U.S. patent application number 13/541302 was filed with the patent office on 2013-04-18 for neublastin variants.
This patent application is currently assigned to BIOGEN IDEC MA INC.. The applicant listed for this patent is R. Blake Pepinsky, Anthony Rossomando, Laura Silvian. Invention is credited to R. Blake Pepinsky, Anthony Rossomando, Laura Silvian.
Application Number | 20130096065 13/541302 |
Document ID | / |
Family ID | 35968235 |
Filed Date | 2013-04-18 |
United States Patent
Application |
20130096065 |
Kind Code |
A1 |
Rossomando; Anthony ; et
al. |
April 18, 2013 |
NEUBLASTIN VARIANTS
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.
Inventors: |
Rossomando; Anthony; (South
Grafton, MA) ; Silvian; Laura; (Waban, MA) ;
Pepinsky; R. Blake; (Arlington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rossomando; Anthony
Silvian; Laura
Pepinsky; R. Blake |
South Grafton
Waban
Arlington |
MA
MA
MA |
US
US
US |
|
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
35968235 |
Appl. No.: |
13/541302 |
Filed: |
July 3, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11573773 |
Jul 24, 2007 |
8263553 |
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PCT/US2005/029637 |
Aug 18, 2005 |
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13541302 |
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60602825 |
Aug 19, 2004 |
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60694067 |
Jun 24, 2005 |
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Current U.S.
Class: |
514/17.7 ;
435/320.1; 435/358; 435/69.1; 530/324; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/185 20130101;
A61K 38/00 20130101; A61P 25/00 20180101; C07K 14/47 20130101; Y02A
50/30 20180101; C07K 14/495 20130101; A61P 25/02 20180101; A61P
25/04 20180101; A61P 43/00 20180101; C07K 19/00 20130101; A61K
47/60 20170801; C07K 14/4756 20130101 |
Class at
Publication: |
514/17.7 ;
530/324; 530/350; 536/23.5; 435/320.1; 435/358; 435/69.1 |
International
Class: |
C07K 14/47 20060101
C07K014/47; C07K 19/00 20060101 C07K019/00 |
Claims
1. A polypeptide comprising an amino acid sequence at least 80%
identical to amino acids 15-113 of SEQ ID NO:1, wherein the amino
acid sequence comprises at least one amino acid substitution,
relative to SEQ ID NO:1, selected from the group consisting of: an
amino acid other than arginine at the position corresponding to
position 48 of SEQ ID NO:1; an amino acid other than arginine at
the position corresponding to position 49 of SEQ ID NO:1; and an
amino acid other than arginine at the position corresponding to
position 51 of SEQ ID NO:1, wherein the polypeptide, when
dimerized, binds to a complex containing GFRalpha3 and RET.
2-16. (canceled)
17. A polypeptide comprising amino acids 15-113 of SEQ ID NO:2,
amino acids 15-113 of SEQ ID NO:3, amino acids 15-113 of SEQ ID
NO:4, amino acids 15-113 of SEQ ID NO:5, amino acids 15-113 of SEQ
ID NO:8, or amino acids 15-113 of SEQ ID NO:9.
18-19. (canceled)
20. A dimer comprising two polypeptides according to claim 1.
21. A conjugate comprising the polypeptide of claim 1 conjugated to
a non-naturally occurring polymer.
22. A fusion protein comprising the polypeptide of claim 1 and a
heterologous amino acid sequence.
23. A pharmaceutical composition comprising the polypeptide of
claim 1 a pharmaceutically acceptable carrier or excipient.
24. A nucleic acid comprising a sequence that encodes the
polypeptide of claim 1.
25. An expression vector comprising the nucleic acid of claim
24.
26. A cell comprising the expression vector of claim 25.
27. A method of making a polypeptide, the method comprising:
providing the cell of claim 26, and culturing the cell under
conditions that permit expression of the nucleic acid.
28. A method of treating or preventing a nervous system disorder in
a mammal, the method comprising administering to the mammal a
therapeutically effective amount of the pharmaceutical composition
of claim 23.
29. A method of treating neuropathic pain in a mammal, the method
comprising administering to the mammal a therapeutically effective
amount of the pharmaceutical composition of claim 23.
30. A method of activating the RET receptor in a mammal, the method
comprising administering to the mammal an effective amount of the
polypeptide of claim 1.
31. A polypeptide comprising an amino acid sequence at least 80%
identical to amino acids 15-113 of SEQ ID NO:1, wherein the amino
acid sequence comprises at least one amino acid substitution,
relative to SEQ ID NO:1, selected from the group consisting of: an
amino acid other than serine at the position corresponding to
position 20 of SEQ ID NO:1; an amino acid other than glutamine at
the position corresponding to position 21 of SEQ ID NO:1; an amino
acid other than histidine at the position corresponding to position
32 of SEQ ID NO:1; an amino acid other than arginine at the
position corresponding to position 33 of SEQ ID NO:1; an amino acid
other than arginine at the position corresponding to position 39 of
SEQ ID NO:1; an amino acid other than serine at the position
corresponding to position 46 of SEQ ID NO:1; an amino acid other
than arginine at the position corresponding to position 68 of SEQ
ID NO:1; an amino acid other than glycine at the position
corresponding to position 72 of SEQ ID NO:1; an amino acid other
than serine at the position corresponding to position 73 of SEQ ID
NO:1; and an amino acid other than valine at the position
corresponding to position 94 of SEQ ID NO:1, wherein the
polypeptide, when dimerized, binds to a complex containing
GFRalpha3 and RET.
32-34. (canceled)
35. A polypeptide comprising an amino acid sequence at least 80%
identical to SEQ ID NO:1, wherein the amino acid sequence comprises
at least one amino acid substitution, relative to SEQ ID NO:1,
selected from the group consisting of: an amino acid other than
arginine at the position corresponding to position 7 of SEQ ID
NO:1; an amino acid other than arginine at the position
corresponding to position 9 of SEQ ID NO:1; and an amino acid other
than arginine at the position corresponding to position 14 of SEQ
ID NO:1, wherein the polypeptide, when dimerized, binds to a
complex containing GFRalpha3 and RET.
36-47. (canceled)
48. A dimer comprising two polypeptides according to claim 31.
49. A conjugate comprising the polypeptide of claim 31 conjugated
to a non-naturally occurring polymer.
50. A fusion protein comprising the polypeptide of claim 31 and a
heterologous amino acid sequence.
51. A pharmaceutical composition comprising the polypeptide of
claim 31 and a pharmaceutically acceptable carrier or
excipient.
52. A nucleic acid comprising a sequence that encodes the
polypeptide of claim 31.
53. An expression vector comprising the nucleic acid of claim
52.
54. A cell comprising the expression vector of claim 53.
55. A method of making a polypeptide, the method comprising:
providing the cell of claim 54, and culturing the cell under
conditions that permit expression of the nucleic acid.
56. A method of treating or preventing a nervous system disorder in
a mammal, the method comprising administering to the mammal a
therapeutically effective amount of the pharmaceutical composition
of claim 51.
57. A method of treating neuropathic pain in a mammal, the method
comprising administering to the mammal a therapeutically effective
amount of the pharmaceutical composition of claim 51.
58. A method of activating the RET receptor in a mammal, the method
comprising administering to the mammal an effective amount of the
polypeptide of claim 31.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from provisional
application No. 60/602,825, filed Aug. 19, 2004 and provisional
application No. 60/694,067, filed Jun. 24, 2005. The entire content
of each of these prior applications is incorporated herein by
reference in its entirety.
TECHNICAL FIELD
[0002] The invention relates to protein chemistry, molecular
biology, and neurobiology.
BACKGROUND
[0003] 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).
[0004] 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 (GFRalpha1-4). Neublastin binds to GFRalpha3 together
with RET forming a ternary signaling complex (Baudet et al. 2000,
Development, 127:4335; Baloh et al., 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, neublastin has clinical application in the
general treatment of neuropathy and more specifically in the
treatment of neuropathic pain.
[0005] 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 of seven conserved cysteine
residues with similar spacing which form the structure of a
cysteine knot (Saarma, 1999, Microsc. Res. Tech., 45:292). Each
monomer contains two disulfide bonds that form a closed loop
structure encircling the third disulfide to form a tight knot
structure. The seventh cysteine contained within each monomer forms
an intermolecular disulfide bond, covalently linking the monomers
to form the final dimer product (Rattenholl et al 2000, J. Mol.
Biol., 305:523).
[0006] 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 (see e.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
[0007] 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 bioavailability of the
variants.
[0008] 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 ID
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 GFRalpha3 and RET.
[0009] 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 ID NO:1 can be substituted with non-conservative amino
acid residues (e.g., glutamic acid).
[0010] 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.
[0011] Also disclosed is a polypeptide containing amino acids
15-113 of SEQ ID NO:2, amino acids 15-113 of SEQ ID NO:3, amino
acids 15-113 of SEQ ID NO:4, amino acids 15-113 of SEQ ID NO:5,
amino acids 15-113 of SEQ ID NO:8, or amino acids 15-113 of SEQ ID
NO:9. In some embodiments, the polypeptide contains amino acids
10-113 of SEQ ID 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 ID 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 ID NO:4, the amino acid sequence of
SEQ ID NO:5, the amino acid sequence of SEQ ID NO:8, or the amino
acid sequence of SEQ ID NO:9.
[0012] Also disclosed is a polypeptide containing an amino acid
sequence at least 80% identical to amino acids 15-113 of SEQ ID
NO:1, wherein the amino acid sequence comprises at least one amino
acid substitution, relative to SEQ ID NO:1, selected from the group
consisting of: (i) an amino acid other than serine at the position
corresponding to position 20 of SEQ ID 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 ID 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
ID 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 ID 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 GFRalpha3 and RET. 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.
[0013] 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 amino acid substitution,
relative to SEQ ID NO:1, selected from the group consisting of: (i)
an amino acid other than arginine at the position corresponding to
position 7 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 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.
[0014] The invention also features conjugates containing a
polypeptide described herein conjugated to a non-naturally
occurring polymer. An exemplary polymer is a water-soluble
synthetic polymer such as a polyalkylene glycol (e.g., polyethylene
glycol).
[0015] The invention also features a fusion protein containing a
polypeptide described herein and a heterologous amino acid
sequence.
[0016] The invention also features a dimer containing two of the
polypeptides, conjugates, or fusion proteins described herein.
[0017] The invention also features a pharmaceutical composition
containing a polypeptide, dimer, conjugate, or fusion protein
described herein and a pharmaceutically acceptable carrier or
excipient.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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).
[0026] 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.
[0027] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] 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.
[0029] FIG. 2A 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] FIG. 5 is a graph depicting the results of a heparin binding
ELISA using wild type human NBN113, wild type human NBN104, Arg48E,
Arg49E, Arg51E, and Arg48, 49E.
[0034] FIG. 6 is a graph depicting the results of KIRA analysis of
wild type rat NBN113, Arg51E, Arg48E, Arg49E, and rat NBN113.
[0035] FIG. 7A is a graph depicting the results of KIRA analysis of
wild type human Neublastin and Arg48,49E mutant human Neublastin
(113 amino acid form).
[0036] 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), Arg48,51E human Neublastin (113 amino acid form),
and Arg49,51E human Neublastin (113 amino acid form).
[0037] FIG. 8 is a graph depicting the results of ternary complex
analysis of wild type human Neublastin, Arg48E, Arg49E, Arg51E,
Arg48,49E, and Arg48,49,51E Neublastin forms.
[0038] FIG. 9 is a graph depicting the results of ternary complex
analysis of wild type Neublastin, Arg48E, Arg49E, Arg51E,
Arg48,49E, and Arg48,49,51E Neublastin forms.
[0039] 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).
[0040] 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).
[0041] FIG. 12 is a graph depicting the results of pharmacokinetics
analysis of 2.times.10K PEGylated Arg48,49E Neublastin following a
single bolus subcutaneous 7 mg/kg (data presented are extrapolated
down to 1 mg/kg) injection and 2.times.10K PEGylated Arg48,49E
Neublastin administered intravenously at 1 mg/kg (Neublastin plasma
concentrations were determined using the Neublastin detection
ELISA).
[0042] FIG. 13 is a graph depicting relative Neublastin expression
levels in CHO cells transfected with plasmids encoding wild type
Neublastin or Arg48,49E.
[0043] 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
[0044] 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 Polypeptides
[0045] Mature wild type human Neublastin is 113 amino acids in
length and has the following amino acid sequence:
TABLE-US-00001 (SEQ ID NO: 1)
AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCRR
ARSPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTW
RTVDRLSATACGCLG.
[0046] 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:1, at (i) an arginine residue
at one or more of positions 48, 49, or 51, and/or (ii) one or more
of Ser 46, Ser 73, Gly 72, Arg 39, Gln 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.
[0047] 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 non-conservative 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.
[0048] A biologically active variant Neublastin polypeptide, when
dimerized, binds to a ternary complex containing GFRalpha3 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 WO00/01815 and
in Example 7.
[0049] A variant Neublastin polypeptide can also be assessed to
evaluate its ability to trigger the Neublastin signaling cascade.
For example, the Kinase Receptor Activation (KIRA) 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).
[0050] 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.
[0051] 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 ID NO: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).
[0052] 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. In 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.
[0053] FIG. 1 is an alignment of the wild type human, mouse, and
rat pre pro Neublastin polypeptides. The vertical lines in FIG. 1
indicate the start of the mature 113 amino acid form (left vertical
line) and 104 amino acid form (right vertical line) of Neublastin.
The RRXR 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.
[0054] 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.
[0055] 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.
[0056] 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, Ile,
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, Ile, Phe or Tip) is substituted
for, or by, one having a smaller side chain (e.g., Ala, Ser) or no
side chain (e.g., Gly).
[0057] 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.
TABLE-US-00002 TABLE 1 Variant Neublastin Polypeptides SEQ Position
Length of ID NO Substituted Polypeptide Amino Acid Sequence 2 Arg
48 113 AGGPGSRARAAGARGCRLRSQLVPVRA LGLGHRSDELVRFRFCSGSCERARSPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT RYEAVSFMDVNSTWRTVDRLSATACGC LG 3 Arg
49 113 AGGPGSRARAAGARGCRLRSQLVPVRA LGLGHRSDELVRFRFCSGSCREARSPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT RYEAVSFMDVNSTWRTVDRLSATACGC LG 4 Arg
51 113 AGGPGSRARAAGARGCRLRSQLVPVRA LGLGHRSDELVRFRFCSGSCRRAESPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT RYEAVSFMDVNSTWRTVDRLSATACGC LG 5 Arg
48 and 113 AGGPGSRARAAGARGCRLRSQLVPVRA Arg 49
LGLGHRSDELVRFRFCSGSCEEARSPHD LSLASLLGAGALRPPPGSRPVSQPCCRPT
RYEAVSFMDVNSTWRTVDRLSATACGC LG 6 Arg 48 and 99
GCRLRSQLVPVRALGLGHRSDELVRFRF Arg 49 CSGSCEEARSPHDLSLASLLGAGALRPPP
GSRPVSQPCCRPTRYEAVSFMDVNSTW RTVDRLSATACGCLG 7 Arg 48 and 104
AAGARGCRLRSQLVPVRALGLGHRSDE Arg 49 LVRFRFCSGSCEEARSPHDLSLASLLGA
GALRPPPGSRPVSQPCCRPTRYEAVSFM DVNSTWRTVDRLSATACGCLG 8 Arg 49 and 113
AGGPGSRARAAGARGCRLRSQLVPVRA Arg 51 LGLGHRSDELVRFRFCSGSCREAESPHD
LSLASLLGAGALRPPPGSRPVSQPCCRPT RYEAVSFMDVNSTWRTVDRLSATACGC LG 9 Arg
48 and 113 AGGPGSRARAAGARGCRLRSQLVPVRA Arg 51
LGLGHRSDELVRFRFCSGSCERAESPHD LSLASLLGAGALRPPPGSRPVSQPCCRPT
RYEAVSFMDVNSTWRTVDRLSATACGC LG
[0058] 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 an N 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).
[0059] 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).
[0060] 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).
[0061] 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 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.
[0062] 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,
Calif.).
Pharmaceutical Compositions
[0063] 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.).
[0064] 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,
intra-articular, intra-arterial, sub-arachnoid, bronchial,
lymphatic, vaginal, and intra-uterine administration.
[0065] 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 be presented in unit-dose or multi-dose form.
[0066] An exemplary formulation contains a variant Neublastin
polypeptide described herein and the following buffer components:
sodium succinate (e.g., 10 mM); NaCl (e.g., 75 mM); and L-arginine
(e.g., 100 mM).
[0067] 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.
[0068] 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 .mu.g/kg to 1000 .mu.g/kg body
weight of the subject, per dose. In another example, the dosage is
from 1 .mu.g/kg to 100 .mu.g/kg body weight of the subject, per
dose. In another example, the dosage is from 1 .mu.g/kg to 30
.mu.g/kg body weight of the subject, per dose, e.g., from 3
.mu.g/kg to 10 .mu.g/kg body weight of the subject, per dose.
[0069] 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.
[0070] 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
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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., U.S. Pat. No. 5,834,029.
[0075] 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.
[0076] 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.
[0077] 1. Treatment of Neuropathic Pain
[0078] 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 ((1-aminomethyl)cyclohexane acetic acid) or
pregabalin (S-(+)-4-amino-3-(2-methylpropyl)butanoic acid).
[0079] 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.
[0080] 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 B6 intoxication,
hexacarbon intoxication, amiodarone, chloramphenicol, disulfuram,
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 ddI, 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), (l) 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 (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.
[0081] 2. Treatment of Tactile Allodynia
[0082] 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.
[0083] 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, anti-arrhythmics, 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
((1-aminomethyl)cyclohexane acetic acid) or pregabalin
(S-(+)-4-amino-3-(2-methylpropyl)butanoic acid).
[0084] 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,
ddI, DDC, d4T, foscarnet, dapsone, metronidazole, and
isoniazid.
[0085] 3. Treatment for Reduction of Loss of Pain Sensitivity
[0086] 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.
[0087] In 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.
[0088] 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.
[0089] 4. Treatment of Viral Infections and Viral-Associated
Neuropathies
[0090] 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). In an alternative embodiment, a
variant Neublastin polypeptide is administered to reduce the
severity of neuropathic pain, should it appear.
[0091] 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). Post-herpetic neuralgia can last for a month or more,
and may appear several months after any rash-like symptoms have
disappeared.
[0092] 5. Treatment of Painful Diabetic Neuropathy
[0093] 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 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.
[0094] 6. Treatment of Nervous System Disorders
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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
[0099] 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: Arg14, Arg48,
Arg49, and Arg51. 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.
[0100] The Neublastin crystallization data also revealed that the
following amino acid residues provide supplementary interactions
with either the triad of sulfate ions or with one or more of three
other sulfate ions that interact with Neublastin: Ser 46, Ser 73,
Gly 72, Arg 39, Gln 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 (Arg14 contributes to the heparin-binding
site that is mainly centered in the hinge region of the
protein).
[0101] 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 NO: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
[0102] 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.
[0103] 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 .about.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 Arg51E (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.
[0104] 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
Arg51E, 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 Chromatography
[0105] 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.
[0106] 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. 3A, 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
[0107] 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.
[0108] Wild-type human Neublastin (40 ug) or the Arg48E mutant was
pre-mixed with CHO cells (10.sup.6 cells) at a cell density which
completely binds both Neublastin forms along with increasing
amounts of 16 kDa heparin sulfate and incubated at 37.degree. 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).
[0109] 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 wild-type 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
Polypeptides
[0110] 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 heparin-binding
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 ID
NO:1) forms to variant Neublastin polypeptides containing a single
amino acid substitution (Arg48E, Arg49E, and Arg51E; SEQ ID
NOS:2-4) as well as a double substitution (Arg48, 49E; SEQ ID
NO:5).
[0111] Both wild-type forms of Neublastin bound heparin with an
EC50 of .about.1 ng/ml heparin (FIG. 5). Arg49E and Arg52E bound
less efficiently, with an apparent EC50 of .about.10 ng/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 .about.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
[0112] 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 wild-type Neublastin were
subjected to Kinase Receptor Activation (KIRA) analysis.
[0113] 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.
[0114] When the Arg48,49E double mutant (SEQ ID NO: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
Arg48,49E double mutant (SEQ ID 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
[0115] 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 (GFRalpha3 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 GFRalpha3 added first, followed by
Neublastin, and then RET (FIG. 9).
[0116] 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 Arg51E and a
triple mutant (Arg48, 49 and 51 all substituted to glutamate)
demonstrated the weakest receptor binding.
[0117] 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 Arg51E mutant. Arg49E and wild-type
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
[0118] 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
[0119] Human Neublastin exhibits poor pharmacokinetics (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.
[0120] 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 .about.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.
[0121] 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.
[0122] 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.
[0123] 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. 2.times.10K PEGylated human double mutant
neublastin was purified to homogeneity, and subjected to structural
and biological analysis prior to PK analysis.
[0124] 2.times.10K 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,
2.times.10K 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
[0125] 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).
[0126] 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 ( ). The
amino acids at positions 48 and 49 are underlined.
TABLE-US-00003 SigPep-NBN (wild type): (SEQ ID NO: 13)
matgsrtslllafgllclswlqegsa{circumflex over (
)}AAGARGCRLRSQLVPVRALGLG
HRSDELVRFRFCSGSCRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC
CRPTRYEAVSFMDVNSTWRTVDRLSATACGCLG. SigPep-NBN (Arg48, 49E): (SEQ ID
NO: 14) matgsrtslllafgllclswlqegsa{circumflex over (
)}AAAGARGCRLRSQLVPVRALGL
GHRSDELVRFRFCSGSCEEARSPHDLSLASLLGAGALRPPPGSRPVSQP
CCRPTRYEAVSFMDVNSTWRTVDRLSATACGCLG.
[0127] 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.
[0128] 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).
[0129] 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
[0130] 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.
Sequence CWU 1
1
141113PRTHomo sapiens 1Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala
Gly Ala Arg Gly Cys1 5 10 15Arg Leu Arg Ser Gln Leu Val Pro Val Arg
Ala Leu Gly Leu Gly His 20 25 30Arg Ser Asp Glu Leu Val Arg Phe Arg
Phe Cys Ser Gly Ser Cys Arg 35 40 45Arg Ala Arg Ser Pro His Asp Leu
Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro Pro
Gly Ser Arg Pro Val Ser Gln Pro Cys65 70 75 80Cys Arg Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95Thr Trp Arg Thr
Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu 100 105
110Gly2113PRTArtificial SequenceExemplary variant of human
Neublastin polypeptide 2Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala
Gly Ala Arg Gly Cys1 5 10 15Arg Leu Arg Ser Gln Leu Val Pro Val Arg
Ala Leu Gly Leu Gly His 20 25 30Arg Ser Asp Glu Leu Val Arg Phe Arg
Phe Cys Ser Gly Ser Cys Glu 35 40 45Arg Ala Arg Ser Pro His Asp Leu
Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro Pro
Gly Ser Arg Pro Val Ser Gln Pro Cys65 70 75 80Cys Arg Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95Thr Trp Arg Thr
Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu 100 105
110Gly3113PRTArtificial SequenceExemplary variant of human
Neublastin polypeptide 3Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala
Gly Ala Arg Gly Cys1 5 10 15Arg Leu Arg Ser Gln Leu Val Pro Val Arg
Ala Leu Gly Leu Gly His 20 25 30Arg Ser Asp Glu Leu Val Arg Phe Arg
Phe Cys Ser Gly Ser Cys Arg 35 40 45Glu Ala Arg Ser Pro His Asp Leu
Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro Pro
Gly Ser Arg Pro Val Ser Gln Pro Cys65 70 75 80Cys Arg Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95Thr Trp Arg Thr
Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu 100 105
110Gly4113PRTArtificial SequenceExemplary variant of human
Neublastin polypeptide 4Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala
Gly Ala Arg Gly Cys1 5 10 15Arg Leu Arg Ser Gln Leu Val Pro Val Arg
Ala Leu Gly Leu Gly His 20 25 30Arg Ser Asp Glu Leu Val Arg Phe Arg
Phe Cys Ser Gly Ser Cys Arg 35 40 45Arg Ala Glu Ser Pro His Asp Leu
Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro Pro
Gly Ser Arg Pro Val Ser Gln Pro Cys65 70 75 80Cys Arg Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95Thr Trp Arg Thr
Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu 100 105
110Gly5113PRTArtificial SequenceExemplary variant of human
Neublastin polypeptide 5Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala
Gly Ala Arg Gly Cys1 5 10 15Arg Leu Arg Ser Gln Leu Val Pro Val Arg
Ala Leu Gly Leu Gly His 20 25 30Arg Ser Asp Glu Leu Val Arg Phe Arg
Phe Cys Ser Gly Ser Cys Glu 35 40 45Glu Ala Arg Ser Pro His Asp Leu
Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro Pro
Gly Ser Arg Pro Val Ser Gln Pro Cys65 70 75 80Cys Arg Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95Thr Trp Arg Thr
Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu 100 105
110Gly699PRTArtificial SequenceExemplary variant of human
Neublastin polypeptide 6Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val
Arg Ala Leu Gly Leu1 5 10 15Gly His Arg Ser Asp Glu Leu Val Arg Phe
Arg Phe Cys Ser Gly Ser 20 25 30Cys Glu Glu Ala Arg Ser Pro His Asp
Leu Ser Leu Ala Ser Leu Leu 35 40 45Gly Ala Gly Ala Leu Arg Pro Pro
Pro Gly Ser Arg Pro Val Ser Gln 50 55 60Pro Cys Cys Arg Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val65 70 75 80Asn Ser Thr Trp Arg
Thr Val Asp Arg Leu Ser Ala Thr Ala Cys Gly 85 90 95Cys Leu
Gly7104PRTArtificial SequenceExemplary variant of human Neublastin
polypeptide 7Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln Leu
Val Pro Val1 5 10 15Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu
Val Arg Phe Arg 20 25 30Phe Cys Ser Gly Ser Cys Glu Glu Ala Arg Ser
Pro His Asp Leu Ser 35 40 45Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu
Arg Pro Pro Pro Gly Ser 50 55 60Arg Pro Val Ser Gln Pro Cys Cys Arg
Pro Thr Arg Tyr Glu Ala Val65 70 75 80Ser Phe Met Asp Val Asn Ser
Thr Trp Arg Thr Val Asp Arg Leu Ser 85 90 95Ala Thr Ala Cys Gly Cys
Leu Gly 1008113PRTArtificial SequenceExemplary variant of human
Neublastin polypeptide 8Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala
Gly Ala Arg Gly Cys1 5 10 15Arg Leu Arg Ser Gln Leu Val Pro Val Arg
Ala Leu Gly Leu Gly His 20 25 30Arg Ser Asp Glu Leu Val Arg Phe Arg
Phe Cys Ser Gly Ser Cys Arg 35 40 45Glu Ala Glu Ser Pro His Asp Leu
Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro Pro
Gly Ser Arg Pro Val Ser Gln Pro Cys65 70 75 80Cys Arg Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95Thr Trp Arg Thr
Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu 100 105
110Gly9113PRTArtificial SequenceExemplary variant of human
Neublastin polypeptide 9Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala
Gly Ala Arg Gly Cys1 5 10 15Arg Leu Arg Ser Gln Leu Val Pro Val Arg
Ala Leu Gly Leu Gly His 20 25 30Arg Ser Asp Glu Leu Val Arg Phe Arg
Phe Cys Ser Gly Ser Cys Glu 35 40 45Arg Ala Glu Ser Pro His Asp Leu
Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro Pro
Gly Ser Arg Pro Val Ser Gln Pro Cys65 70 75 80Cys Arg Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95Thr Trp Arg Thr
Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu 100 105
110Gly10220PRTHomo sapiens 10Met Glu Leu Gly Leu Gly Gly Leu Ser
Thr Leu Ser His Cys Pro Trp1 5 10 15Pro Arg Arg Gln Pro Ala Leu Trp
Pro Thr Leu Ala Ala Leu Ala Leu 20 25 30Leu Ser Ser Val Ala Glu Ala
Ser Leu Gly Ser Ala Pro Arg Ser Pro 35 40 45Ala Pro Arg Glu Gly Pro
Pro Pro Val Leu Ala Ser Pro Ala Gly His 50 55 60Leu Pro Gly Gly Arg
Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg65 70 75 80Pro Pro Pro
Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro 85 90 95Ser Ala
Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly 100 105
110Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln
115 120 125Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp
Glu Leu 130 135 140Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg
Ala Arg Ser Pro145 150 155 160His Asp Leu Ser Leu Ala Ser Leu Leu
Gly Ala Gly Ala Leu Arg Pro 165 170 175Pro Pro Gly Ser Arg Pro Val
Ser Gln Pro Cys Cys Arg Pro Thr Arg 180 185 190Tyr Glu Ala Val Ser
Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val 195 200 205Asp Arg Leu
Ser Ala Thr Ala Cys Gly Cys Leu Gly 210 215 22011224PRTMus musculus
11Met Glu Leu Gly Leu Ala Glu Pro Thr Ala Leu Ser His Cys Leu Arg1
5 10 15Pro Arg Trp Gln Ser Ala Trp Trp Pro Thr Leu Ala Val Leu Ala
Leu 20 25 30Leu Ser Cys Val Thr Glu Ala Ser Leu Asp Pro Met Ser Arg
Ser Pro 35 40 45Ala Ala Arg Asp Gly Pro Ser Pro Val Leu Ala Pro Pro
Thr Asp His 50 55 60Leu Pro Gly Gly His Thr Ala His Leu Cys Ser Glu
Arg Thr Leu Arg65 70 75 80Pro Pro Pro Gln Ser Pro Gln Pro Ala Pro
Pro Pro Pro Gly Pro Ala 85 90 95Leu Gln Ser Pro Pro Ala Ala Leu Arg
Gly Ala Arg Ala Ala Arg Ala 100 105 110Gly Thr Arg Ser Ser Arg Ala
Arg Thr Thr Asp Ala Arg Gly Cys Arg 115 120 125Leu Arg Ser Gln Leu
Val Pro Val Ser Ala Leu Gly Leu Gly His Ser 130 135 140Ser Asp Glu
Leu Ile Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg145 150 155
160Ala Arg Ser Gln His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly
165 170 175Ala Leu Arg Ser Pro Pro Gly Ser Arg Pro Ile Ser Gln Pro
Cys Cys 180 185 190Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp
Val Asn Ser Thr 195 200 205Trp Arg Thr Val Asp His Leu Ser Ala Thr
Ala Cys Gly Cys Leu Gly 210 215 22012224PRTRattus norvegicus 12Met
Glu Leu Gly Leu Gly Glu Pro Thr Ala Leu Ser His Cys Leu Arg1 5 10
15Pro Arg Trp Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu
20 25 30Leu Ser Ser Val Thr Glu Ala Ser Leu Asp Pro Met Ser Arg Ser
Pro 35 40 45Ala Ser Arg Asp Val Pro Ser Pro Val Leu Ala Pro Pro Thr
Asp Tyr 50 55 60Leu Pro Gly Gly His Thr Ala His Leu Cys Ser Glu Arg
Ala Leu Arg65 70 75 80Pro Pro Pro Gln Ser Pro Gln Pro Ala Pro Pro
Pro Pro Gly Pro Ala 85 90 95Leu Gln Ser Pro Pro Ala Ala Leu Arg Gly
Ala Arg Ala Ala Arg Ala 100 105 110Gly Thr Arg Ser Ser Arg Ala Arg
Ala Thr Asp Ala Arg Gly Cys Arg 115 120 125Leu Arg Ser Gln Leu Val
Pro Val Ser Ala Leu Gly Leu Gly His Ser 130 135 140Ser Asp Glu Leu
Ile Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg145 150 155 160Ala
Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly 165 170
175Ala Leu Arg Ser Pro Pro Gly Ser Arg Pro Ile Ser Gln Pro Cys Cys
180 185 190Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn
Ser Thr 195 200 205Trp Arg Thr Val Asp His Leu Ser Ala Thr Ala Cys
Gly Cys Leu Gly 210 215 22013130PRTArtificial SequenceSynthetically
generated peptide 13Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala
Phe Gly Leu Leu1 5 10 15Cys Leu Ser Trp Leu Gln Glu Gly Ser Ala Ala
Ala Gly Ala Arg Gly 20 25 30Cys Arg Leu Arg Ser Gln Leu Val Pro Val
Arg Ala Leu Gly Leu Gly 35 40 45His Arg Ser Asp Glu Leu Val Arg Phe
Arg Phe Cys Ser Gly Ser Cys 50 55 60Arg Arg Ala Arg Ser Pro His Asp
Leu Ser Leu Ala Ser Leu Leu Gly65 70 75 80Ala Gly Ala Leu Arg Pro
Pro Pro Gly Ser Arg Pro Val Ser Gln Pro 85 90 95Cys Cys Arg Pro Thr
Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn 100 105 110Ser Thr Trp
Arg Thr Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys 115 120 125Leu
Gly 13014130PRTArtificial SequenceSynthetically generated peptide
14Met Ala Thr Gly Ser Arg Thr Ser Leu Leu Leu Ala Phe Gly Leu Leu1
5 10 15Cys Leu Ser Trp Leu Gln Glu Gly Ser Ala Ala Ala Gly Ala Arg
Gly 20 25 30Cys Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly
Leu Gly 35 40 45His Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser
Gly Ser Cys 50 55 60Glu Glu Ala Arg Ser Pro His Asp Leu Ser Leu Ala
Ser Leu Leu Gly65 70 75 80Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser
Arg Pro Val Ser Gln Pro 85 90 95Cys Cys Arg Pro Thr Arg Tyr Glu Ala
Val Ser Phe Met Asp Val Asn 100 105 110Ser Thr Trp Arg Thr Val Asp
Arg Leu Ser Ala Thr Ala Cys Gly Cys 115 120 125Leu Gly 130
* * * * *