increased potency and bioavailabilty as compared to single amino acid mutants
and/or wild type Neublastin. For example, the double mutant was found to exhibit an
approximately 185-fold increase in serum exposure as compared to wild type Neublastin.
In addition, this double mutant was found to exhibit an over five fold increase in
expression in vitro as compared to wild type Neublastin, thereby facilitating large scale
production of the protein.
The advantages and unexpected properties of the variant Neublastin polypeptides
allow for treatment of subjects using lower doses of protein and/or allow for lengthened
intervals between administrations (as compared to treatments with the wild type protein).
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 present invention, the
exemplary methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are incorporated by reference
in their entirety. In case of conflict, the present application, including definitions, will
control. The materials, methods, and examples are illustrative only and not intended to
be limiting.
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
Brief Description of the Drawings
Fig. 1 is an alignment of wild type human (SEQ ID NO: 10), mouse (SEQ ID
NO: 11), and rat (SEQ ID NO: 12) pre pro Neublastin polypeptides. The left and right
vertical lines indicate, respectively, the start of the mature 113 amino and 104 amino acid
forms. The RRXR heparin binding motif is boxed.
Fig. 2 A depicts a cationic elution profile of wild-type Neublastin (Peak D) and
three single Arg-to-Glu substitution mutants (Peaks A, B, and C) (sloping line represents
the theoretical sodium chloride concentration for any given volume eluted from the
column). Data are a representation of the OD280 values of the eluted sample.
Fig. 2B depicts a Heparin Sepharose elution profile of wild-type Neublastin
(Peak H) and three single Arg-to-Glu substitution mutants (Peaks E, F, and G) (sloping
line represents the theoretical sodium chloride concentration for any given volume eluted
from the column). Data are a representation of the OD280 values of the eluted sample.
Figs. 3A-3B are photographs depicting SDS/PAGE of wild-type Neublastin
following anionic chromatography in the presence (2A) or absence (2 B) of heparin.
Fig. 4 is a graph depicting the results of a Neublastin CHO cell binding assay.
Following SDS/PAGE and desitometry, OD values of Neublastin wild type and Arg48E
mutant bands were plotted against the heparin concentration in each sample.
Fig. 5 is a graph depicting the results of a heparin binding ELISA using wild type
human NBN113, wild type human NBN104, Arg48E, Arg49E, ArgSlE, and Arg48,49E.
Fig. 6 is a graph depicting the results of KIRA analysis of wild type ratNBNl 13,
ArgSlE, Arg48E, Arg49E, andratNBN113.
Fig. 7 A is a graph depicting the results of KIRA analysis of wild type human
Neublastin and Arg48,49E mutant human Neublastin (113 amino acid form).
Fig. 7B is a graph depicting the results of KIRA analysis of wild type human
Neublastin, Arg48,49E mutant human Neublastin (104 amino acid form), Arg48,5 IE
human Neublastin (113 amino acid form), and Arg49,5 IE human Neublastin (113 amino
acid form).
Fig. 8 is a graph depicting the results of ternary complex analysis of wild type
human Neublastin, Arg48E, Arg49E, ArgSlE, Arg48,49E, and Arg48,49,5 IE Neublastin
forms.
Fig. 9 is a graph depicting the results of ternary complex analysis of wild type
Neublastin, Arg48E, Arg49E, ArgSlE, Arg48,49E, and Arg48,49,5IE Neublastin forms.
Fig. 10 is a graph depicting the results of pharmacokinetics analysis of wild type
Neublastin and Arg48,49E following a single bolus 7 mg/kg subcutaneous injection
(Neublastin plasma concentrations were determined using the Neublastin detection
ELISA).
Fig. 11 is a graph depicting the results of pharmacokinetics analysis of wild type
Neublastin and Arg48,49E following a single bolus 1 mg/kg intravenous injection
(Neublastin plasma concentrations were determined using the Neublastin detection
ELISA).
Fig. 12 is a graph depicting the results of pharmacokinetics analysis of 2X10K
PEGylated Arg48,49E Neublastin following a single bolus subcutaneous 7mg/kg (data
presented are extrapolated down to 1 mg/kg) injection and 2X10K PEGylated Arg48,49E
Neublastin administered intravenously at 1 mg/kg (Neublastin plasma concentrations
were determined using the Neublastin detection ELISA).
Fig. 13 is a graph depicting relative Neublastin expression levels in CHO cells
transfected with plasmids encoding wild type Neublastin or Arg48,49E.
Fig. 14 is a graph depicting relative Neublastin expression levels in the leading
Arg48,49E double mutant transfected CHO cell lines and a leading wild type Neublastin
transfected CHO cell line.
Detailed Description
The present invention provides variant Neublastin polypeptides having
substitutions at selected amino acid residues. As disclosed in the accompanying
Examples, specific residues in the wild type Neublastin polypeptide have been found to
be important for heparin binding. Because heparin binding is believed to contribute to
clearance of Neublastin in vivo, substitutions at one or more of these specific residues are
expected to decrease heparin binding and thereby increase serum exposure of the variant
polypeptide.
Variant Neublastin Polvpeptides
Mature wild type human Neublastin is 113 amino acids in length and has the
following amino acid sequence: AGGPGSRARAAGARGCRLRSQLVPVRALGLGHR
SDELVRFRFCSGSCRRARSPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAV
SFMDVNSTWRTVDRLSATACGCLG (SEQ ID NO:1).
Disclosed herein are polypeptides that have substitutions at one or more selected
amino acid residues of the Neublastin polypeptide. Mutations at one or more of these
residues are expected to result in a variant Neublastin polypeptide having reduced or
absent heparin binding ability as compared to wild type Neublastin. A variant Neublastin
polypeptide contains an amino acid substitution, relative to SEQ ID NO:13 at (i) an
arginine residue at one or more of positions 48,49, or 51, and/or (ii) one or more of
Ser 46, Set 73, Gly 72, Arg 39, Gin 21, Ser 20, Arg 68, Arg 33, His 32, Val 94, Arg 7,
Arg 9, or Arg 14. Unless otherwise stated, any reference herein to a Neublastin amino
acid reside by position number refers to the numbering of residues relative to SEQ ID
NO:1.
A Neublastin amino acid residue designated for substitution (e.g., an arginine
residue at position 48,49, and/or 51) can be substituted with a non-conservative amino
acid residue (e.g., glutamic acid) or a conservative or amino acid residue. As detailed in
the accompanying Examples, substitution of Arg48, Arg 49, and/or Arg 51 with a nonconservative
amino acid can result in a variant Neublastin polypeptide that has reduced
heparin binding activity but retained (or even enhanced) Neublastin biological activity.
Exemplary amino acids that can be substituted an amino acid residue identified herein
(e.g., an arginine residue at position 48, 49, and/or 51) include glutamic acid, aspartic
acid, and alanine.
A biologically active variant Neublastin polypeptide, when dimerized, binds to a
ternary complex containing GFRalphaS and RET. Any method for detecting binding to
this complex can be used to evaluate the biological activity a variant Neublastin
polypeptide. Exemplary assays for detecting the ternary complex-binding ability of a
variant Neublastin polypeptide are described in WOOO/01815 and in Example 7.
A variant Neublastin polypeptide can also be assessed to evaluate its ability to
trigger the Neublastin signaling cascade. For example, the Kinase Receptor Activation
(KERA) assay described in Example 6 can be used to assess the ability of a variant
Neublastin polypeptide to induce RET autophosphorylation (See also, Sadick et al., 1996,
Anal Biochem., 235(2):207).
In addition to the specific amino acid substitutions identified herein, a variant
Neublastin polypeptide can also contain one or more additions, substitutions, and/or
deletions at other amino acid positions, as detailed in the following sections.
A variant Neublastin polypeptide can, in addition to having one or more of the
amino acid substitutions described herein, also vary in length. Although the mature
human Neublastin polypeptide (SEQ ID NO: 1) consists of the carboxy terminal 113
amino acids of pre pro Neublastin, not all of the 113 amino acids are required to achieve
useful Neublastin biological activity. Amino terminal truncation is permissible. Thus, a
variant Neublastin polypeptide can contain one or more of the amino acid substitutions
described herein in the context of the carboxy terminal 99, 100,101, 102, 103,104, 105,
106,107, 108,109,110, 111, 112, or 113 amino acids of SEQ IDNO:1 (i.e., its length
can be 99,100, 101, 102,103,104, 105,106,107, 108,109, 110, 111, 112, or 113 amino
acids).
A variant Neublastin polypeptide can, in addition to having one or more of the
amino acid substitutions described herein (and optionally having a truncation described
herein), also vary in sequence, m particular, certain amino acid substitutions can be
introduced into the Neublastin sequence without appreciable loss of a Neublastin
biological activity. In exemplary embodiments, a polypeptide (i) contains one or more of
the amino acid substitutions described herein, and (ii) is at least 70%, 80%, 85%, 90%,
95%, 98% or 99% identical to SEQ ID NO:1 (or 70%, 80%, 85%, 90%, 95%, 98% or
99% identical to amino acids 15-113 of SEQ ID NO: 1). A variant Neublastin
polypeptide differing in sequence from SEQ ID NO:1 (or differing in sequence from
amino acids 15-113 of SEQ ID NO:1) may include one or more conservative amino acid
substitutions, one or more non-conservative amino acid substitutions, and/or one or more
deletions or insertions.
Fig. 1 is an alignment of the wild type human, mouse, and rat pre pro Neublastin
polypeptides. The vertical lines in Fig.l indicate the start of the mature 113 amino acid
form (left vertical line) and 104 amino acid form (right vertical line) of Neublastin. The
KRXR heparin binding motif is boxed. This alignment of naturally occurring, bioactive
forms of Neublastin indicates specific exemplary residues (i.e., those that are not
conserved among the human, mouse, and rat forms) that can be substituted without
eliminating bioactivity.
Percent identity between amino acid sequences can be determined using the
BLAST 2.0 program. Sequence comparison can be performed using an ungapped
alignment and using the default parameters (Blossom 62 matrix, gap existence cost of 11,
per residue gap cost of 1, and a lambda ratio of 0.85). The mathematical algorithm used
in BLAST programs is described in Altschul et al., 1997, Nucleic Acids Research
25:3389-3402.
10
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.
Non-conservative substitutions include those in which (i) a residue having an
electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an
electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is
substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val), (iii) a
cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a
bulky hydrophobic or aromatic side chain (e.g., Val, He, Phe or Trp) is substituted for, or
by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).
Exemplary variant Neublastin polypeptides are disclosed in Table 1. Amino acid
residues of the variant Neublastin polypeptides that are mutated as compared to the
corresponding wild type position are bolded and underlined. In addition, the Neublastin
polypeptide (113, 99, or 104 amino acids in length) used as the background for the
substitution is depicted in Table 1.
Ammo Acid Sequence
AGGPGSRARAAGARGCRLRSQLVPVRA
LGLGHRSDELVRFRFCSGSCERARSPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT
RYEAVSFMDVNSTWRTVDRLSATACGC
LG
AGGPGSRARAAGARGCRLRSQLVPVRA
LGLGHRSDELVRFRFCSGSCREARSPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT
RYEAVSFMDVNSTWRTVDRLSATACGC
LG
AGGPGSRARAAGARGCRLRSQLVPVRA
LGLGHRSDELVRFRFCSGSCRRAESPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT
RYEAVSFMDVNSTWRTVDRLSATACGC
LG
AGGPGSRARAAGARGCRLRSQLVPVRA
LGLGHRSDELVRFRFCSGSCEEARSPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT
RYEAVSFMDVNSTWRTVDRLSATACGC
LG
GCRLRSQLVPVRALGLGHRSDELVRFRF
CSGSCEEARSPHDLSLASLLGAGALRPPP
GSRPVSQPCCRPTRYEAVSFMDVNSTW
RTVDRLSATACGCLG
AAGARGCRLRSQLVPVRALGLGHRSDE
LVRFRFCSGSCEEARSPHDLSLASLLGA
GALRPPPGSRPVSQPCCRPTRYEAVSFM
DVNSTWRTVDRLSATACGCLG
AGGPGSRARAAGARGCRLRSQLVPVRA
LGLGHRSDELVRFRFCSGSCREAESPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT
RYEAVSFMDVNSTWRTVDRLSATACGC
LG
AGGPGSRARAAGARGCRLRSQLVPVRA
LGLGHRSDELVRFRFCSGSCERAESPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT
RYEAVSFMDVNSTWRTVDRLSATACGC
LG
12
A variant Neublastin polypeptide can be optionally coupled to a polymer (e.g., a
polyalkylene glycol moiety such as a polyethylene glycol moiety). In some
embodiments, the polymer is coupled to the polypeptide at a site on the Neublastin
polypeptide that is anN terminus. In some embodiments, the variant Neublastin
polypeptide includes at least one amino acid substitution with respect to SEQ ID NO: 1 (or
with respect to amino acids 15-113 of SEQ ID NO:1), which provides an internal polymer
conjugation site to which a polymer can be conjugated. In some embodiments, the
polymer is coupled to the variant Neublastin polypeptide at a residue (numbered in
accordance with the sequence of SEQ ID NO: 1) selected from the group consisting of
position 14, position 39, position 68, and position 95. Exemplary Neublastin variants that
provide internal polymer conjugation sites are described in WO 02/060929 and WO
04/069176 (the contents of which are incorporated herein by reference).
A polypeptide can optionally contain heterologous amino acid sequences in
addition to a variant Neublastin polypeptide. "Heterologous," as used when referring to
an amino acid sequence, refers to a sequence that originates from a source foreign to the
particular host cell, or, if from the same host cell, is modified from its original form.
Exemplary heterologous sequences include a heterologous signal sequence (e.g., native
rat albumin signal sequence, a modified rat signal sequence, or a human growth hormone
signal sequence) or a sequence used for purification of a variant Neublastin polypeptide
(e.g., a histidine tag).
Neublastin polypeptides can be isolated using methods known in the art.
Naturally occurring Neublastin polypeptides can be isolated from cells or tissue sources
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, variant Neublastin polypeptides are produced by
recombinant DNA techniques. For example, a nucleic acid molecule encoding a variant
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
13
cells. When expressed in a recombinant cell, the cell is preferably cultured under
conditions allowing for expression of a variant Neublastin polypeptide. The variant
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.
Variant 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 variant Neublastin polypeptide. Many site-directed mutagenesis
kits are commercially available. One such kit is the "Transformer Site Directed
Mutagenesis Kit" sold by Clontech Laboratories (Palo Alto, CA).
Pharmaceutical Compositions
A variant Neublastin polypeptide can be incorporated into a pharmaceutical
composition containing a therapeutically effective amount of the polypeptide and one or
more adjuvants, excipients, carriers, and/or diluents. Acceptable diluents, carriers and
excipients typically do not adversely affect a recipient's homeostasis (e.g., electrolyte
balance). Acceptable carriers include biocompatible, inert or bioabsorbable salts,
buffering agents, oligo- or polysaccharides, polymers, viscosity-improving agents,
preservatives and the like. One exemplary carrier is physiologic saline (0.15 M NaCl, pH
7.0 to 7.4). Another exemplary carrier is 50 mM sodium phosphate, 100 mM sodium
chloride. Further details on techniques for formulation and administration of
pharmaceutical compositions can be found in, e.g., REMINGTON'S
PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.).
Administration of a pharmaceutical composition containing a variant Neublastin
polypeptide can be systemic or local. Pharmaceutical compositions can be formulated
such that they are suitable for parenteral and/or non-parenteral administration. Specific
administration modalities include subcutaneous, intravenous, intramuscular,
intraperitoneal transdermal, intrathecal, oral, rectal, buccal, topical, nasal, ophthalmic,
infra-articular, infra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and
infra-uterine administration.
Formulations suitable for parenteral administration conveniently contain a sterile
aqueous preparation of the variant Neublastin polypeptide, which preferably is isotonic
with the blood of the recipient (e.g., physiological saline solution). Formulations may presented in unit-dose or multi-dose form.
An exemplary formulation contains a variant Neublastin polypeptide described
herein and the following buffer components: sodium succinate (e.g., 10 mM); Nad
(e.g., 75 mM); and L-arginine (e.g., 100 mM).
Formulations suitable for oral administration may be presented as discrete units
such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount
of the variant Neublastin polypeptide; or a suspension in an aqueous liquor or a
non-aqueous liquid, such as a syrup, an elixir, an emulsion, or a draught.
Therapeutically effective amounts of a pharmaceutical composition may be
administered to a subject in need thereof in a dosage regimen ascertainable by one of skill
in the art. For example, a composition can be administered to the subject, e.g.,
systemically at a dosage from 0.01|j.g/kg to 1000 ug/kg body weight of the subject, per
dose, hi another example, the dosage is from 1 ug/kg to 100 |J,g/kg body weight of the
subject, per dose. In another example, the dosage is from 1 |J.g/kg to 30 ug/kg body
weight of the subject, per dose, e.g., from 3 |ig/kg to 10 ug/kg body weight of the subject,
per dose.
In order to optimize therapeutic efficacy, a variant Neublastin polypeptide is first
administered at different dosing regimens. The unit dose and regimen depend on factors
that include, e.g., the species of mammal, 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 variant Neublastin polypeptide 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. In addition, the frequency of dosing may be
varied depending on whether the treatment is prophylactic or therapeutic.
Methods of Treatment
Variant Neublastin polypeptides are useful for modulating metabolism, growth,
differentiation, or survival of a nerve or neuronal cell. In particular, variant Neublastin
polypeptides can be used to treat or alleviate a disorder or disease of a living animal, e.g.,
a human, which disorder or disease is responsive to the activity of a neurotrophic agent.
The variant Neublastin polypeptides disclosed herein (and pharmaceutical
compositions comprising same) can be used in methods for treating a disorder
characterized by damage 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 vestibuloacoustic complex of the eighth cranial nerve; the
ventrolateral pole of the maxillomandibular lobe of the trigeminal ganglion; and the
mesencephalic trigeminal nucleus.
In some embodiments, sensory and/or autonomic system neurons can be treated.
In particular, nociceptive and mechanoreceptive neurons can be treated, more particularly
A-delta 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
variant Neublastin polypeptides 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. Such nerve guidance channels are
disclosed, e.g., United States Patent No. 5,834,029.
In some embodiments, the variant Neublastin polypeptides (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.
In some embodiments, the variant Neublastin polypeptides (and pharmaceutical
compositions comprising same) are used for treating neuropathic pain, for treating tactile
allodynia, for reducing loss of pain sensitivity associated with neuropathy, for treating
viral infections and viral-associated neuropathies, for treating painful diabetic
neuropathy, and for treating nervous system disorders. The methods are discussed in
detail in the following subsections.
1. Treatment of Neuropathic Pain
The variant Neublastin polypeptides disclosed herein (and pharmaceutical
compositions comprising same) can be used in methods for treating neuropathic pain in a
subject comprising administering to the subject an effective amount of a variant
Neublastin polypeptide 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 anti-inflammatory drugs (NSAIDS). In
one embodiment, the analgesia-inducing compound is an anticonvulsant. In another
embodiment, the analgesia-inducing compound is gabapentin
((l-aminomethyl)cyclohexane acetic acid) orpregabalin (S-(+)-4-amino-3-
(2-methylpropyl) butanoic acid).
The variant Neublastin polypeptides disclosed herein (and pharmaceutical
compositions comprising same) can be used in the treatment of pain associated with
peripheral neuropathies. Among the peripheral neuropathies which can be treated 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 variant Neublastin polypeptides disclosed herein (and pharmaceutical
compositions comprising same) can be used in the treatment of 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 B 6 intoxication, hexacarbon intoxication, amiodarone,
chloramphenicol, disulfiram, isoniazide, gold, lithium, metronidazole, misonidazole,
nitrofurantoin), (d) drug-induced neuropathies, including therapeutic drug-induced
neuropathic pain (such as caused by anti-cancer agents, particularly anti-cancer agents
selected from the group consisting of taxol, taxotere, cisplatin, nocodazole, vincristine,
vindesine and vinblastine; and such as caused by anti-viral agents, particularly anti-viral
agents selected from the group consisting of ddl, DDC, d4T, foscarnet, dapsone,
metronidazole, and isoniazid), (e) vitamin-deficiency-induced 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 postherpetic
neuralgia), a human immunodeficiency virus (HIV), and a papilloma virus), (n)
auto-immune neuropathies (including but not limited to Guillain-Barre syndrome, chronic
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 variant Neublastin polypeptides disclosed herein (and pharmaceutical
compositions comprising same) can be used in the treatment of tactile allodynia in a
subject. The term "tactile allodynia" typically refers to the condition in a subject where
pain is evoked by stimulation of the skin (e.g. touch) that is normally innocuous.
In some embodiments, tactile allodynia is treated by administering to the subject a
pharmaceutically effective amount of a variant Neublastin polypeptide. In a related
embodiment, tactile allodynia may be treated by administering to a subject an effective
amount of a variant Neublastin polypeptide either alone, or by administering to the
subject an effective amount of a variant Neublastin polypeptide with an effective amount
of an analgesia-inducing compound selected from the group consisting of opioids, antiarrhythmics,
topical analgesics, local anaesthetics, anticonvulsants, antidepressants,
corticosteroids and NSAIDS. In one embodiment, the analgesia-inducing compound is
an anticonvulsant In another preferred embodiment, the analgesia-inducing compound is
gabapentin ((l-aminomethyl)cyclohexane acetic acid) or pregabalin (S-(+)-4-amino-3-
(2-methylpropyl)butanoic acid).
In some embodiments, a variant Neublastin polypeptide 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, vincristine, vindesine and vinblastine. Anti-viral agents include,
but are not limited to, ddl, DDC, d4T, foscamet, dapsone, metronidazole, and isoniazid.
3. Treatment for Reduction of Loss of Pain Sensitivity
In another embodiment, variant Neublastin polypeptides disclosed herein (and
pharmaceutical compositions comprising same) can be used in a method for reducing the
loss of pain sensitivity in a subject afflicted with a neuropathy. In one embodiment, the
neuropathy is diabetic neuropathy. In some embodiments, the loss of pain sensitivity is a
loss in thermal pain sensitivity. This methods include both prophylactic and therapeutic
treatment.
hi prophylactic treatment, a variant Neublastin polypeptide is administered to a
subject at risk of developing loss of pain sensitivity (such a subject would be expected to
be a subject with an early stage neuropathy). The treatment with a variant Neublastin
polypeptide under such circumstances would serve to treat at-risk patients preventively.
In therapeutic treatment, a variant Neublastin polypeptide is administered to a
subject who has experienced loss of pain sensitivity as a result of affliction with a
neuropathy (such a subject would be expected to be a subject with a late stage
neuropathy). The treatment with a variant Neublastin polypeptide 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 variant Neublastin polypeptide 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), hi an alternative embodiment, a
variant Neublastin polypeptide 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
the body, which is a common complication of a herpes zoster infection (shingles). Postherpetic
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 diabetesassociated
symptoms. During treatment, a variant Neublastin polypeptide is administered
to prevent appearance of neuropathic pain. In an alternative embodiment, a variant
Neublastin polypeptide is administered to reduce the severity of neuropathic pain that has
already appeared.
6. Treatment of Nervous System Disorders
The variant Neublastin polypeptides disclosed herein (and pharmaceutical
compositions comprising same) can be used in the treatment or prevention of 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 variant Neublastin polypeptide, a
composition containing a variant Neublastin polypeptide, or a complex that includes a
stable, aqueous soluble conjugated variant Neublastin polypeptide coupled to a
polyalkylene 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 variant Neublastin polypeptide is useful for treating 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. Variant Neublastin polypeptides are useful in the treatment of
neurodegenerative disease, e.g., cerebral ischemic neuronal damage; neuropathy, e.g.,
peripheral neuropathy, Alzheimer's disease, Huntington's disease, Parkinson's disease,
amyotrophic lateral sclerosis (ALS). Such variant Neublastin polypeptides can be used in
the treatment of impaired memory, e.g., memory impairment associated with dementia.
The following are examples of the practice of the invention. They are not to be
construed as limiting the scope of the invention in any way.
Examples
Example 1: Design and Synthesis of Variant Neublastin Polypeptides
Human Neublastin was crystallized and its structure revealed a triad of sulfate
ions interacting with the following four Neublastin residues in close proximity to each
other: Argl4, Arg48, Arg49, and ArgSl. Based upon the presence of this triad and their
relative spacing to one another, it was postulated that this region of Neublastin could be a
potential heparin sulfate-binding domain. Subsequently, a previously solved heparin
sulfate structure was docked (in-silico) to Neublastin at the site of the sulfate triad.
Heparin sulfate fit precisely in this position, suggesting that this region within Neublastin
has potential for heparin sulfate binding.
The Neublastin crystallization data also revealed that the following amino acid
residues provide supplementary interactions with either the triad of sulfate ions or with
21
one or more of three other sulfate ions that interact with Neublastin: Ser 46, Ser 73, Gly
72, Arg 39, Gin 21, Ser 20, Arg 68, Arg 33, His 32, and Val 94. In addition to the sulfate
binding sites revealed by the crystal structure, Neublastin contains a heparin sulfate
binding site consensus sequence (GPGSRAR) at residues 3-9 at its N-terminus. This
region was unstructured in the crystal structure but may become ordered upon binding
glycosaminoglycans. The region is likely to be close in space to the three sulfate cluster
observed in the crystal structure (Argl4 contributes to the heparin-binding site that is
mainly centered in the hinge region of the protein).
To investigate the biological relevance of the potential heparin sulfate-binding
domain, three individual single amino acid residue substitutions were made within the
mature 113 amino acid human Neublastin (SEQ ID NO: 1). The arginine residues at each
of position 48 (variant named "Arg48E"; SEQ ID N0:2), position 49 (variant named
"Arg49E"; SEQ ID NO:3), and position 51 (variant named "Arg51E"; SEQ ID NO:4)
were replaced by glutamate (i.e., three different single amino acid variant constructs were
prepared) with the intention of changing the residue charge from one that would attract
sulfate to one that would repel, and to potentially stabilize surrounding arginine residues.
Proteins were refolded and purified from E. coli inclusion bodies (see WO 04/069176).
Each Neublastin variant was subjected to analysis to verify structural integrity and
confirm the correct residue substitution. All three mutants were structurally comparable
to the wild-type human Neublastin.
Example 2: Cationic and Heparin Sepharose Chromatography
The variant Neublastin polypeptides were subjected to further biochemical
analysis to determine the effect each mutation had on heparin binding. Heparin
Sepharose and cationic chromatography were both employed. Since wild-type human
Neublastin is a basic protein with an apparent pi of 11.31, Neublastin binds efficiently to
cationic-based resins. A single conversion of arginine to glutamate decreases the
apparent pi to 10.88. However, this lower pi was not expected to dramatically alter
cationic resin-binding nor was it expected to alter the elution profile of the mutants
compared to that of the wild-type control.
Each of the mutants (along with the wild-type Neublastin control) was subjected
to cationic chromatography. The samples were loaded onto resin in buffer containing
5 mM phosphate pH 6.5 and 150 mM sodium chloride followed by elution with a linear
salt gradient starting at 150 mM and ending with 1M sodium chloride. Wild-type
Neublastin eluted at ~800 mM sodium chloride (Fig. 2A; Peak D), whereas each of the
three mutants eluted within a salt range of approximately 500 mM, thus reflecting their
lower pi. Arg49E and Arg5 IE (Fig. 2A; Peaks B and C) eluted with slightly higher salt
than was required to elute Arg48E (Fig. 2A; Peak A) (520 mM vs 490 mM, respectively).
This difference suggested that Arg48 is more surface accessible and contributes more to
cationic binding than that of the other two mutations.
To determine whether the Arg-to-Glu substitutions had an effect on heparin
binding, each of the three mutants (along with wild-type human Neublastin) was
subjected to Heparin Sepharose chromatography (Fig. 2B). Binding and elution
conditions were similar to those used for cationic chromatography. However, the
observed elution profile was significantly different from the cationic resin elution profile.
Wild-type Neublastin eluted at approximately 720 mM sodium chloride (Fig. 2B;
Peak H) whereas ArgSlE, Arg49E, and Arg48E eluted at 570 mM (Fig. 2B; Peak G),
510 mM (Fig. 2B; Peak F), and 450 mM (Fig. 2B; Peak E) sodium chloride, respectively.
Arg48E appeared to have a particularly dramatic effect on heparin binding. Taken
together, these chromatography profiles suggested that each mutation decreases
Neublastin's apparent affinity for heparin.
Example 3: Anionic Chromatographv •
At standard pH conditions of 6.5 and a sodium chloride concentration of 150 mM,
Neublastin does not bind to anionic resins. In contrast, heparin sulfate does bind to
anionic resins under these same conditions.
When Neublastin was pre-mixed in a 1:1 molar ratio with 16-kDa-heparin sulfate
and applied to an anionic matrix using the above conditions, Neublastin bound and eluted
with 600 mM sodium chloride (Fig. 3B, lanes marked "FT"), suggesting that Neublastin
was binding the anionic matrix through its interaction with heparin sulfate. In the
absence of heparin, Neublastin did not bind to the anionic resin (Fig. 3 A, lanes marked
"FT") and no Neublastin eluted with 600 mM sodium chloride (Fig. 3A, lanes marked
"Elution"). These data provide further evidence of Neublastin's ability to bind to
heparin.
Example 4: Chinese Hamster Ovary Cell Binding Studies
Neublastin has been shown previously to bind non-specifically to the surface of
Chinese Hamster Ovary (CHO) cells. A Neublastin CHO cell-binding assay was
established to determine whether this interaction is mediated, at least in part, through
Neublastin's binding to cell surface heparin sulfate molecules.
Wild-type human Neublastin (40 ug) or the Arg48E mutant was pre-mixed with
CHO cells (106 cells) at a cell density which completely binds both Neublastin forms
along with increasing amounts of 16 kDa heparin sulfate and incubated at 37°C for
4 hours. Following incubation, the cells were pelleted by centrifugation and remaining
non-bound Neublastin in the supernatant was subjected to SDS/PAGE analysis. After
quantification of each protein band by densitometry, the resulting optical density value
was plotted against the heparin concentration in each sample (Fig. 4).
At the two lower heparin concentrations, both the wild type and the mutant
Neublastin forms had equal amounts of protein identified in the supernatant. However,
as the heparin concentration increased to 0.5 ug/ml and higher, more wild-type
Neublastin was identified in the supernatant than that of the Arg48E mutant. This
observation suggested that heparin can compete with cell surface-bound heparin for wildtype
Neublastin binding (i.e., binding of heparin to wild-type Neublastin results in its
removal from the cell surface), whereas heparin cannot as readily compete off the
Arg48E mutant. At the highest heparin concentration (50 ug/ml), the Arg48E mutant
began to elute off the cell surface, suggesting an ionic interaction between heparin and
the Arg48E mutant might be responsible for this observation.
Example 5: Heparin Binding of Wild Type Neublastin and Variant Neublastin
Polyp eptides
To further investigate the role of the identified arginine triad as a heparin-binding
site of Neublastin, a heparin binding ELISA was established. In brief, an anti-Neublastin
monoclonal antibody was coated onto a 96-well plate, followed by washing and the
addition of one of the Neublastin forms. Biotinylated heparin was then added to the
plate. Following an additional wash step, the Neublastin/Heparin complex was identified
using a Strepavidin-HRP conjugate with a chemiluminescent substrate. This heparinbinding
ELISA was used to compare wild type human Neublastin 113 amino acid (SEQ
ID NO:1) and 104 amino acid (amino acids 10-113 of SEQ ED NO:1) forms to variant
Neublastin polypeptides containing a single amino acid substitution (Arg48E, Arg49E,
and ArgSlE; SEQ ID NOS:2-4) as well as a double substitution (Arg48, 49E; SEQ ID
NO:5).
Both wild-type forms of Neublastin bound heparin with an EC50 of ~1 ng/ml
heparin (Fig. 5). Arg49E and ArgSlE bound less efficiently, with an apparent EC50 of
~10ng/ml, but maximum binding remained the same (Fig. 5). Of the three single point
mutations, Arg48E had the most dramatic effect on heparin binding, with an apparent
EC50 of ~ 100 ng/ml, but still achieved the same maximum heparin binding value when
compared to the unmodified Neublastin forms (Fig. 5). The Arg48E mutant was thus one
hundred fold less efficient in binding heparin as compared to the unmodified Neublastin
forms and ten fold less efficient as compared to the other single substitution mutants.
When both Arg48 and Arg49 were substituted with glutamate, heparin binding was
almost eliminated, resulting in a seven-fold decrease in maximum heparin binding, but
the EC50 remained within range of the single point mutants. These results suggest that
Arg48 plays an important role in heparin binding due to its central location in the putative
heparin-binding site.
Example 6: Kinase Receptor Activation Analysis of Wild-Type Neublastin and Heparin
Binding Mutants
To determine whether heparin-binding site mutations have an effect on Neublastin
receptor signaling in a cell-based bioassay, mutant Neublastin forms along with the wildtype
Neublastin were subjected to Kinase Receptor Activation (ORA) analysis.
Each of the single Arg-to-Glu substitution mutants appeared identical to the
unmodified control with respect to KIRA activity, suggesting that these mutants are
structurally similar to the wild-type and are capable of activating the Neublastin receptor
and signaling cascade (Fig. 6). Furthermore, these data suggest that heparin binding to
Neublastin may not be required for receptor activation.
When the Arg48,49E double mutant (SEQ ID N0:5; 113 amino acid form) was
subjected to KIRA analysis, its apparent EC50 was shifted to the left by approximately
one order of magnitude with an increase in its maximum receptor activation when
compared with the wild-type human Neublastin control (Fig. 7A). Similarly, the
Arg48,49B double mutant (SEQ ED NO:7; 104 amino acid form) also exhibited increased
potency as compared to wild-type Neublastin (Fig. 7B). Each of the Arg48,51E and
Arg49,51E double mutants (SEQ ID NO:9 and SEQ ID NO:8, respectively; 113 amino
acid forms) appeared similar to the unmodified Neublastin control with respect to KIRA
activity (Fig. 7B).
Example 7: Ternary Complex Analysis
Wild-type human Neublastin and each of the heparin mutants were subjected to
ternary complex analysis using two slightly different protocols. The first protocol
combined Neublastin's receptor components (GFRalphaS and RET) along with
Neublastin in a pool before addition to an ELISA plate coated with capture antibody
(Fig. 8). The second protocol added these components sequentially to an ELISA plate
with GFRalphaS added first, followed by Neublastin, and then RET (Fig. 9).
When the components were added together as a pool, maximum binding was
achieved with both Arg48E and Arg48,49E, suggesting that these Neublastin forms have
the highest affinity for their receptor. Wild-type Neublastin appeared to bind with a
similar affinity to that of the Arg49E mutant, whereas the ArgSlE and a triple mutant
(Arg48, 49 and 51 all substituted to glutamate) demonstrated the weakest receptor
binding.
When the receptor components were added sequentially, Arg48E showed the best
receptor binding. However under these conditions, the double mutant weakly bound to its
receptor with an affinity that appeared similar to the ArgSlE mutant. Arg49E and wildtype
Neublastin had an affinity for the receptor that was midway between the observed
maximum and minimum binding. The triple mutant did not bind under these conditions.
Overall, these data suggest that Arg48 plays a pivotal role in affecting Neublastin's
affinity for its receptor.
Example 8: Near and Far UV CD Analysis
To further investigate the effects of the double mutations on Neublastin's
secondary and tertiary structure, the Arg48,49E double mutant was subjected to both
Near and Far UV CD analysis. Although subtle differences were detected in the
secondary and tertiary structures, the conformation of the double mutant was very close
to that of the wild-type Neublastin.
Example 9: Pharmacokinetic Analysis of the Neublastin Arg48. 49E Double Mutant
Human Neublastin exhibits poor pharmacokmetics (PK) when administered to
rats intravenously (IV) or subcutaneously (SC), with an overall bioavailability of less
than 1%. Heparin-based clearance may be one of the reasons for this low bioavailability.
To determine whether heparin-based clearance participates in human Neublastin's rapid
clearance from the rat, the Arg48,49E double mutant (along with the wild-type control)
was subjected to PK analysis.
Both forms were administered separately in rats at 7 mg/kg SC. Serum samples
were collected starting at 1 hour, completed at 96 hours, and analyzed for Neublastin
(Fig. 10). The observed area under the curve (AUC) for wild-type Neublastin was ~109
whereas the observed AUC for the double mutant was 20,145. This represented a 185-
fold increase in AUC for the double mutant (compared to wild-type Neublastin) and a
significant increase in serum exposure.
Both the wild type and double mutant Neublastin were also subjected to PK
analysis following IV administration (1 mg/kg). The initial plasma concentration of the
double mutant was approximately six-fold higher (diamonds) than that of the wild-type
control (squares) at five minutes following injection but quickly approached wild type
levels within one hour (Fig. 11). These data suggest that the double mutation in
Neublastin, aids in increasing serum exposure but does not affect the overall clearance
rate.
27
Taken together with the SC observation, heparin-binding appears to be especially
relevant following SC administration, perhaps resulting in a depot-like effect. Once
Neublastin enters circulation, the rate at which the double mutant and wild type
molecules are cleared is approximately the same.
To address the rate at which Neublastin is cleared from circulation in the rat, both
the wild type and double mutant forms of Neublastin were PEGylated with 10 kDa PEG
using SPA-based coupling chemistry. Since Neublastin is a homo-dimer with no native
lysine residues, the 10-kDa moieties specifically labeled the amino terminus of each
monomer. 2X1 OK PEGylated human double mutant neublastin was purified to
homogeneity, and subjected to structural and biological analysis prior to PK analysis.
2X10K PEG Arg48,49E double mutant was injected either IV (1 mg/kg) or SC (7
mg/kg) into rats and serum collected at various time points for analysis. Following IV
administration, 2X1 OK PEG double mutant achieved the theoretical Cmax of 10 ug/ml
(diamonds) with typical alpha and beta phases (Fig. 12). SC administration of the
PEGylated double mutant demonstrated a Cmax of 40 ng/ml at 24 hours injection
(Fig. 12). Once the drug reached circulation, the apparent rate of clearance paralleled
that of IV dose. Bioavailability of this construct was approximately 10% compared to
less than 1% for the non-PEGylated or PEGylated wild type human Neublastin.
Example 10: Expression of a Neublastin Heparin-Binding Mutant in Chinese Hamster
Ovary Cells
Plasmid constructs encoding wild type and mutant Neublastin were expressed in
CHO cells and the amount of secreted soluble protein was measured by ELISA. The
plasmid constructs used in these experiments encoded a fusion protein containing the
human growth hormone signal peptide (SigPep) (with or without an intron included in the
plasmid) fused to (i) the carboxy terminal 104 amino acids of wild type human
Neublastin, or (ii) the Arg48,49E double mutant (104 amino acid form).
The following are the amino acid sequences of the Neublastin fusion proteins
used in these experiments. The Neublastin sequences are in upper case type. The human
growth hormone signal peptide sequences are in lower case type. The junction of the
signal peptide and Neublastin sequences is indicated with a carat (A). The amino acids at
positions 48 and 49 are underlined.
SigPep-NBN (wild type): matgsrtslllafgllclswlqegsaAAAGARGCRLRSQLVPV
RALGLGHRSDELVRFRFCSGSCRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC
CRPTRYEAVSFMDVNSTWRTVDPxLSATACGCLG (SEQ ID NO:13).
SigPep-NBN (Arg48,49E): rnatgsrtslllafgllclswlqegsaAAAGARGCRLRSQLVP
VRALGLGHRSDELVRFRFCSGSCEEARSPHDLSLASLLGAGALRPPPGSRPVSQP
CCRPTRYEAVSFMDVNSTWRTVDRLSATACGCLG (SEQ IDNO:14).
CHO cells were transfected with plasmids encoding each of the foregoing forms
of Neublastin and cultured in 384-well plates. After several weeks, wells that contained
growing cells were transferred to fresh 96-well culture plates. Conditioned medium was
analyzed by ELISA to measure the titer of soluble Neublastin. The cumulative
absorbance data for each plasmid tested (mean value with one standard deviation as error
bars) was detected.
Transfection of CHO cells with plasmids encoding the Arg48,49E double mutant
resulted in a significantly increased number of cell lines exhibiting high expression levels
of recombinant protein, as compared to cells transfected with plasmids encoding wild
type Neublastin (Fig. 13).
The leading cell lines from each transfection were further expanded. Fixed
numbers of cells were cultured for three days and total cell count, viability, and titer were
determined. The titers of Neublastin expressed from the leading Arg48,49E double
mutant cell lines were roughly five-fold greater than those of a leading wild type
Neublastin cell line (Fig. 14).
Other Embodiments
While the invention has been described in conjunction with the detailed
description thereof, the foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the following claims.
What is claimed is:





WE CLAIM:
1. A polypeptide comprising an amino acid sequence that is at least 80% identical to amino acids 15-113 of SEQ ED NO:l, wherein the amino acid sequence comprises an amino acid other than arginine substituted at the positions corresponding to position 48 and position 49 of SEQ ID NO:l, wherein the polypeptide, when dimerized, binds to a complex containing GFRalpha3 and RET, and wherein the polypeptide exhibits decreased heparin binding as compared to the wild-type neublastin protein of SEQ ID NO:l.
2. The polypeptide as claimed in claim 1, wherein the arginine residue at position 48 and the arginine residue at position 49 of SEQ ID NO:l are substituted with non-conservative amino acid residues.
3. The polypeptide as claimed in claim 1, wherein the arginine residue at position 48 and the arginine residue at position 49 of SEQ ID NO: 1 are each substituted with glutamic acid.
4. The polypeptide as claimed in any one of claims 1 to 3, wherein the amino acid sequence is at least 90% identical to amino acids 15-113 of SEQ ID NO:l.
5. The polypeptide as claimed in any one of claims 1 to 3, wherein the amino acid sequence is at least 95% identical to amino acids 15-113 of SEQ ID NO: 1.
6. The polypeptide as claimed in any one of claims 1 to 3, wherein the amino acid sequence is at least 98% identical to amino acids 15-113 of SEQ ID NO: 1.
7. The polypeptide as claimed in claim 1, wherein the polypeptide comprises amino acids 15-113 of SEQ ID NO:5.
8. The polypeptide as claimed in claim 1, wherein the polypeptide comprises amino acids 10-113 of SEQ ID NO:5.
9. The polypeptide as claimed in claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:5.
10. The polypeptide as claimed in claim 1, wherein the polypeptide consists of amino acids 15-113 of SEQ ID NO:5.
11. The polypeptide as claimed in claim 1, wherein the polypeptide consists of amino acids 10-113 of SEQ ID NO:5.
12. The polypeptide as claimed in claim 1, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:5 or 7.
13. A dimer comprising two polypeptides as claimed in any one of claims 1 to 12.
14. A conjugate comprising the polypeptide as claimed in any one of claims 1 to 12 conjugated to a non-naturally occurring polymer.
15. The conjugate as claimed in claim 14, wherein the non-naturally occurring polymer is a polyalkylene glycol.
16. The conjugate as claimed in claim 15, wherein the polyalkylene glycol is polyethylene glycol.
17. The conjugate as claimed in any one of claims 14 to 16, wherein the non-naturally occurring polymer is coupled to the polypeptide at the amino terminus.
18. The conjugate as claimed in any one of claims 14 to 16, wherein the non-naturally occurring polymer is coupled to the polypeptide at an internal polymer conjugation site.
19. A fusion protein comprising the polypeptide as claimed in any one of claims 1 to 12 and a heterologous amino acid sequence.
20. A pharmaceutical composition comprising the polypeptide of any one as claimed in claims 1 to 12, the dimer of claim 13, the conjugate of any one of claims 14 to 18, or the fusion protein of claim 19 and a pharmaceutically acceptable carrier or excipient.
21. A nucleic acid comprising a sequence that encodes the polypeptide as claimed in any one of claims 1 to 12.
22. An expression vector comprising the nucleic acid as claimed in claim 21.


Documents:

1294-delnp-2007-Abstract-(19-02-2013).pdf

1294-delnp-2007-abstract.pdf

1294-delnp-2007-claims.pdf

1294-delnp-2007-Correspondence Others-(19-02-2013).pdf

1294-DELNP-2007-Correspondence-Others.pdf

1294-delnp-2007-description (complete).pdf

1294-delnp-2007-drawings.pdf

1294-delnp-2007-Drawomgs-(19-02-2013).pdf

1294-DELNP-2007-Form-1.pdf

1294-delnp-2007-Form-2-(19-02-2013).pdf

1294-delnp-2007-form-2.pdf

1294-delnp-2007-Form-3-(19-02-2013).pdf

1294-delnp-2007-form-3.pdf

1294-delnp-2007-form-5.pdf

1294-delnp-2007-GPA-(19-02-2013).pdf

1294-delnp-2007-gpa.pdf

1294-delnp-2007-pct-101.pdf

1294-delnp-2007-pct-304.pdf

1294-delnp-2007-Petition-137-(19-02-2013).pdf


Patent Number 258802
Indian Patent Application Number 1294/DELNP/2007
PG Journal Number 07/2014
Publication Date 14-Feb-2014
Grant Date 07-Feb-2014
Date of Filing 19-Feb-2007
Name of Patentee BIOGEN IDEC MA INC.
Applicant Address 14 CAMBRIDGE CENTER, CAMBRIDGE, MASSACHUSETTS 02142, USA
Inventors:
# Inventor's Name Inventor's Address
1 ANTHONY ROSSOMANDO 7, PRATT STREET, SOUTH GRAFTON, MASSACHUSETTS 01560, USA
2 LAURA SILVIAN 1325 BEACON STREET, WABAN MASSACHUSETTS 02468, USA
3 BLAKE R. PEPINSKY 30 FALMOUTH ROAD, ARLINGTON, MASSACHUSETTS 02474, USA
PCT International Classification Number A61K 38/17
PCT International Application Number PCT/US2005/029637
PCT International Filing date 2005-08-18
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/694,067 2005-06-24 U.S.A.
2 60/602,825 2004-08-19 U.S.A.