U.S. patent application number 13/228784 was filed with the patent office on 2012-01-12 for tweak binding antibodies.
This patent application is currently assigned to Biogen Idec MA Inc.. Invention is credited to Linda C. Burkly, Ellen Garber, Alexey Lugovskoy.
Application Number | 20120009178 13/228784 |
Document ID | / |
Family ID | 37482143 |
Filed Date | 2012-01-12 |
United States Patent
Application |
20120009178 |
Kind Code |
A1 |
Burkly; Linda C. ; et
al. |
January 12, 2012 |
TWEAK BINDING ANTIBODIES
Abstract
Anti-Tweak antibodies are described.
Inventors: |
Burkly; Linda C.; (West
Newton, MA) ; Garber; Ellen; (Cambridge, MA) ;
Lugovskoy; Alexey; (Woburn, MA) |
Assignee: |
Biogen Idec MA Inc.
|
Family ID: |
37482143 |
Appl. No.: |
13/228784 |
Filed: |
September 9, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11944709 |
Nov 26, 2007 |
8048422 |
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13228784 |
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PCT/US2006/019706 |
May 25, 2006 |
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11944709 |
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60685149 |
May 27, 2005 |
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Current U.S.
Class: |
424/130.1 ;
530/387.1; 530/387.3; 530/391.1; 530/391.3; 530/391.7 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 43/00 20180101; C07K 2317/24 20130101; A61P 25/00 20180101;
C07K 2317/76 20130101; A61P 37/06 20180101; A61P 37/02 20180101;
A61P 25/28 20180101; A61P 9/10 20180101; A61P 35/00 20180101; A61P
19/02 20180101; A61K 2039/505 20130101; C07K 16/2875 20130101; A61P
37/00 20180101; A61P 9/00 20180101 |
Class at
Publication: |
424/130.1 ;
530/387.1; 530/391.1; 530/391.3; 530/391.7; 530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 29/00 20060101 A61P029/00; A61P 25/00 20060101
A61P025/00; C07K 16/18 20060101 C07K016/18 |
Claims
1. An antibody or antigen binding fragment thereof comprising heavy
chain complementarity determining regions (CDRs) 1-3 that have the
amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and
SEQ ID NO: 3, respectively and light chain CDRs 1-3 that have the
amino acid sequences set forth in SEQ ID NO: 8, SEQ ID NO: 9, and
SEQ ID NO: 10, respectively, wherein the CDRs are modified at zero
to four amino acid residues per CDR.
2. The antibody or antigen binding fragment of claim 1 further
comprising a heavy chain variable domain comprising the amino acid
sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,
SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID
NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,
SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID
NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47,
SEQ ID NO: 48, or SEQ ID NO: 49.
3. The antibody or antigen binding fragment of claim 1 further
comprising a light chain variable domain comprising an amino acid
sequence set forth in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID
NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.
4. The antibody or antigen binding fragment of claim 1, wherein the
heavy chain variable domain varies from the sequence set forth in
SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID
NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35,
SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID
NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44,
SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, and SEQ
ID NO: 49 by fewer than eight amino acid substitutions, insertions
or deletions.
5. The antibody or antigen binding fragment of claim 1, wherein the
light chain variable domain varies from the sequence set forth in
SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID
NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,
and SEQ ID NO: 26 by fewer than eight amino acid substitutions,
insertions or deletions.
6. The antibody or antigen binding fragment of claim 1, wherein the
heavy chain variable domain comprises an amino acid sequence
selected from the group consisting of: a) an amino acid sequence
that is at least 95% identical to the sequence set forth in SEQ ID
NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31,
SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID
NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40,
SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID
NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO:
49; and b) an amino acid sequence that is at least 90% identical to
the sequence set forth in SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:
29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ
ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:
38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ
ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO:
47, SEQ ID NO: 48, or SEQ ID NO: 49.
7. The antibody or antigen binding fragment of claim 1, wherein the
light chain variable domain comprises an amino acid sequence
selected from the group consisting of: a) an amino acid sequence
that is at least 95% identical to the sequence set forth in SEQ ID
NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ
ID NO: 26; and b) an amino acid sequence that is at least 90%
identical to the sequence set forth in SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ
ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.
8. An antibody or antigen binding fragment thereof comprising a
heavy chain variable domain as set forth in claim 6 and a light
chain variable domain as set forth in claim 7.
9. The antibody or antigen binding fragment of claim 1, wherein the
heavy chain variable domain comprises an amino acid sequence
selected from the group consisting of a) an amino acid sequence set
forth in SEQ ID NO: 50; b) an amino acid sequence that is at least
95% identical to the sequence set forth in SEQ ID NO: 50; and c) an
amino acid sequence that is at least 90% identical to the sequence
set forth in SEQ ID NO: 50.
10. The antibody or antigen binding fragment of claim 1, wherein
the light chain variable domain comprises an amino acid sequence
selected from the group consisting of a) an amino acid sequence set
forth in SEQ ID NO: 51; b) an amino acid sequence that is at least
95% identical to the sequence set forth in SEQ ID NO: 51; and c) an
amino acid sequence that is at least 90% identical to the sequence
set forth in SEQ ID NO: 51.
11. The antibody or antigen binding fragment of claim 1 further
comprising a heavy and light chain variable domain comprising: 1) a
heavy chain variable domain comprising an amino acid sequence
selected from the group consisting of: a) an amino acid sequence
set forth in SEQ ID NO: 50; b) an amino acid sequence that is at
least 95% identical to the sequence set forth in SEQ ID NO: 50; and
c) an amino acid sequence that is at least 90% identical to the
sequence set forth in SEQ ID NO: 50; and 2) a light chain variable
domain comprising an amino acid sequence selected from the group
consisting of a) an amino acid sequence set forth in SEQ ID NO: 51;
b) an amino acid sequence that is at least 95% identical to the
sequence set forth in SEQ ID NO: 51; and c) an amino acid sequence
that is at least 90% identical to the sequence set forth in SEQ ID
NO: 51.
12. The antibody or antigen binding fragment of claim 1 further
comprising a heavy chain comprising an amino acid sequence selected
from the group consisting of a) an amino acid sequence set forth in
SEQ ID NOs: 59, or 64; b) an amino acid sequence that is at least
95% identical to the sequence set forth in SEQ ID NOs: 59, or 64;
and c) an amino acid sequence that is at least 90% identical to the
sequence set forth in SEQ ID NOs: 59 or 64.
13. The antibody or antigen binding fragment of claim 1 further
comprising a light chain comprising an amino acid sequence selected
from the group consisting of: a) an amino acid sequence set forth
in SEQ ID NOs: 61, 63, 66, or 68; b) an amino acid sequence that is
at least 95% identical to the sequence set forth in SEQ ID NOs: 61,
63, 66, or 68; and c) an amino acid sequence that is at least 90%
identical to the sequence set forth in SEQ ID NOs: 61, 63, 66, or
68.
14. The antibody or antigen binding fragment of claim 1 further
comprising a heavy and light chain comprising an amino acid
sequence selected from the group consisting of: 1) a heavy chain
comprising an amino acid sequence selected from the group
consisting of: a) an amino acid sequence set forth in SEQ ID NOs:
59, or 64; b) an amino acid sequence that is at least 95% identical
to the sequence set forth in SEQ ID NOs: 59, or 64; and c) an amino
acid sequence that is at least 90% identical to the sequence set
forth in SEQ ID NOs: 59 or 64; and 2) a light chain comprising an
amino acid sequence selected from the group consisting of: a) an
amino acid sequence set forth in SEQ ID NOs: 61, 63, 66, or 68; b)
an amino acid sequence that is at least 95% identical to the
sequence set forth in SEQ ID NOs: 61, 63, 66, or 68; and c) an
amino acid sequence that is at least 90% identical to the sequence
set forth in SEQ ID NOs: 61, 63, 66, or 68.
15. The antibody or antigen binding fragment of claim 1, wherein
the heavy chain variable domain sequence is at least 95% identical
to the amino acid sequence of the corresponding variable domain of
SEQ ID NO: 59, or SEQ ID NO: 64, and the light chain variable
domain sequence is at least 95% identical to the amino acid
sequence of the corresponding variable domain of SEQ ID NO: 61, SEQ
ID NO: 63, SEQ ID NO: 66; or SEQ ID NO: 68.
16. The antibody or antigen binding fragment of claim 1, wherein
the heavy chain variable domain sequence is identical to the amino
acid sequence of the corresponding variable domain of SEQ ID NO:
59, or SEQ ID NO: 64, and wherein the light chain variable domain
sequence is identical to the amino acid sequence of the
corresponding variable domain of SEQ ID NO: 61, SEQ ID NO: 63, SEQ
ID NO: 66; or SEQ ID NO: 68.
17. An antibody or antigen binding fragment thereof comprising a
variable heavy domain comprising the amino acids set forth in SEQ
ID NO: 59 and a variable light domain comprising the amino acids
set forth in SEQ ID NO: 61.
18. An antibody or antigen binding fragment thereof comprising a
variable heavy domain comprising the amino acids set forth in SEQ
ID NO: 59 and a variable light domain comprising the amino acids
set forth in SEQ ID NO: 63.
19. The antibody or antigen binding fragment of any one of claims
1-18, wherein the antibody or antigen binding fragment is linked to
one or more other functional molecules.
20. The antibody or antigen binding fragment of claim 19, wherein
the one or more other functional molecules is selected from the
group consisting of another antibody or antigen binding fragment
thereof, toxins, radioisotopes, polymers, and cytotoxic or
cytostatic agents.
21. The antibody or antigen binding fragment of claim 20, wherein
the antigen binding fragment is an scFv.
22. The antibody or antigen binding fragment of claim 1 further
comprising a heavy chain framework region comprising FR1, FR2, and
FR3, wherein FR1, FR2, and FR3 comprise an amino acid sequence that
is at least 85% identical to the heavy chain framework selected
from the group consisting of: a germline V segment sequence
selected from the group consisting of: DP-25, DP-1, DP-12, DP-9,
DP-7, DP-31, DP-32, DP-33, DP-58, and DP-54; a V.sub.H I subgroup
germline sequence; a V.sub.H III subgroup germline sequence; and a
V gene which is compatible with the canonical structure class
1-3.
23. The antibody or antigen binding fragment of claim 22 wherein
the framework sequence comprises one or more of the following
residues according to Kabat numbering: Ala, Gly, Thr, or Val at
position 26; Gly at position 26; Tyr, Phe, or Gly at position 27;
Phe, Val, Ile, or Leu at position 29; Met, He, Leu, Val, Thr, Trp,
or Ile at position 34; Arg, Thr, Ala, or Lys at position 94; Gly,
Ser, Asn, or Asp at position 54; Arg at position 71.
24. The antibody or antigen binding fragment of claim 1 further
comprising a light chain framework regions FR1, FR2, FR3, wherein
FR1, FR2, and FR3 comprise an amino acid sequence that is at least
85% identical to the light chain framework selected from the group
consisting of: a V.kappa. II subgroup germline sequence; a germline
V segment sequence selected from the group consisting of: A17, A1,
A 18, A2, A19/A3, and A23; a V.kappa. I subgroup germline sequence
including DPK9; a V gene which is compatible with the canonical
structure class 4-1.
25. The antibody or antigen binding fragment of claim 24, wherein
the framework sequence comprises one or more of the following
residues according to Kabat numbering: Val, Leu, or Ile at position
2; Ser or Pro at position 25; Ile or Leu at position 27b; Gly at
position 29; Phe or Leu at position 33; Ile or Val at position 48;
Phe at position 71.
26. The antibody or antigen binding fragment of claim 22, wherein
the framework regions of the heavy chain variable domain sequence
are at least 95% identical, collectively, to the DP54 germ line
sequence.
27. The antibody or antigen binding fragment of claim 24, wherein
the framework regions of the light chain variable domain sequence
are at least 95% identical, collectively, to the sequence of a
V.kappa. I subgroup germline sequence.
28. The antibody or antigen binding fragment of claim 22, wherein
the framework regions of the heavy chain variable domain sequence
are at least 95% identical, collectively, to the sequence of a
V.sub.H I subgroup germline sequence.
29. A pharmaceutical composition comprising the antibody or antigen
binding fragment according to any one of claim 1-18, or 22-28 and a
pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application, filed under 35 U.S.C. .sctn.111, is a
continuation claiming priority under 35 U.S.C. .sctn.120 of
International Application No. PCT/US2006/019706, filed on May 25,
2006, which claims priority to U.S. Application Ser. No.
60/685,149, filed on May 27, 2005. The contents of all the
foregoing applications are hereby incorporated by reference.
BACKGROUND
[0002] The tumor-necrosis factor (TNF)-related cytokines are a
superfamily of proteins that have an array of functions, including
ones implicated in immune regulation and apoptosis regulation.
TWEAK (TNF-like weak inducer of apoptosis) is one member of this
superfamily.
SUMMARY
[0003] Anti-TWEAK antibodies can be used to treat a variety of
conditions and disorders, e.g., an inflammatory disorder, a
neuronal disorder or other disorder described herein. When used to
treat a human subject, the antibody is preferably a human,
humanized or otherwise effectively human antibody.
[0004] In one aspect, the disclosure features a protein that
includes a first and a second immunoglobulin variable domain
sequence and that binds to TWEAK, e.g., human TWEAK. The protein
can bind to TWEAK, e.g., with an affinity corresponding to a
K.sub.D of less than 10.sup.-7 M, e.g., 10.sup.-8, 10.sup.-9,
10.sup.-10, 10.sup.-11 M or better. The protein is also referred to
herein as an "anti-TWEAK antibody." The first and second
immunoglobulin variable domain sequences can include at least a
sufficient portion of an immunoglobulin variable domain to form an
antigen binding site that binds to TWEAK. Typically, the first and
second immunoglobulin variable domain sequences correspond to
immunoglobulin variable domain sequences of a heavy and light
chain, respectively, e.g., a paired or otherwise compatible heavy
and light chain.
[0005] The antibody can bind to an epitope on TWEAK which includes
at least one, two, three or four amino acid residues from an
epitope on TWEAK recognized by P2D10, to a peptide from TWEAK that
is bound by P2D10 (e.g., a peptide less than 25, 20, or 15 amino
acids in length) or to a region of TWEAK recognized by P2D10. For
example, the antibody specifically binds to an epitope, e.g., a
linear or a conformational epitope, of TWEAK, in particular human
TWEAK, e.g., the soluble region of TWEAK. The antibody may compete
with P2D10 for binding to TWEAK, e.g., to human TWEAK. The antibody
may competitively inhibit binding of P2D10 to TWEAK, e.g., human
TWEAK. In one embodiment, the antibody may bind to an epitope which
overlaps with that of P2D10, e.g., includes at least one, two,
three or four amino acids in common with the P2D10 epitope, or an
epitope which, when bound, sterically prevents TWEAK interaction
with P2D10.
[0006] For example, the anti-TWEAK antibody can bind to TWEAK and
modulate, e.g., inhibit, an interaction (e.g., binding) between
TWEAK and a TWEAK receptor, e.g., Fn14 (e.g., human Fn14). The
antibody may also reduce signaling activity of a TWEAK receptor.
The antibody may target TWEAK, sequester TWEAK, and/or modulate the
in vivo stability of TWEAK.
[0007] In one embodiment, the antibody specifically binds to at
least a part of the interaction site on TWEAK that contacts Fn14
(e.g., human Fn14). The antibody may compete with Fn14 for binding
to TWEAK, e.g., to human TWEAK. The antibody may competitively
inhibit binding of Fn14 to TWEAK. The antibody may interact with an
epitope on TWEAK which, when bound, sterically prevents interaction
between TWEAK and Fn14 (e.g., between human TWEAK and human
Fn14).
[0008] In one embodiment, the antibody can inhibit one or more
TWEAK-associated activities with an IC.sub.50 of about 50 nM to 5
pM, typically about 100 to 250 pM or less. For example, the
antibody can inhibit the ability of TWEAK to promote endothelial
cell proliferation or neovascularization. In one embodiment, the
anti-TWEAK antibody reduces at least one TWEAK-associated activity,
e.g., such that the antibody can modulate an inflammatory condition
when administered to a subject.
[0009] In other embodiments, the antibody can associate with TWEAK
with kinetics in the range of 10.sup.3 to 10.sup.8
M.sup.-1s.sup.-1, typically 10.sup.4 to 10.sup.7 M.sup.-1s.sup.-1.
In yet another embodiment, the antibody has dissociation kinetics
in the range of 10.sup.-2 to 10.sup.-6 s.sup.-1, typically
10.sup.-2 to 10.sup.-5s.sup.-1. In one embodiment, the antibody
binds to TWEAK, e.g., human TWEAK, with an affinity and/or kinetics
similar to (e.g., within a factor of five or ten of) monoclonal
antibody P2D10, or modified forms thereof, e.g., chimeric forms or
humanized forms thereof (e.g., a humanized form described herein).
The affinity and binding kinetics of the anti-TWEAK antibody can be
tested, e.g., using biosensor technology (BIACORE.TM.).
[0010] In one embodiment, the antibody is an antigen-binding
fragment of a full length antibody, e.g., a Fab, F(ab')2, Fv or a
single chain Fv fragment. Typically, the antibody is a full length
antibody. The antibody can be a monoclonal antibody or a
mono-specific antibody. For example, the antibody is in a
composition that includes less than 20 other species of anti-TWEAK
antibodies, e.g., in a composition that does not include another
species of anti-TWEAK antibody.
[0011] The antibody can be effectively human. An "effectively
human" antibody is an antibody that includes a sufficient number of
human amino acid positions such that the antibody does not elicit
an immunogenic response in a normal human. Preferably, the protein
does not evoke a neutralizing antibody response, e.g., the human
anti-murine antibody (HAMA) response. HAMA can be problematic in a
number of circumstances, e.g., if the antibodies are desired to be
administered repeatedly, e.g., in treatment of a chronic or
recurrent disease condition. A HAMA response can make repeated
antibody administration potentially ineffective because of an
increased antibody clearance from the serum (see, e.g., Saleh et
al., Cancer Immunol. Immunother., 32:180-190 (1990)) and also
because of potential allergic reactions (see, e.g., LoBuglio et al.
(1986) Hybridoma, 5:5117-5123).
[0012] For example, the antibody can be a human, humanized,
CDR-grafted, chimeric, mutated, affinity matured, deimmunized,
synthetic or otherwise in vitro-generated antibody, and
combinations thereof. In one embodiment, the anti-TWEAK antibody is
a humanized antibody.
[0013] The heavy and light chains of the anti-TWEAK antibody can be
substantially full-length. The protein can include at least one,
and preferably two, complete heavy chains, and at least one, and
preferably two, complete light chains) or can include an
antigen-binding fragment (e.g., a Fab, F(ab')2, Fv or a single
chain Fv fragment). In yet other embodiments, the antibody has a
heavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3,
IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from,
e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g.,
human IgG1). Typically, the heavy chain constant region is human or
a modified form of a human constant region. In another embodiment,
the antibody has a light chain constant region chosen from, e.g.,
kappa or lambda, particularly, kappa (e.g., human kappa).
[0014] In one embodiment, the protein includes at least one, two
and preferably three CDRs from the light or heavy chain variable
region of an antibody disclosed herein, e.g., P2D10. In this
context, CDRs refer to CDRs as defined by Chothia's hypervariable
loops. For example, the protein includes, in the heavy chain
variable domain sequence, at least one, two, or three of the
following sequences within a CDR region:
[0015] GFTFSRYAMS (CDR1) (SEQ ID NO:1),
[0016] EISSGGSYPYYPDTVTG (CDR2) (SEQ ID NO:2),
[0017] VLYYDYDGDRIEVMDY (CDR3) (SEQ ID NO:3), or a CDR having an
amino acid sequence that differs by no more than 4, 3, 2.5, 2, 1.5,
1, or 0.5 alterations (e.g., substitutions, insertions or
deletions) for every 10 amino acids (e.g., the number of
differences being proportional to the CDR length) relative to a
sequence listed above, e.g., at least one alteration but not more
than two, three, or four per CDR. The heavy chain variable domain
sequence may include these CDR sequences particularly in CDR3, or
in at least two CDRs, e.g., CDR1 and CDR3, CDR2 and CDR3, or in all
three CDRs.
[0018] The protein can include, in the heavy chain variable domain
sequence, at least one, two, or three of the following sequences
within a CDR region (amino acids in parentheses represent
alternatives for the particular position):
TABLE-US-00001 (SEQ ID NO: 4), (i)
G-(YF)-(NT)-F-(STDN)-(RY)-Y-A-(MIL)-(HS); (SEQ ID NO: 5) (ii)
Y-Y-(PV)-D-(TS)-V-(TK)-G; and (SEQ ID NO: 6) (iii)
(VL)-(IL)-(YF)-(YF)-D-(YF)-D; or (SEQ ID NO: 7).
(DE)-(RK)-(ILVM)-(EQD)-(VAL)-M-(DE);
[0019] The protein can include, in the light chain variable domain
sequence, at least one, two, or three of the following sequences
within a CDR region:
[0020] RSSQSLVSSKGNTYLH; (CDR1) (SEQ ID NO:8),
[0021] KVSNRFS; (CDR2) (SEQ ID NO:9), and
[0022] SQSTHFPRT; (CDR3) (SEQ ID NO:10), or a CDR having an amino
acid sequence that differs by no more than 4, 3, 2.5, 2, 1.5, 1, or
0.5 alterations (e.g., substitutions, insertions or deletions) for
every 10 amino acids (e.g., the number of differences being
proportional to the CDR length) relative to a sequence listed
above, e.g., at least one alteration but not more than two, three,
or four per CDR. The light chain variable domain sequence may
include these CDR sequences particularly in CDR3, or in at least
two CDRs, e.g., CDR1 and CDR3, CDR2 and CDR3, or in all three
CDRs.
[0023] The protein can include, in the light chain variable domain
sequence, at least one, two, or three of the following sequences
within a CDR region (amino acids in parentheses represent
alternatives for the particular position):
TABLE-US-00002 (SEQ ID NO: 11), (i)
(RK)-S-S-Q-S-(LI)-(KV)-S-S-(KR)-G-N-(TN)- Y-L-(EHDNQY); or (SEQ ID
NO: 12) (RK)-S-S-Q-S-(LI)-V-S-S-(KR)-G-N-(TN)- Y-L-H; (SEQ ID NO:
13), (ii) (KE)-(LVI)-S-(NYS)-(RW)-(FAD)-S; or (SEQ ID NO: 14),
K(LVI)-S-(NYS)-R-(FAD)-S; and (SEQ ID NO: 15) (iii)
(SM)-Q-(GSA)-(ST)-(HEQ)-(FWL)-P; or (SEQ ID NO: 16).
S-Q-(GSA)-(SIT)-(HEQ)-F-P;
[0024] In one preferred embodiment, the protein includes all six
CDR's from P2D10 or closely related CDRs, e.g., CDRs which are
identical or which have at least one amino acid alteration, but not
more than two, three or four alterations (e.g., substitutions,
deletions, or insertions), or other CDR described herein.
[0025] In still another example, the protein includes at least one,
two, or three CDR regions that have the same canonical structures
and the corresponding Chothia CDR regions of P2D10, e.g., the same
canonical structures as at least CDR1 and/or CDR2 of the heavy
and/or light chain variable domains of P2D10.
[0026] The protein can include one of the following sequences:
TABLE-US-00003 (SEQ ID NO: 17)
DIVMTQTPLSLPVTPGEPASISCRSSQSLVSSKGNTYLHWYLQKPGQSP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
18) DIVMTQTPLSLPVTPGEPASISCRSSQSLVSSKGNTYLHWYLQKPGQSP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
19) DVVMTQSPLSLPVTLGQPASISCRSSQSLVSSKGNTYLHWFQQRPGQSP
RRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
20) DVVMTQSPLSLPVTLGQPASISCRSSQSLVSSKGNTYLHWFQQRPGQSP
RRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
21) DIVMTQTPLSLSVTPGQPASISCRSSQSLVSSKGNTYLHWYLQKPGQSP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
22) DIVMTQTPLSLSVTPGQPASISCRSSQSLVSSKGNTYLHWYLQKPGQPP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
23) DIVMTQSPLSLPVTPGEPASISCRSSQSLVSSKGNTYLHWYLQKPGQSP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
24) DIVMTQSPLSLPVTPGEPASISCRSSQSLVSSKGNTYLHWYLQKPGQSP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
25) DIVMTQTPLSSPVTLGQPASISCRSSQSLVSSKGNTYLHWLQQRPGQPP
RLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCSQSTH FPRT (SEQ ID NO:
26) DIQMTQSPSSLSASVGDRVTITCRSSQSLVSSKGNTYLHWYQQKPGKAP
KLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCSQSTH FPRT
or a sequence that has fewer than eight, seven, six, five, four,
three, or two alterations (e.g., substitutions, insertions or
deletions, e.g., conservative substitutions or a substitution for
an amino acid residue at a corresponding position in P2D10,
huP2D10-L1, or huP2D10-L2). Exemplary substitutions are at one of
the following Kabat positions: 2, 4, 6, 35, 36, 38, 44, 47, 49, 62,
64-69, 85, 87, 98, 99, 101, and 102. The substitutions can, for
example, substitute one or more amino acids from P2D10 into
corresponding positions in a framework region, e.g., a human
framework region, e.g., in FR2 (e.g., at position 46 to Phe
according to consecutive numbering) and in FR3 (e.g., at position
87 to Phe).
[0027] The protein can include one of the following sequences in
the heavy chain variable domain:
TABLE-US-00004 (SEQ ID NO: 27)
QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSWVRQAPGQGLEWMG
EISSGGSYPYYPDTVTGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 28) QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSWVRQAPGQRLEWMG
EISSGGSYPYYPDTVTGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 29) QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSWVRQATGQGLEWMG
EISSGGSYPYYPDTVTGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 30) QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSWVRQAPGQGLEWMG
EISSGGSYPYYPDTVTGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 31) QVQLVQSGAEVKKPGASVKVSCKVSGFTFSRYAMSWVRQAPGKGLEWMG
EISSGGSYPYYPDTVTGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT VLYYDYDGDRIE (SEQ
ID NO: 32) QMQLVQSGAEVKKTGSSVKVSCKASGFTFSRYAMSWVRQAPGQALEWMG
EISSGGSYPYYPDTVTGRVTITRDRSMSTAYMELSSLRSEDTAMYYCAR VLYYDYDGDRIE (SEQ
ID NO: 33) QVQLVQSGAEVKKPGASVKVSCKASGFTFSRYAMSWVRQAPGQGLEWMG
EISSGGSYPYYPDTVTGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 34) QMQLVQSGPEVKKPGTSVKVSCKASGFTFSRYAMSWVRQARGQRLEWIG
EISSGGSYPYYPDTVTGRVTITRDMSTSTAYMELSSLRSEDTAVYYCAA VLYYDYDGDRIE (SEQ
ID NO: 35) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVA
EISSGGSYPYYPDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 36) EVQLVESGGGLVQPGRSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVS
EISSGGSYPYYPDTVTGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK DVLYYDYDGDRIE
(SEQ ID NO: 37) QVQLVESGGGLVKPGGSLRLSCAASGFTFSRYAMSWIRQAPGKGLEWVS
EISSGGSYPYYPDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 38) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVG
EISSGGSYPYYPDTVTGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT VLYYDYDGDRIE (SEQ
ID NO: 39) EVQLVESGGGVVRPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVS
EISSGGSYPYYPDTVTGRFTISRDNAKNSLYLQMNSLRAEDTALYHCAR VLYYDYDGDRIE (SEQ
ID NO: 40) EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVS
EISSGGSYPYYPDTVTGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 41) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVS
EISSGGSYPYYPDTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK VLYYDYDGDRIE (SEQ
ID NO: 42) QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVA
EISSGGSYPYYPDTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK VLYYDYDGDRIE (SEQ
ID NO: 43) QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVA
EISSGGSYPYYPDTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 44) QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVA
EISSGGSYPYYPDTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK VLYYDYDGDRIE (SEQ
ID NO: 45) QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVA
EISSGGSYPYYPDTVTGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 46) EVQLVESGGVVVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVS
EISSGGSYPYYPDTVTGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAK DVLYYDYDGDRIE
(SEQ ID NO: 47) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVS
EISSGGSYPYYPDTVTGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR VLYYDYDGDRIE (SEQ
ID NO: 48) EVQLVESGGGLVQPGRSLRLSCTASGFTFSRYAMSWFRQAPGKGLEWVG
EISSGGSYPYYPDTVTGRFTISRDGSKSIAYLQMNSLKTEDTAVYYCTR VLYYDYDGDRIE (SEQ
ID NO:49) EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEYVS
EISSGGSYPYYPDTVTGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCAR VLYYDYDGDRIE
[0028] or a sequence that has fewer than eight, seven, six, five,
four, three, or two alterations (e.g., substitutions, insertions or
deletions, e.g., conservative substitutions or a substitution for
an amino acid residue at a corresponding position in P2D10).
Exemplary substitutions are at one of the following Kabat
positions: 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106,
and 107. The substitutions can, for example, substitute one or more
amino acids from P2D10 into corresponding positions in a framework
region, e.g., a human framework region.
[0029] In one embodiment, the heavy chain framework (e.g., FR1,
FR2, FR3, individually, or a sequence encompassing FR1, FR2, and
FR3, but excluding CDRs) includes an amino acid sequence, which is
at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identical to the
heavy chain framework of one of the following germline V segment
sequences: DP-25, DP-1, DP-12, DP-9, DP-7, DP-31, DP-32, DP-33,
DP-58, DP-54, other VH I subgroup germline sequence, other VH III
subgroup germline sequence, or another V gene which is compatible
with the canonical structure class 1-3 (see, e.g., Chothia et al.
(1992) J. Mol. Biol. 227:799-817; Tomlinson et al. (1992) J. Mol.
Biol. 227:776-798). Other frameworks compatible with the canonical
structure class 1-3 include frameworks with the one or more of the
following residues according to Kabat numbering: Ala, Gly, Thr, or
Val at position 26; Gly at position 26; Tyr, Phe, or Gly at
position 27; Phe, Val, Ile, or Leu at position 29; Met, Ile, Leu,
Val, Thr, Trp, or Ile at position 34; Arg, Thr, Ala, Lys at
position 94; Gly, Ser, Asn, or Asp at position 54; and Arg at
position 71.
[0030] In one embodiment, the light chain framework (e.g., FR1,
FR2, FR3, individually, or a sequence encompassing FR1, FR2, and
FR3, but excluding CDRs) includes an amino acid sequence, which is
at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identical to the
light chain framework of a V.kappa. II subgroup germline sequence
or one of the following germline V segment sequences: A17, A1, A18,
A2, A19/A3, A23, a V.kappa. I subgroup germline sequence (e.g., a
DPK9 sequence), or another V gene which is compatible with the
canonical structure class 4-1 (see, e.g., Tomlinson et al. (1995)
EMBO J. 14:4628). Other frameworks compatible with the canonical
structure class 4-1 include frameworks with the one or more of the
following residues according to Kabat numbering: Val or Leu or Ile
at position 2; Ser or Pro at position 25; Ile or Leu at position
27b; Gly at position 29; Phe or Leu at position 33; and Phe at
position 71. Further, according to the Kabat numbering, position 48
can be Ile or Val.
[0031] In another embodiment, the light chain framework (e.g., FR1,
FR2, FR3, individually, or a sequence encompassing FR1, FR2, and
FR3, but excluding CDRs) includes an amino acid sequence, which is
at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identical to the
light chain framework of a V.kappa. I subgroup germline sequence,
e.g., a DPK9 sequence.
[0032] In one embodiment, the light or the heavy chain variable
framework (e.g., the region encompassing at least FR1, FR2, FR3,
and optionally FR4) can be chosen from: (a) a light or heavy chain
variable framework including at least 80%, 90%, 95%, or preferably
100% of the amino acid residues from a human light or heavy chain
variable framework, e.g., a light or heavy chain variable framework
residue from a human mature antibody, a human germline sequence, a
human consensus sequence, or a human antibody described herein; (b)
a light or heavy chain variable framework including from 20% to
80%, 40% to 60%, 60% to 90%, or 70% to 95% of the amino acid
residues from a human light or heavy chain variable framework,
e.g., a light or heavy chain variable framework residue from a
human mature antibody, a human germline sequence, a human consensus
sequence; (c) a non-human framework (e.g., a rodent framework); or
(d) a non-human framework that has been modified, e.g., to remove
antigenic or cytotoxic determinants, e.g., deimmunized, or
partially humanized. In one embodiment, the heavy chain variable
domain sequence includes human residues or human consensus sequence
residues at one or more of the following positions (preferably at
least five, ten, twelve, or all): (in the FR of the variable domain
of the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L,
63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L,
and/or (in the FR of the variable domain of the heavy chain) 2H,
4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H,
70H, 73H, 74H, 75H, 78H, 91H, 92H, 93H, and/or 103H (according to
the Kabat numbering).
[0033] In one embodiment, the protein includes at least one
non-human CDR, e.g., a murine CDR, e.g., a CDR from P2D10, or a
mutant thereof, and at least one framework which differs from a
framework of P2D10 by at least one amino acid, e.g., at least 5, 8,
10, 12, 15, or 18 amino acids. For example, the proteins include
one, two, three, four, five, or six such non-human CDR's and
includes at least one amino acid difference in at least three of HC
FR1, HC FR2, HC FR3, LC FR1, LC FR2, and LC FR3.
[0034] In one embodiment, the heavy or light chain variable domain
sequence of the protein includes an amino acid sequence, which is
at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identical to a
variable domain sequence of an antibody described herein, e.g.,
P2D10, huP2D10-1, or huP2D10-2; or which differs at least 1 or 5
residues, but less than 40, 30, 20, or 10 residues, from a variable
domain sequence of an antibody described herein, e.g., P2D10,
huP2D10-1, or huP2D10-2.
[0035] In one embodiment, one or both of the variable domains
include amino acid positions in the framework region that are
variously derived from both a murine antibody (e.g., P2D10) and a
humanized antibody (e.g., 56-84m and K107) or germline sequence.
For example, the variable domain will include a number of positions
at which the amino acid is identical to both the murine antibody
and the human antibody (or germline sequence) because the two are
identical at that position. Of the remaining framework positions
where the murine and human differ, at least 50, 60, 70, 80, or 90%
of the positions of the variable domain are preferably identical to
the human antibody (or germline sequence) rather than the murine.
None, or at least one, two, three, or four of such remaining
framework positions may be identical to the murine antibody rather
than to the human antibody. For example, in HC FR1, one or two such
positions can be murine; in HC FR2, one or two such positions can
be murine; in FR3, one, two, three, or four such positions can be
murine; in LC FR1, one, two, three, or four such positions can be
murine; in LC FR2, one or two such positions can be murine; in LC
FR3, one or two such positions can be murine.
[0036] In one embodiment, the heavy or light chain variable domain
sequence of the protein includes an amino acid sequence encoded by
a nucleic acid sequence described herein or a nucleic acid that
hybridizes to a nucleic acid sequence described herein (e.g., a
specific nucleic acid sequence or a nucleic acid sequence that
encodes an amino acid sequence described herein) or its complement,
e.g., under low stringency, medium stringency, high stringency, or
very high stringency conditions.
[0037] The anti-TWEAK antibody can be derivatized or linked to
another functional molecule, e.g., another peptide, protein, or
compound. For example, the antibody can be functionally linked
(e.g., by chemical coupling, genetic fusion, non-covalent
association or otherwise) to one or more other molecular entities,
such as another antibody (e.g., a bispecific or a multi-specific
antibody), toxins, radioisotopes, polymers, cytotoxic or cytostatic
agents, among others.
[0038] In another aspect, the disclosure provides compositions,
e.g., pharmaceutical compositions, that include a pharmaceutically
acceptable carrier and an anti-TWEAK antibody, e.g., an anti-Tweak
antibody described herein.
[0039] In yet another embodiment, the anti-TWEAK antibody (e.g., a
pharmaceutical composition thereof) is administered to a subject
who needs an anti-TWEAK antibody therapy or whose condition would
be ameliorated by the antibody. For example, the anti-TWEAK
antibody can be administered to a subject who has or is at risk for
an inflammatory disorder, immune disorder, autoimmune disorder,
neuronal disorder, a neoplastic disorder, or other disorder
described herein. In one embodiment, an anti-TWEAK antibody
described herein is used for the preparation of a medicament for
the treatment of an inflammatory disorder, immune disorder,
autoimmune disorder, neuronal disorder, a neoplastic disorder, or
other disorder described herein.
[0040] In another aspect, the disclosure features a method of
treating a TWEAK-associated disorder, in a subject. The method
includes: administering to the subject an anti-TWEAK antibody, in
an amount sufficient to treat (e.g., improve or prevent) the
TWEAK-associated disorder. The anti-TWEAK antibody can be
administered to the subject, alone or in combination with other
therapeutic modalities as described herein. In one embodiment, the
subject is a mammal, e.g., a human, e.g., a human having a
TWEAK-associated disorder, e.g., a disorder disclosed herein. The
antibody can be used to ameliorate one or more symptoms of such
disorders. The term "treating" refers to administering a therapy in
an amount, manner, and/or mode effective to improve or prevent a
condition, symptom, or parameter associated with a disorder (e.g.,
a disorder described herein) or to prevent onset, progression, or
exacerbation of the disorder, to either a statistically significant
degree or to a degree detectable to one skilled in the art.
Accordingly, treating can achieve therapeutic and/or prophylactic
benefits. An effective amount, manner, or mode can vary depending
on the subject and may be tailored to the subject. In one
embodiment, an anti-TWEAK antibody described herein is used for the
preparation of a medicament for the treatment of a TWEAK-associated
disorder.
[0041] In another aspect, the disclosure features a method of
modulating interaction between TWEAK and a TWEAK receptor protein.
For example, an anti-TWEAK antibody can be used to reduce or
inhibit binding, between TWEAK and a TWEAK receptor, such as Fn14.
The method comprises contacting TWEAK or a complex that contains
TWEAK with the antibody. The method can be used on cells in vitro
e.g., in culture, e.g. in vitro or ex vivo. For example, TWEAK
receptor-expressing cells can be cultured in vitro in culture
medium and the contacting step can be effected by adding an
anti-TWEAK antibody to the culture medium. Alternatively, the
method can be performed on cells present in a subject, e.g., as
part of an in vivo (e.g., therapeutic or prophylactic) protocol.
For example, the anti-TWEAK antibody can be delivered locally or
systemically. In one embodiment, an anti-TWEAK antibody described
herein is used for the preparation of a medicament for of
modulating interaction between TWEAK and a TWEAK receptor
protein.
[0042] The method can include contacting TWEAK with the TWEAK
receptor complex, or subunit thereof, under conditions that allow
an interaction between TWEAK and the TWEAK receptor complex, or
subunit thereof, to occur to thereby form a TWEAK/TWEAK receptor
mixture. Generally, the anti-TWEAK antibody is provided in an
effective amount, e.g., so that contacting the TWEAK/TWEAK receptor
mixture with the anti-TWEAK antibody modulates, e.g., interferes
with (e.g., inhibits, blocks or otherwise reduces) the interaction
between TWEAK and the receptor protein or at least one function of
TWEAK, e.g., TWEAK mediated signaling.
[0043] The disclosure also features nucleic acids comprising
nucleotide sequences, which encode heavy and light chain variable
regions of the anti-TWEAK antibodies, e.g., as described herein.
For example, the disclosure features a first and second nucleic
acid encoding heavy and light chain variable regions, respectively,
of P2D10. In another aspect, the disclosure features host cells and
vectors containing the nucleic acids described herein.
[0044] The disclosure also features the epitope of TWEAK, e.g.,
human TWEAK, recognized by P2D10 and proteins able to interact with
the epitope. For example, proteins and peptides that include the
epitope can be used to generate or screen for other binding
compounds that interact with the epitope, e.g., proteins such as
antibodies or small molecules. For example, a peptide that includes
the epitope can be used as an immunogen or as a target for
screening an expression library. It is also possible to evaluate
compounds for their ability to interact with the peptide, or, by
mapping or structure determination, to evaluate compounds for their
ability to interact with the epitope, e.g., in the context of a
mature TWEAK. An exemplary evaluation includes determining if the
compound can interact with TWEAK in the presence of a competing
P2D10 antibody.
[0045] Methods for delivering or targeting an agent, e.g., a
therapeutic (including a genetic agent) or a cytotoxic agent, with
an anti-TWEAK antibody (e.g., P2D10 or other antibody described
herein) to a TWEAK-expressing cell or structure in vivo are also
disclosed.
[0046] As used herein, the term "antibody" refers to a protein that
includes at least one immunoglobulin variable region, e.g., an
amino acid sequence that provides an immunoglobulin variable domain
or an immunoglobulin variable domain sequence. For example, an
antibody can include a heavy (H) chain variable region (abbreviated
herein as VH), and a light (L) chain variable region (abbreviated
herein as VL). In another example, an antibody includes two heavy
(H) chain variable regions and two light (L) chain variable
regions. The term "antibody" encompasses antigen-binding fragments
of antibodies (e.g., single chain antibodies, Fab fragments,
F(ab').sub.2 fragments, Fd fragments, Fv fragments, and dAb
fragments) as well as full length antibodies, e.g., full length,
immunoglobulins of types IgA, IgG (e.g., IgG1, IgG2, IgG3, IgG4),
IgE, IgD, IgM (as well as subtypes thereof). The term "full length
antibody" refers to an antibody having at least 96% of the length
of a natural antibody that is processed to remove any signal
sequences. A full length antibody can include the complete length
of the natural antibody, e.g., residues from the amino-terminal
residue of a natural antibody (e.g., a IgG1, IgG2, IgG3, IgG4) to
its carboxy-terminal residue.
[0047] An "immunoglobulin variable domain sequence" refers to an
amino acid sequence which can form the structure of an
immunoglobulin variable domain. For example, the sequence may
include all or part of the amino acid sequence of a
naturally-occurring variable domain. For example, the sequence may
or may not include one, two or more N- or C-terminal amino acids,
or may include other alterations that are compatible with formation
of the protein structure.
[0048] An "isolated composition" refers to a composition that is
removed from at least 90% of at least one component of a natural
sample from which the isolated composition can be obtained.
Compositions produced artificially or naturally can be
"compositions of at least" a certain degree of purity if the
species or population of species of interest is at least 5, 10, 25,
50, 75, 80, 90, 95, 98, or 99% pure on a weight-weight basis.
[0049] An "epitope" refers to the site on a target compound that is
bound by an antibody. In the case where the target compound is a
protein, for example, an epitope may refer to the amino acids
(particularly amino acid side chains) that are bound by the
antibody. Overlapping epitopes include at least one common amino
acid residue, e.g., at at least 2, 3, 4 or 5 common amino acid
residues.
[0050] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described
in that reference and either can be used. Specific hybridization
conditions referred to herein are as follows: 1) low stringency
hybridization conditions in 6.times. sodium chloride/sodium citrate
(SSC) at about 45.degree. C., followed by two washes in
0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature
of the washes can be increased to 55.degree. C. for low stringency
conditions); 2) medium stringency hybridization conditions in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; 3) high stringency
hybridization conditions in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C.; and preferably 4) very high stringency hybridization
conditions are 0.5M sodium phosphate, 7% SDS at 65.degree. C.,
followed by one or more washes at 0.2.times.SSC, 1% SDS at
65.degree. C. High stringency conditions (3) are the preferred
conditions and the ones that should be used unless otherwise
specified.
[0051] A "TWEAK-associated disorder" is any disorder in which TWEAK
contributes to etiology or a disorder whose condition, symptoms, or
risk of onset is altered by provision of a TWEAK blocking
agent.
[0052] 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 described herein,
suitable methods and materials are described below. In addition,
embodiments of the invention described with respect to Chothia CDRs
may also be implemented using Kabat CDRs.
[0053] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions, controls. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
DETAILED DESCRIPTION
[0054] P2D10 is an exemplary murine antibody that specifically
binds to human TWEAK and inhibits TWEAK function. Variants of the
P2D10 antibody are also disclosed, including exemplary humanized
variants. These antibodies, other anti-TWEAK antibodies, and other
TWEAK blocking agents can be used to treat or prevent
TWEAK-mediated disorders, e.g., inflammatory disorders and other
disorders disclosed herein.
[0055] Anti-TWEAK Antibodies
[0056] This disclosure includes the sequences of specific examples
of anti-TWEAK antibodies, such as P2D10, huP2D10-1, and huP2D10-2.
Particular antibodies, such as these, can be made, for example, by
preparing and expressing synthetic genes that encode the recited
amino acid sequences or by mutating human germline genes to provide
a gene that encodes the recited amino acid sequences. Moreover,
these antibodies and other anti-TWEAK antibodies can be produced,
e.g., using one or more of the following methods.
[0057] Numerous methods are available for obtaining antibodies,
particularly human antibodies. One exemplary method includes
screening protein expression libraries, e.g., phage or ribosome
display libraries. Phage display is described, for example, U.S.
Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; WO
92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO
92/01047; WO 92/09690; and WO 90/02809. The display of Fab's on
phage is described, e.g., in U.S. Pat. Nos. 5,658,727; 5,667,988;
and 5,885,793.
[0058] In addition to the use of display libraries, other methods
can be used to obtain a TWEAK-binding antibody. For example, the
TWEAK protein or a peptide thereof can be used as an antigen in a
non-human animal, e.g., a rodent, e.g., a mouse, hamster, or
rat.
[0059] In one embodiment, the non-human animal includes at least a
part of a human immunoglobulin gene. For example, it is possible to
engineer mouse strains deficient in mouse antibody production with
large fragments of the human Ig loci. Using the hybridoma
technology, antigen-specific monoclonal antibodies derived from the
genes with the desired specificity may be produced and selected.
See, e.g., XENOMOUSE.TM., Green et al. (1994) Nature Genetics
7:13-21, U.S. 2003-0070185, WO 96/34096, and WO 96/33735.
[0060] In another embodiment, a monoclonal antibody is obtained
from the non-human animal, and then modified, e.g., humanized or
deimmunized. Winter describes an exemplary CDR-grafting method that
may be used to prepare humanized antibodies described herein (U.S.
Pat. No. 5,225,539). All or some of the CDRs of a particular human
antibody may be replaced with at least a portion of a non-human
antibody. It may only be necessary to replace the CDRs required for
binding or binding determinants of such CDRs to arrive at a useful
humanized antibody that binds to TWEAK.
[0061] Humanized antibodies can be generated by replacing sequences
of the Fv variable region that are not directly involved in antigen
binding with equivalent sequences from human Fv variable regions.
General methods for generating humanized antibodies are provided by
Morrison, S. L. (1985) Science 229:1202-1207, by Oi et al. (1986)
BioTechniques 4:214, and by U.S. Pat. No. 5,585,089; U.S. Pat. No.
5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 5,859,205; and
U.S. Pat. No. 6,407,213. Those methods include isolating,
manipulating, and expressing the nucleic acid sequences that encode
all or part of immunoglobulin Fv variable regions from at least one
of a heavy or light chain. Sources of such nucleic acid are well
known to those skilled in the art and, for example, may be obtained
from a hybridoma producing an antibody against a predetermined
target, as described above, from germline immunoglobulin genes, or
from synthetic constructs. The recombinant DNA encoding the
humanized antibody can then be cloned into an appropriate
expression vector.
[0062] Human germline sequences, for example, are disclosed in
Tomlinson, I. A. et al. (1992) J. Mol. Biol. 227:776-798; Cook, G.
P. et al. (1995) Immunol. Today 16: 237-242; Chothia, D. et al.
(1992) J. Mol. Bio. 227:799-817; and Tomlinson et al. (1995) EMBO J
14:4628-4638. The V BASE directory provides a comprehensive
directory of human immunoglobulin variable region sequences
(compiled by Tomlinson, I. A. et al. MRC Centre for Protein
Engineering, Cambridge, UK). These sequences can be used as a
source of human sequence, e.g., for framework regions and CDRs.
Consensus human framework regions can also be used, e.g., as
described in U.S. Pat. No. 6,300,064.
[0063] A non-human TWEAK-binding antibody may also be modified by
specific deletion of human T cell epitopes or "deimmunization" by
the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the
heavy and light chain variable regions of an antibody can be
analyzed for peptides that bind to MHC Class II; these peptides
represent potential T-cell epitopes (as defined in WO 98/52976 and
WO 00/34317). For detection of potential T-cell epitopes, a
computer modeling approach termed "peptide threading" can be
applied, and in addition a database of human MHC class II binding
peptides can be searched for motifs present in the V.sub.H and
V.sub.L sequences, as described in WO 98/52976 and WO 00/34317.
These motifs bind to any of the 18 major MHC class II DR allotypes,
and thus constitute potential T cell epitopes. Potential T-cell
epitopes detected can be eliminated by substituting small numbers
of amino acid residues in the variable regions, or preferably, by
single amino acid substitutions. As far as possible, conservative
substitutions are made. Often, but not exclusively, an amino acid
common to a position in human germline antibody sequences may be
used. After the deimmunizing changes are identified, nucleic acids
encoding V.sub.H and V.sub.L can be constructed by mutagenesis or
other synthetic methods (e.g., de novo synthesis, cassette
replacement, and so forth). A mutagenized variable sequence can,
optionally, be fused to a human constant region, e.g., human IgG1
or kappa constant regions.
[0064] In some cases, a potential T cell epitope will include
residues which are known or predicted to be important for antibody
function. For example, potential T cell epitopes are usually biased
towards the CDRs. In addition, potential T cell epitopes can occur
in framework residues important for antibody structure and binding.
Changes to eliminate these potential epitopes will in some cases
require more scrutiny, e.g., by making and testing chains with and
without the change. Where possible, potential T cell epitopes that
overlap the CDRs can be eliminated by substitutions outside the
CDRs. In some cases, an alteration within a CDR is the only option,
and thus variants with and without this substitution can be tested.
In other cases, the substitution required to remove a potential T
cell epitope is at a residue position within the framework that
might be critical for antibody binding. In these cases, variants
with and without this substitution are tested. Thus, in some cases
several variant deimmunized heavy and light chain variable regions
are designed and various heavy/light chain combinations are tested
to identify the optimal deimmunized antibody. The choice of the
final deimmunized antibody can then be made by considering the
binding affinity of the different variants in conjunction with the
extent of deimmunization, particularly, the number of potential T
cell epitopes remaining in the variable region. Deimmunization can
be used to modify any antibody, e.g., an antibody that includes a
non-human sequence, e.g., a synthetic antibody, a murine antibody
other non-human monoclonal antibody, or an antibody isolated from a
display library.
[0065] Other methods for humanizing antibodies can also be used.
For example, other methods can account for the three dimensional
structure of the antibody, framework positions that are in three
dimensional proximity to binding determinants, and immunogenic
peptide sequences. See, e.g., WO 90/07861; U.S. Pat. Nos.
5,693,762; 5,693,761; 5,585,089; 5,530,101; and 6,407,213; Tempest
et al. (1991) Biotechnology 9:266-271. Still another method is
termed "humaneering" and is described, for example, in U.S.
2005-008625.
[0066] The antibody can include a human Fc region, e.g., a
wild-type Fc region or an Fc region that includes one or more
alterations. In one embodiment, the constant region is altered,
e.g., mutated, to modify the properties of the antibody (e.g., to
increase or decrease one or more of: Fc receptor binding, antibody
glycosylation, the number of cysteine residues, effector cell
function, or complement function). For example, the human IgG1
constant region can be mutated at one or more residues, e.g., one
or more of residues 234 and 237. Antibodies may have mutations in
the CH2 region of the heavy chain that reduce or alter effector
function, e.g., Fc receptor binding and complement activation. For
example, antibodies may have mutations such as those described in
U.S. Pat. Nos. 5,624,821 and 5,648,260. Antibodies may also have
mutations that stabilize the disulfide bond between the two heavy
chains of an immunoglobulin, such as mutations in the hinge region
of IgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol.
Immunol. 30:105-08). See also, e.g., U.S. 2005-0037000.
[0067] Affinity Maturation. In one embodiment, an anti-TWEAK
antibody is modified, e.g., by mutagenesis, to provide a pool of
modified antibodies. The modified antibodies are then evaluated to
identify one or more antibodies which have altered functional
properties (e.g., improved binding, improved stability, reduced
antigenicity, or increased stability in vivo). In one
implementation, display library technology is used to select or
screen the pool of modified antibodies. Higher affinity antibodies
are then identified from the second library, e.g., by using higher
stringency or more competitive binding and washing conditions.
Other screening techniques can also be used.
[0068] In some implementations, the mutagenesis is targeted to
regions known or likely to be at the binding interface. If, for
example, the identified binding proteins are antibodies, then
mutagenesis can be directed to the CDR regions of the heavy or
light to chains as described herein. Further, mutagenesis can be
directed to framework regions near or adjacent to the CDRs, e.g.,
framework regions, particularly within 10, 5, or 3 amino acids of a
CDR junction. In the case of antibodies, mutagenesis can also be
limited to one or a few of the CDRs, e.g., to make step-wise
improvements.
[0069] In one embodiment, mutagenesis is used to make an antibody
more similar to one or more germline sequences. One exemplary
germlining method can include: identifying one or more germline
sequences that are similar (e.g., most similar in a particular
database) to the sequence of the isolated antibody. Then mutations
(at the amino acid level) can be made in the isolated antibody,
either incrementally, in combination, or both. For example, a
nucleic acid library that includes sequences encoding some or all
possible germline mutations is made. The mutated antibodies are
then evaluated, e.g., to identify an antibody that has one or more
additional germline residues relative to the isolated antibody and
that is still useful (e.g., has a functional activity). In one
embodiment, as many germline residues are introduced into an
isolated antibody as possible.
[0070] In one embodiment, mutagenesis is used to substitute or
insert one or more germline residues into a CDR region. For
example, the germline CDR residue can be from a germline sequence
that is similar (e.g., most similar) to the variable region being
modified. After mutagenesis, activity (e.g., binding or other
functional activity) of the antibody can be evaluated to determine
if the germline residue or residues are tolerated. Similar
mutagenesis can be performed in the framework regions.
[0071] Selecting a germline sequence can be performed in different
ways. For example, a germline sequence can be selected if it meets
a predetermined criteria for selectivity or similarity, e.g., at
least a certain percentage identity, e.g., at least 75, 80, 85, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5% identity, relative to
the donor non-human antibody. The selection can be performed using
at least 2, 3, 5, or 10 germline sequences. In the case of CDR1 and
CDR2, identifying a similar germline sequence can include selecting
one such sequence. In the case of CDR3, identifying a similar
germline sequence can include selecting one such sequence, but may
include using two germline sequences that separately contribute to
the amino-terminal portion and the carboxy-terminal portion. In
other implementations, more than one or two germline sequences are
used, e.g., to form a consensus sequence.
[0072] In other embodiments, the antibody may be modified to have
an altered glycosylation pattern (i.e., altered from the original
or native glycosylation pattern). As used in this context,
"altered" means having one or more carbohydrate moieties deleted,
and/or having one or more glycosylation sites added to the original
antibody. Addition of glycosylation sites to the presently
disclosed antibodies may be accomplished by altering the amino acid
sequence to contain glycosylation site consensus sequences; such
techniques are well known in the art. Another means of increasing
the number of carbohydrate moieties on the antibodies is by
chemical or enzymatic coupling of glycosides to the amino acid
residues of the antibody. These methods are described in, e.g., WO
87/05330, and Aplin and Wriston (1981) CRC Crit. Rev. Biochem.
22:259-306. Removal of any carbohydrate moieties present on the
antibodies may be accomplished chemically or enzymatically as
described in the art (Hakimuddin et al. (1987) Arch. Biochem.
Biophys. 259:52; Edge et al. (1981) Anal. Biochem. 118:131; and
Thotakura et al. (1987) Meth. Enzymol. 138:350). See, e.g., U.S.
Pat. No. 5,869,046 for a modification that increases in vivo half
life by providing a salvage receptor binding epitope.
[0073] In one embodiment, an antibody has CDR sequences that differ
only insubstantially from those of P2D10. Insubstantial differences
include minor amino acid changes, such as substitutions of 1 or 2
out of any of typically 5-7 amino acids in the sequence of a CDR,
e.g., a Chothia or Kabat CDR. Typically an amino acid is
substituted by a related amino acid having similar charge,
hydrophobic, or stereochemical characteristics. Such substitutions
would be within the ordinary skills of an artisan. Unlike in CDRs,
more substantial changes in structure framework regions (FRs) can
be made without adversely affecting the binding properties of an
antibody. Changes to FRs include, but are not limited to,
humanizing a nonhuman-derived framework or engineering certain
framework residues that are important for antigen contact or for
stabilizing the binding site, e.g., changing the class or subclass
of the constant region, changing specific amino acid residues which
might alter an effector function such as Fc receptor binding (Lund
et al. (1991) J. Immun. 147:2657-62; Morgan et al. (1995)
Immunology 86:319-24), or changing the species from which the
constant region is derived.
[0074] The anti-TWEAK antibodies can be in the form of full length
antibodies, or in the form of fragments of antibodies, e.g., Fab,
F(ab').sub.2, Fd, dAb, and scFv fragments. Additional forms include
a protein that includes a single variable domain, e.g., a camel or
camelized domain. See, e.g., U.S. 2005-0079574 and Davies et al.
(1996) Protein Eng. 9(6):531-7.
[0075] Antibody Production. Some antibodies, e.g., Fab's, can be
produced in bacterial cells, e.g., E. coli cells. Antibodies can
also be produced in eukaryotic cells. In one embodiment, the
antibodies (e.g., scFv's) are expressed in a yeast cell such as
Pichia (see, e.g., Powers et al. (2001) J Immunol Methods.
251:123-35), Hanseula, or Saccharomyces.
[0076] In one preferred embodiment, antibodies are produced in
mammalian cells. Exemplary mammalian host cells for expressing an
antibody include Chinese Hamster Ovary (CHO cells) (including
dhfr.sup.- CHO cells, described in Urlaub and Chasin (1980) Proc.
Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable
marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol.
159:601-621), lymphocytic cell lines, e.g., NSO myeloma cells and
SP2 cells, COS cells, and a cell from a transgenic animal, e.g., a
transgenic mammal. For example, the cell is a mammary epithelial
cell.
[0077] In addition to the nucleic acid sequence encoding the
diversified immunoglobulin domain, the recombinant expression
vectors may carry additional sequences, such as sequences that
regulate replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017). For example, typically the selectable marker gene
confers resistance to drugs, such as G418, hygromycin, or
methotrexate, on a host cell into which the vector has been
introduced.
[0078] In an exemplary system for antibody expression, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr.sup.-
CHO cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to enhancer/promoter regulatory
elements (e.g., derived from SV40, CMV, adenovirus and the like,
such as a CMV enhancer/AdMLP promoter regulatory element or an SV40
enhancer/AdMLP promoter regulatory element) to drive high levels of
transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that
have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the antibody heavy and light
chains and the antibody is recovered from the culture medium.
Standard molecular biology techniques are used to prepare the
recombinant expression vector, transfect the host cells, select for
transformants, culture the host cells and recover the antibody from
the culture medium. For example, some antibodies can be isolated by
affinity chromatography with a Protein A or Protein G coupled
matrix.
[0079] For antibodies that include an Fc domain, the antibody
production system preferably synthesizes antibodies in which the Fc
region is glycosylated. For example, the Fc domain of IgG molecules
is glycosylated at asparagine 297 in the CH2 domain. This
asparagine is the site for modification with biantennary-type
oligosaccharides. It has been demonstrated that this glycosylation
is required for effector functions mediated by Fc.gamma. receptors
and complement Clq (Burton and Woof (1992) Adv. Immunol. 51:1-84;
Jefferis et al. (1998) Immunol. Rev. 163:59-76). In one embodiment,
the Fc domain is produced in a mammalian expression system that
appropriately glycosylates the residue corresponding to asparagine
297. The Fc domain or other region of the antibody can also include
other eukaryotic post-translational modifications.
[0080] Antibodies can also be produced by a transgenic animal. For
example, U.S. Pat. No. 5,849,992 describes a method of expressing
an antibody in the mammary gland of a transgenic mammal. A
transgene is constructed that includes a milk-specific promoter and
nucleic acids encoding the antibody of interest and a signal
sequence for secretion. The milk produced by females of such
transgenic mammals includes, secreted-therein, the antibody of
interest. The antibody can be purified from the milk, or for some
applications, used directly.
[0081] Characterization
[0082] The binding properties of an antibody may be measured by any
standard method, e.g., one of the following methods: BIACORE.TM.
analysis, Enzyme Linked Immunosorbent Assay (ELISA), Fluorescence
Resonance Energy Transfer (FRET), x-ray crystallography, sequence
analysis and scanning mutagenesis. The ability of a protein to
inhibit one or more activities of TWEAK can be evaluated in vitro
or in an animal model of a disorder, e.g., a disorder described
herein. Preferably, the antibody has a statistically significant
effect that indicates that the antibody inhibits one or more
activities of TWEAK.
[0083] In one embodiment, an antibody is evaluated for inhibition
of the ability of TWEAK to stimulate production of IL-8, MMP-1,
PGE2, IL-6, IP-10 and RANTES in dermal fibroblasts. See
Chicheportiche et al. (2002) Arthritis Res. 4(2):126-133 for
suitable assay conditions.
[0084] In another embodiment, an antibody is evaluated for its
ability to inhibit TWEAK from stimulating the proliferation of an
endothelial cell. See, e.g., U.S. 2003-0211993 which describes a
proliferation assay (as well as other useful assays) as follows:
HVEC are plated in 96-well microtiter plates at subconfluence (4000
cells per well) and cultured overnight in CS-C Medium without
addition of supplier growth supplements. Media is replaced with
complete Media, or with basal media. Cells are cultured in basal
media with or without TWEAK (100 ng/ml), bFGF using a 1/500 to
1/1000 dilution of bFGF growth supplement (Clonetics) or 1 ng/ml
(R&D Systems), VEGF (10 ng/ml) or combinations of these
factors. Where indicated, 10 .mu.g/ml of the antibody being tested
or a control antibody is also added. Cells are incubated at
37.degree. C. with 5% CO.sub.2 for three days and proliferation was
measured by pulsing with .sup.3H-Thymidine for the last 10 hours of
culture. Cell-bound radioactivity can be measured with a
BETAPLATE.TM. (EG&G Wallac, Gaithersburg, Md.). A decrease in
proliferation mediated by TWEAK or the combination of TWEAK and
bFGF can indicate that the antibody is effective at blocking TWEAK
activity.
[0085] Surface Plasmon Resonance (SPR). The binding interaction of
a protein of interest and a target (e.g., TWEAK) can be analyzed
using SPR. SPR or Biomolecular Interaction Analysis (BIA) detects
biospecific interactions in real time, without labeling any of the
interactants. Changes in the mass at the binding surface
(indicative of a binding event) of the BIA chip result in
alterations of the refractive index of light near the surface (the
optical phenomenon of surface plasmon resonance (SPR)). The changes
in the refractivity generate a detectable signal, which are
measured as an indication of real-time reactions between biological
molecules. Methods for using SPR are described, for example, in
U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer
Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345;
Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line
resources provide by BIAcore International AB (Uppsala, Sweden).
Information from SPR can be used to provide an accurate and
quantitative measure of the equilibrium dissociation constant
(K.sub.d), and kinetic parameters, including K.sub.on and
K.sub.off, for the binding of a biomolecule to a target.
[0086] Epitopes can also be directly mapped by assessing the
ability of different antibodies to compete with each other for
binding to TWEAK (e.g., human TWEAK, particularly soluble human
TWEAK) using BIAcore chromatographic techniques (Pharmacia
BlAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May
1994); see also Johne et al. (1993) J. Immunol. Methods,
160:191-198). Additional general guidance for evaluating
antibodies, e.g., in Western blots and immunoprecipitation assays,
can be found in Antibodies: A Laboratory Manual, ed. by Harlow and
Lane, Cold Spring Harbor press (1988)).
[0087] TWEAK-Associated Disorders
[0088] An anti-TWEAK antibody (such as an antibody described
herein) can be used to treat a variety of disorders, such as a
TWEAK-associated disorder. For example, the antibody can be used to
treat inflammatory, immune, or autoimmune disorders in patients, as
well as neoplastic disorders. Examples of inflammatory
TWEAK-associated disorders include rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis, inflammatory bowel disease
(including ulcerative colitis and Crohn's disease), psoriasis, or
inflammatory myositis. Still other examples of inflammatory
disorders that can be treated include Langerhans-cell
histiocytosis, adult respiratory distress syndrome/bronchiolitis
obliterans, Wegener's granulomatosis, vasculitis, cachexia,
stomatitis, idiopathic pulmonary fibrosis, dermatomyositis or
polymyositis, non-infectious scleritis, chronic sarcoidosis with
pulmonary involvement, myelodysplastic syndromes/refractory anemia
with excess blasts, ulcerative colitis, moderate to severe chronic
obstructive pulmonary disease, and giant cell arteritis.
[0089] A subject who is at risk for, diagnosed with, or who has one
of these disorders can be administered an anti-TWEAK antibody in an
amount and for a time to provide an overall therapeutic effect. The
anti-TWEAK antibody can be administered alone or in combination
with other agents. For example, U.S. Ser. No. 60/679,518 describes
methods for administering a TWEAK blocking agent in combination
with a TNF-.alpha. blocking agent. In the case of a combination
therapy, the amounts and times of administration can be those that
provide, e.g., a synergistic therapeutic effect. Further, the
administration of the TWEAK blocking agent (with or without the
second agent) can be used as a primary, e.g., first line treatment,
or as a secondary treatment, e.g., for subjects who have an
inadequate response to an previously administered therapy (i.e., a
therapy other than one with a TWEAK blocking agent).
[0090] Rheumatoid Arthritis (RA)
[0091] An anti-TWEAK antibody (such as an antibody described
herein) can be used to treat rheumatoid arthritis and related
disorders. Rheumatoid arthritis ("RA") is a chronic inflammatory
disease that causes pain, swelling, stiffness, and loss of
function, primarily in joints. RA frequently begins in the
synovium, the membrane that surrounds a joint creating a protective
sac. In many individuals suffering from RA, leukocytes infiltrate
from the circulation into the synovium causing continuous abnormal
inflammation (e.g., synovitis). Consequently, the synovium becomes
inflamed, causing warmth, redness, swelling, and pain. The collagen
in the cartilage is gradually destroyed, narrowing the joint space
and eventually damaging bone. The inflammation causes erosive bone
damage in the affected area. During this process, the cells of the
synovium grow and divide abnormally, making the normally thin
synovium thick and resulting in a joint that is swollen and puffy
to the touch.
[0092] As RA progresses, abnormal synovial cells can invade and
destroy the cartilage and bone within the joint. The surrounding
muscles, ligaments, and tendons that support and stabilize the
joint can become weak and unable to work normally. RA also may
cause more generalized bone loss that may lead to osteoporosis,
making bones fragile and more prone to fracture. All of these
effects cause the pain, impairment and deformities associated with
RA. Regions that can be effected include the wrists, knuckles,
knees and the ball of the foot. Often, many joints may be involved,
and even the spine can be affected. In about 25% of people with RA,
inflammation of small blood vessels can cause rheumatoid nodules,
or lumps, under the skin, that often form close to the joints. As
the disease progresses, fluid may also accumulate, particularly in
the ankles. Many patients with RA also develop anemia, or a
decrease in the normal number of red blood cells.
[0093] RA encompasses a number of disease subtypes, such as Felty's
syndrome, seronegative RA, "classical" RA, progressive and/or
relapsing RA, and RA with vasculitis. Some experts classify the
disease into type 1 or type 2. Type 1, the less common form, lasts
a few months at most and leaves no permanent disability. Type 2 is
chronic and lasts for years, sometimes for life. RA can also
manifest as subcutaneous rheumatoid nodules, visceral nodules,
vasculitis causing leg ulcers or mononeuritis multiplex, pleural or
pericardial effusions, lymphadenopathy, Felty's syndrome, Sjogren's
syndrome, and episcleritis. These disease subtypes and also
subjects showing one or more of the above symptoms can be treated
using the antibodies described herein.
[0094] RA can be assessed by a variety of clinical measures. Some
exemplary indicia include the total Sharp score (TSS), Sharp
erosion score, and the HAQ disability index. The methods herein can
be used to achieve an improvement for at least one of these
indicia. The therapeutic properties of an anti-TWEAK antibody for
treating RA can be evaluated in an animal model, e.g., using the
mouse collagen-induced arthritis (mCIA) model (see e.g., Stuart et
al., J. Clin. Invest. 69:673-683 (1982).
[0095] Multiple Sclerosis
[0096] An anti-TWEAK antibody (such as an antibody described
herein) can be used to treat multiple sclerosis (MS) and related
disorders. MS is a central nervous system disease that is
characterized by inflammation and loss of myelin sheaths.
[0097] Patients having MS may be identified by criteria
establishing a diagnosis of clinically definite MS as defined by
the workshop on the diagnosis of MS (Poser et al., Ann. Neurol.
(1983) 13:227). Briefly, an individual with clinically definite MS
has had two attacks and clinical evidence of either two lesions or
clinical evidence of one lesion and paraclinical evidence of
another, separate lesion. Definite MS may also be diagnosed by
evidence of two attacks and oligoclonal bands of IgG in
cerebrospinal fluid or by combination of an attack, clinical
evidence of two lesions and oligoclonal band of IgG in
cerebrospinal fluid.
[0098] Effective treatment of multiple sclerosis may be examined in
several different ways. The following parameters can be used to
gauge effectiveness of treatment. Three main criteria are used:
EDSS (extended disability status scale), appearance of
exacerbations or MRI (magnetic resonance imaging). The EDSS is a
means to grade clinical impairment due to MS (Kurtzke (1983)
Neurology 33:1444). Eight functional systems are evaluated for the
type and severity of neurologic impairment. Briefly, prior to
treatment, patients are evaluated for impairment in the following
systems: pyramidal, cerebella, brainstem, sensory, bowel and
bladder, visual, cerebral, and other. Follow-ups are conducted at
defined intervals. The scale ranges from 0 (normal) to 10 (death
due to MS). A decrease of in EDSS indicates an effective treatment
(Kurtzke (1994) Ann. Neurol. 36:573-79).
[0099] An exemplary animal model for multiple sclerosis is the
experimental autoimmune encephalitis (EAE) mouse model, e.g., as
described in Tuohy et al. (J. Immunol. (1988) 141: 1126-1130),
Sobel et al. (J. Immunol. (1984) 132: 2393-2401), and Traugott
(Cell Immunol. (1989) 119: 114-129). Mice can be administered an
antibody described herein prior to EAE induction. The mice are
evaluated for characteristic criteria to determine the efficacy of
the antibody.
[0100] Stroke
[0101] An anti-TWEAK antibody (such as an antibody described
herein) can be used to treat a subject who has experienced a
stroke, e.g., a thromboembolic or hemorrhagic stroke (e.g., within
the last 48, 24, 12, 8, or 2 hours), or to prevent a stroke, e.g.,
in a subject at risk for stroke. Exemplary methods are described in
U.S. Ser. No. 60/653,811. Stroke is a general term for acute brain
damage resulting from disease of blood vessels. Stroke can be
classified into at least two main categories: hemorrhagic stroke
(resulting from leakage of blood outside of the normal blood
vessels) and ischemic stroke (cerebral ischemia due to lack of
blood supply). Some events that can cause ischemic stroke include
thrombosis, embolism, and systemic hypoperfusion (with resultant
ischemia and hypoxia).
[0102] Stroke generally causes neuronal death and injury in the
brain by oxygen deprivation and secondary events. The area of the
brain that dies as a result of the lack of blood supply or other
damage is called an infarct. In some cases, the treatments
described herein can be used to reduce or minimize the size of an
infarct, e.g., by reducing secondary events that cause neuronal
death or injury.
[0103] Obstruction of a cerebral artery resulting from a thrombus
which has built up on the wall of a brain artery is generally
called cerebral thrombosis. In cerebral embolism, the occlusive
material blocking the cerebral artery arises downstream in the
circulation (e.g. an embolus is carried to the cerebral artery from
the heart). Because it is difficult to discern whether a stroke is
caused by thrombosis or embolism, the term thromboembolism is used
to cover both these types of stroke. Systemic hypoperfusion may
arise as a consequence of decreased blood levels, reduced
hematocrit, low blood pressure or inability of the heart to pump
blood adequately.
[0104] Further, an anti-TWEAK antibody can be administered as a
prophylactic stroke therapy, or as a component thereof, e.g., to a
subject who has experienced a transient ischemic attack (TIA) or is
exhibiting symptoms of TIA.
[0105] Neuronal Disorders
[0106] An anti-TWEAK antibody (such as an antibody described
herein) can be used to treat or prevent neuronal disorders such as
mechanical neuronal traumas and neurodegenerative disorders.
Examples of mechanical neuronal traumas include spinal cord injury
(SCl) and traumatic brain injury (TBI). Examples of
neurodegenerative disorders include amyotrophic lateral sclerosis
(ALS), progressive bulbar palsy (PBP), primary lateral sclerosis
(PLS), progressive muscular atrophy (PMA), Parkinson's Disease,
Huntington's Disease (HD), and Alzheimer's Disease. See, e.g., U.S.
Ser. No. 60/653,813. In one embodiment, the neuronal disorder is
primarily characterized by destruction or death of nerve cells,
e.g., of motor neurons (e.g., ALS), of striatal neurons of basal
ganglia and/or cortical neurons (e.g., Huntington's disease), of
substantia nigra neurons (e.g., Parkinson's disease).
[0107] Cancer
[0108] TWEAK and its receptors may be involved in the development
of at least some types of cancer, e.g., a pancreatic cancer. An
anti-TWEAK antibody (such as an antibody described herein) can be
used to treat or prevent cancers (e.g., adenocarcinomas) and other
neoplastic disorders. See, e.g., "TREATMENT OF CANCER," U.S. Ser.
No. 60/685,465, filed on May 27, 2005.
[0109] Pharmaceutical Compositions
[0110] An anti-TWEAK antibody (such as an antibody described
herein) can be formulated as a pharmaceutical composition for
administration to a subject, e.g., to treat a disorder described
herein. Typically, a pharmaceutical composition includes a
pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are to physiologically compatible. The composition can include a
pharmaceutically acceptable salt, e.g., an acid addition salt or a
base addition salt (see e.g., Berge, S. M., et al. (1977) J. Pharm.
Sci. 66:1-19).
[0111] Pharmaceutical fonnulation is a well-established art, and is
further described, e.g., in Gennaro (ed.), Remington: The Science
and Practice of Pharmacy, 20.sup.th ed., Lippincott, Williams &
Wilkins (2000) (ISBN: 0683306472); Ansel et al., Pharmaceutical
Dosage Forms and Drug Delivery Systems, 7.sup.th Ed., Lippincott
Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and
Kibbe (ed.), Handbook of Pharmaceutical Excipients American
Pharmaceutical Association, 3.sup.rd ed. (2000) (ISBN:
091733096X).
[0112] The pharmaceutical compositions may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form can depend
on the intended mode of administration and therapeutic application.
Typically compositions for the agents described herein are in the
form of injectable or infusible solutions.
[0113] In one embodiment, the anti-TWEAK antibody is formulated
with excipient materials, such as sodium chloride, sodium dibasic
phosphate heptahydrate, sodium monobasic phosphate, and a
stabilizer. It can be provided, for example, in a buffered solution
at a suitable concentration and can be stored at 2-8.degree. C.
[0114] Such compositions can be administered by a parenteral mode
(e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular
injection). The phrases "parenteral administration" and
"administered parenterally" as used herein mean modes of
administration other than enteral and topical administration,
usually by injection, and include, without limitation, intravenous,
intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous, subcuticular, intraarticular,
subcapsular, subarachnoid, intraspinal, epidural and intrasternal
injection and infusion.
[0115] The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable for stable storage at high concentration. Sterile
injectable solutions can be prepared by incorporating an agent
described herein in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating an agent described herein
into a sterile vehicle that contains a basic dispersion medium and
the required other ingredients from those enumerated above. In the
case of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze drying that yield a powder of an agent described herein
plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0116] In certain embodiments, the anti-TWEAK antibody may be
prepared with a carrier that will protect the compound against
rapid release, such as a controlled release formulation, including
implants, and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known. See, e.g., Sustained
and Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York (1978).
[0117] An anti-TWEAK antibody can be modified, e.g., with a moiety
that improves its stabilization and/or retention in circulation,
e.g., in blood, serum, or other tissues, e.g., by at least 1.5, 2,
5, 10, or 50 fold. The modified antibody can be evaluated to assess
whether it can reach sites of inflammation, e.g., joints.
[0118] For example, the anti-TWEAK antibody can be associated with
(e.g., conjugated to) a polymer, e.g., a substantially
non-antigenic polymer, such as a polyalkylene oxide or a
polyethylene oxide. Suitable polymers will vary substantially by
weight. Polymers having molecular number average weights ranging
from about 200 to about 35,000 Daltons (or about 1,000 to about
15,000, and 2,000 to about 12,500) can be used.
[0119] For example, the anti-TWEAK antibody can be conjugated to a
water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g.,
polyvinylalcohol or polyvinylpyrrolidone. Examples of such polymers
include polyalkylene oxide homopolymers such as polyethylene glycol
(PEG) or polypropylene glycols, polyoxyethylenated polyols,
copolymers thereof and block copolymers thereof, provided that the
water solubility of the block copolymers is maintained. Additional
useful polymers include polyoxyalkylenes such as polyoxyethylene,
polyoxypropylene, and block copolymers of polyoxyethylene and
polyoxypropylene; polymethacrylates; carbomers; and branched or
unbranched polysaccharides.
[0120] In some implementations, the anti-TWEAK antibody can also be
coupled to or otherwise associated with a label or other agent,
e.g., another therapeutic agent such as a cytotoxic or cytostatic
agent, although, in many embodiments, this configuration is
unnecessary. Examples of cytotoxic and chemotherapeutic agents
include taxol, cytochalasin B, gramicidin D, vinblastine,
doxorubicin, daunorubicin, a maytansinoid (e.g., maytansinol or the
DM1 maytansinoid, a sulfhydryl-containing derivative of
maytansine), mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, taxane,
tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof.
[0121] When the anti-TWEAK antibody is used in combination with a
second agent (e.g., an anti-TNF-.alpha. antibody or other agent),
the two agents can be formulated separately or together. The agents
can be formulated or otherwise used in a synergistically effective
amount. It is also possible to use one or both of the agents in
amounts less than would be used for mono-therapy. For example, the
respective pharmaceutical compositions can be mixed, e.g., just
prior to administration, and administered together or can be
administered separately, e.g., at the same or different times.
[0122] It is also possible to use other TWEAK blocking agents,
e.g., agents described in U.S. Ser. No. 60/679,518. The agent may
be any type of compound (e.g., small organic or inorganic molecule,
nucleic acid, protein, or peptide mimetic) that can be administered
to a subject. In one embodiment, the blocking agent is a biologic,
e.g., a protein having a molecular weight of between 5-300 kDa. For
example, a TWEAK blocking agent may inhibit binding of TWEAK to a
TWEAK receptor. Exemplary TWEAK blocking agents, other than
antibodies that bind to TWEAK, include antibodies that bind to
TWEAK-R and soluble forms of the TWEAK-R (e.g., Fn14) that compete
with cell surface TWEAK-R for binding to TWEAK. Other therapeutic
agents described herein can also be provided as a pharmaceutical
composition, e.g., by standard methods or method described
herein.
[0123] Administration
[0124] The anti-TWEAK antibody can be administered to a subject,
e.g., a human subject, by a variety of methods. For many
applications, the route of administration is one of: intravenous
injection or infusion (IV), subcutaneous injection (SC),
intraperitoneally (IP), or intramuscular injection. It is also
possible to use intra-articular delivery. Other modes of parenteral
administration can also be used. Examples of such modes include:
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, transtracheal, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, and
epidural and intrasternal injection. In some cases, administration
may be directly to a site of inflammation, e.g., a joint or other
inflamed site.
[0125] The route and/or mode of administration of the antibody can
also be tailored for the individual case, e.g., by monitoring the
subject, e.g., using tomographic imaging, neurological exam, and
standard parameters associated with the particular disorder, e.g.,
criteria for assessing rheumatoid arthritis.
[0126] The antibody can be administered as a fixed dose, or in a
mg/kg dose. The dose can also be chosen to reduce or avoid
production of antibodies against the anti-TWEAK antibody. Dosage
regimens are adjusted to provide the desired response, e.g., a
therapeutic response or a combinatorial therapeutic effect.
Generally, doses of the anti-TWEAK antibody (and optionally a
second agent) can be used in order to provide a subject with the
agent in bioavailable quantities. For example, doses in the range
of 0.1-100 mg/kg, 0.5-100 mg/kg, 1 mg/kg-100 mg/kg, 0.5-20 mg/kg,
0.1-10 mg/kg, or 1-10 mg/kg can be administered. Other doses can
also be used.
[0127] Dosage unit form or "fixed dose" as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier and
optionally in association with the other agent. Single or multiple
dosages may be given. Alternatively, or in addition, the antibody
may be administered via continuous infusion.
[0128] A anti-TWEAK antibody dose can be administered, e.g., at a
periodic interval over a period of time (a course of treatment)
sufficient to encompass at least 2 doses, 3 doses, 5 doses, 10
doses, or more, e.g., once or twice daily, or about one to four
times per week, or preferably weekly, biweekly, monthly, e.g., for
between about 1 to 12 weeks, preferably between 2 to 8 weeks, more
preferably between about 3 to 7 weeks, and even more preferably for
about 4, 5, or 6 weeks. Factors that may influence the dosage and
timing required to effectively treat a subject, include, e.g., the
severity of the disease or disorder, formulation, route of
delivery, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a compound can
include a single treatment or, preferably, can include a series of
treatments. Animal models can also be used to determine a useful
dose, e.g., an initial dose or a regimen.
[0129] If a subject is at risk for developing an inflammatory
disorder or other disorder described herein, the antibody can be
administered before the full onset of the disorder, e.g., as a
preventative measure. The duration of such preventative treatment
can be a single dosage of the antibody or the treatment may
continue (e.g., multiple dosages). For example, a subject at risk
for the disorder or who has a predisposition for the disorder may
be treated with the antibody for days, weeks, months, or even years
so as to prevent the disorder from occurring or fulminating.
[0130] A pharmaceutical composition may include a "therapeutically
effective amount" of an agent described herein. Such effective
amounts can be determined based on the effect of the administered
agent, or the combinatorial effect of agents if more than one agent
is used. A therapeutically effective amount of an agent may also
vary according to factors such as the disease state, age, sex, and
weight of the individual, and the ability of the compound to elicit
a desired response in the individual, e.g., amelioration of at
least one disorder parameter or amelioration of at least one
symptom of the disorder. A therapeutically effective amount is also
one in which any toxic or detrimental effects of the composition
are outweighed by the therapeutically beneficial effects.
[0131] Devices and Kits for Therapy
[0132] Pharmaceutical compositions that include the anti-TWEAK
antibody can be administered with a medical device. The device can
designed with features such as portability, room temperature
storage, and ease of use so that it can be used in emergency
situations, e.g., by an untrained subject or by emergency personnel
in the field, removed from medical facilities and other medical
equipment. The device can include, e.g., one or more housings for
storing pharmaceutical preparations that include anti-TWEAK
antibody, and can be configured to deliver one or more unit doses
of the antibody. The device can be further configured to administer
a second agent, e.g., an anti-TNF-.alpha. antibody, either as a
single pharmaceutical composition that also includes the anti-TWEAK
antibody or as two separate pharmaceutical compositions.
[0133] For example, the pharmaceutical composition can be
administered with a needleless hypodermic injection device, such as
the devices disclosed in U.S. Pat. No. 5,399,163; 5,383,851;
5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples
of well-known implants and modules include: U.S. Pat. No.
4,487,603, which discloses an implantable micro-infusion pump for
dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which discloses a therapeutic device for administering
medicants through the skin; U.S. Pat. No. 4,447,233, which
discloses a medication infusion pump for delivering medication at a
precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a
variable flow implantable infusion apparatus for continuous drug
delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug
delivery system having multi-chamber compartments; and U.S. Pat.
No. 4,475,196, which discloses an osmotic drug delivery system.
Many other devices, implants, delivery systems, and modules are
also known.
[0134] An anti-TWEAK antibody can be provided in a kit. In one
embodiment, the kit includes (a) a container that contains a
composition that includes the anti-TWEAK antibody, and optionally
(b) informational material. The informational material can be
descriptive, instructional, marketing or other material that
relates to the methods described herein and/or the use of the
agents for therapeutic benefit.
[0135] In an embodiment, the kit also includes a second agent for
treating an inflammatory disorder, e.g., an anti-TNF-.alpha.
antibody. For example, the kit includes a first container that
contains a composition that includes the anti-TWEAK antibody, and a
second container that includes the second agent.
[0136] The informational material of the kits is not limited in its
form. In one embodiment, the informational material can include
information about production of the compound, molecular weight of
the compound, concentration, date of expiration, batch or
production site information, and so forth. In one embodiment, the
informational material relates to methods of administering the
anti-TWEAK antibody, e.g., in a suitable dose, dosage form, or mode
of administration (e.g., a dose, dosage form, or mode of
administration described herein), to treat a subject who has had or
who is at risk for an inflammatory disorder, or other disorder
described herein. The information can be provided in a variety of
formats, include printed text, computer readable material, video
recording, or audio recording, or information that provides a link
or address to substantive material, e.g., on the internet.
[0137] In addition to the antibody, the composition in the kit can
include other ingredients, such as a solvent or buffer, a
stabilizer, or a preservative. The antibody can be provided in any
form, e.g., liquid, dried or lyophilized form, preferably
substantially pure and/or sterile. When the agents are provided in
a liquid solution, the liquid solution preferably is an aqueous
solution. When the agents are provided as a dried form,
reconstitution generally is by the addition of a suitable solvent.
The solvent, e.g., sterile water or buffer, can optionally be
provided in the kit.
[0138] The kit can include one or more containers for the
composition or compositions containing the agents. In some
embodiments, the kit contains separate containers, dividers or
compartments for the composition and informational material. For
example, the composition can be contained in a bottle, vial, or
syringe, and the informational material can be contained in a
plastic sleeve or packet. In other embodiments, the separate
elements of the kit are contained within a single, undivided
container. For example, the composition is contained in a bottle,
vial or syringe that has attached thereto the informational
material in the form of a label. In some embodiments, the kit
includes a plurality (e.g., a pack) of individual containers, each
containing one or more unit dosage forms (e.g., a dosage form
described herein) of the agents. The containers can include a
combination unit dosage, e.g., a unit that includes both the
anti-TWEAK antibody and the second agent, e.g., in a desired ratio.
For example, the kit includes a plurality of syringes, ampules,
foil packets, blister packs, or medical devices, e.g., each
containing a single combination unit dose. The containers of the
kits can be air tight, waterproof (e.g., impermeable to changes in
moisture or evaporation), and/or light-tight.
[0139] The kit optionally includes a device suitable for
administration of the composition, e.g., a syringe or other
suitable delivery device. The device can be provided pre-loaded
with one or both of the agents or can be empty, but suitable for
loading.
[0140] Targetting TWEAK-Expressing Cells
[0141] The anti-TWEAK antibodies described herein can be used to
target a payload to a TWEAK-expressing cell or to a tissue or other
structure associated with TWEAK. For example, the antibodies can be
attached to a virus or virus like particle that can deliver an
exogenous gene (e.g., for gene therapy) or to a liposome, e.g., a
liposome that encapsulates a therapeutic agent or exogenous gene.
An exemplary method for using an antibody to target a virus is
described in Roux et al. (1989) Proc Natl Acad Sci USA (1989)
86:9079-9083. See also, e.g., Curr Gene Ther. (2005) 5:63-70 and
Hum Gene Ther. (2004) 15:1034-1044.
[0142] The anti-TWEAK Abs of this invention may also be attached to
liposomes containing a therapeutic agent such as a chemotherapeutic
agents. Attachment of antibodies to liposomes may be accomplished
by any known cross-linking agent such as heterobifunctional
cross-linking agents that have been widely used to couple toxins or
chemotherapeutic agents to antibodies for targeted delivery. For
example, conjugation to liposomes can be accomplished using the
carbohydrate-directed cross-linking reagent 4-(4-maleimidophenyl)
butyric acid hydrazide (MPBH) (Duzgunes et al. (1992) J. Cell.
Biochem. Abst. Suppl. 16E 77). Liposomes containing antibodies can
also be prepared by well-known methods (See, e.g. DE 3,218,121;
Epstein et al. (1985) Proc. Natl. Acad. Sci. USA, 82:3688-92; Hwang
et al. (1980) Proc. Natl. Acad. Sci. USA, 77:4030-34; U.S. Pat.
Nos. 4,485,045 and 4,544,545).
[0143] Diagnostic Uses
[0144] Anti-TWEAK antibodies can be used in a diagnostic method for
detecting the presence of a TWEAK, in vitro (e.g., a biological
sample, such as tissue, biopsy) or in vivo (e.g., in vivo imaging
in a subject). For example, human or effectively human anti-TWEAK
antibodies can be administered to a subject to detect TWEAK within
the subject. For example, the antibody can be labeled, e.g., with
an MRI detectable label or a radiolabel. The subject can be
evaluated using a means for detecting the detectable label. For
example, the subject can be scanned to evaluate localization of the
antibody within the subject. For example, the subject is imaged,
e.g., by NMR or other tomographic means.
[0145] Examples of labels useful for diagnostic imaging include
radiolabels such as .sup.131I, .sup.111In, .sup.123I, .sup.99mTe,
.sup.32P, .sup.33P, .sup.125I, .sup.3H, .sup.14C, and .sup.188Rh,
fluorescent labels such as fluorescein and rhodamine, nuclear
magnetic resonance active labels, positron emitting isotopes
detectable by a positron emission tomography ("PET") scanner,
chemiluminescers such as luciferin, and enzymatic markers such as
peroxidase or phosphatase. Short-range radiation emitters, such as
isotopes detectable by short-range detector probes, can also be
employed. The protein ligand can be labeled with such reagents
using known techniques. For example, see Wensel and Meares (1983)
Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York for
techniques relating to the radiolabeling of antibodies and Colcher
et al. (1986) Meth. Enzymol. 121: 802-816.
[0146] The subject can be "imaged" in vivo using known techniques
such as radionuclear scanning using e.g., a gamma camera or
emission tomography. See e.g., A. R. Bradwell et al., "Developments
in Antibody Imaging", Monoclonal Antibodies for Cancer Detection
and Therapy, R. W. Baldwin et al., (eds.), pp 65-85 (Academic Press
1985). Alternatively, a positron emission transaxial tomography
scanner, such as designated Pet VI located at Brookhaven National
Laboratory, can be used where the radiolabel emits positrons (e.g.,
.sup.11C, .sup.18F, .sup.15O, and .sup.13N).
[0147] MRI Contrast Agents. Magnetic Resonance Imaging (MRI) uses
NMR to visualize internal features of living subject, and is useful
for prognosis, diagnosis, treatment, and surgery. MRI can be used
without radioactive tracer compounds for obvious benefit. Some MRI
techniques are summarized in EPO 502 814 A. Generally, the
differences related to relaxation time constants T1 and T2 of water
protons in different environments is used to generate an image.
However, these differences can be insufficient to provide sharp
high resolution images.
[0148] The differences in these relaxation time constants can be
enhanced by contrast agents. Examples of such contrast agents
include a number of magnetic agents, paramagnetic agents (which
primarily alter T1) and ferromagnetic or superparamagnetic agents
(which primarily alter T2 response). Chelates (e.g., EDTA, DTPA and
NTA chelates) can be used to attach (and reduce toxicity) of some
paramagnetic substances (e.g., Fe.sup.3+, Mn.sup.2+, Gd.sup.3+).
Other agents can be in the form of particles, e.g., less than 10
.mu.M to about 10 nm in diameter). Particles can have
ferromagnetic, anti-ferromagnetic or superparamagnetic properties.
Particles can include, e.g., magnetite (Fe.sub.3O.sub.4),
.gamma.-Fe.sub.2O.sub.3, ferrites, and other magnetic mineral
compounds of transition elements. Magnetic particles may include
one or more magnetic crystals with and without nonmagnetic
material. The nonmagnetic material can include synthetic or natural
polymers (such as sepharose, dextran, dextrin, starch and the
like).
[0149] The anti-TWEAK antibodies can also be labeled with an
indicating group containing the NMR-active .sup.19F atom, or a
plurality of such atoms inasmuch as (i) substantially all of
naturally abundant fluorine atoms are the .sup.19F isotope and,
thus, substantially all fluorine-containing compounds are
NMR-active; (ii) many chemically active polyfluorinated compounds
such as trifluoracetic anhydride are commercially available at
relatively low cost, and (iii) many fluorinated compounds have been
found medically acceptable for use in humans such as the
perfluorinated polyethers utilized to carry oxygen as hemoglobin
replacements. After permitting such time for incubation, a whole
body MRI is carried out using an apparatus such as one of those
described by Pykett (1982) Scientific American, 246:78-88 to locate
and image TWEAK distribution.
[0150] In another aspect, the disclosure provides a method for
detecting the presence of TWEAK in a sample in vitro (e.g., a
biological sample, such as serum, plasma, tissue, biopsy). The
subject method can be used to diagnose a disorder, e.g., an immune
cell-associated disorder. The method includes: (i) contacting the
sample or a control sample with the anti-TWEAK antibody; and (ii)
evaluating the sample for the presence of TWEAK, e.g., by detecting
formation of a complex between the anti-TWEAK antibody and TWEAK,
or by detecting the presence of the antibody or TWEAK. For example,
the antibody can be immobilized, e.g., on a support, and retention
of the antigen on the support is detected, and/or vice versa. A
control sample can be included. A statistically significant change
in the formation of the complex in the sample relative to the
control sample can be indicative of the presence of TWEAK in the
sample. Generally, an anti-TWEAK antibody can be used in
applications that include fluorescence polarization, microscopy,
ELISA, centrifugation, chromatography, and cell sorting (e.g.,
fluorescence activated cell sorting).
Example 1
[0151] The sequence of the murine P2D10 heavy chain variable
domain, with CDRs underlined, is:
TABLE-US-00005 (SEQ ID NO: 50) 1 EVQLVESGGG LVRPGGSLKL FCAASGFTFS
RYAMSWVRQS PEKRLEWVAE 51 ISSGGSYPYY PDTVTGRFTI SRDNAKNTLY
LEMSSLKSED TAMYYCARVL 101 YYDYDGDRIE VMDYWGQGTA VIVSS
[0152] This is a murine subgroup 3D heavy chain variable
domain.
[0153] The sequence of the murine P2D10 light chain variable
domain, with CDRs underlined, is:
TABLE-US-00006 (SEQ ID NO: 51) 1 DVVMTQSPLS LSVSLGDQAS ISCRSSQSLV
SSKGNTYLHW YLQKPGQSPK 51 FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI
SRVAAEDLGV YFCSQSTHFP 101 RTFGGGTTLE IK
[0154] This is a murine subgroup 2 kappa light chain.
[0155] The following human acceptor frameworks were chosen for
huP2D10: Human 56-84m subgroup 3 heavy chain variable domain (NCBI
database accession number GI:33318898, Scamurra et al., direct
submission). The sequence with CDRs underlined is as follows.:
TABLE-US-00007 (SEQ ID NO: 52) 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS
SYWMSWVRQA PGKGLEWVAN 51 IKQDGSEKYY VDSVKGRFTI SRDNAKNSLY
LQMNSLRAED TAVYYCARDP 101 MTTVVKPSLA TNDYWGQGTL VTVSS
[0156] The sequence of human K107 subgroup 2 light chain variable
domain (NCBI database accession number GI: 21669075, Akahori et
al., direct submission), with CDRs underlined is:
TABLE-US-00008 (SEQ ID NO: 53) 1 DVVMTQSPLS LPVTPGEPAS ISCRSSQSLL
HSNGYNYLDW YLQKPGQSPQ 51 LLIYLGSNRA SGVPDRFSGS GSGTDFTLKI
SRVEAEDVGV YYCMQALQTP 101 LTFGGGTKVE IK
[0157] In the human acceptor sequences shown, the CDRs (which are
underlined) are the same length and canonical classes as those in
the muP2D10 variable domains.
[0158] Shown below is the alignment of the murine P2D10 (top) and
human acceptor 56-84m (bottom) heavy chain variable domains (68.8%
identical):
##STR00001##
[0159] Shown below is the alignment of the murine P2D10 (top) and
human acceptor K107 light (bottom) chain variable domains (75.9%
identical):
##STR00002##
[0160] There are two versions of huP2D10 light chain (L1 and L2).
The antibody huP2D10-1 refers to an antibody that includes huP2D10
H1 and huP2D10 L1. The antibody huP2D10-2 refers to an antibody
that includes huP2D10 H1 and huP2D10 L2.
[0161] Shown below is the alignment of the 56-84m human acceptor
(top) and the huP2D10 H1 heavy chain (bottom) variable domain:
##STR00003##
[0162] CDRs are underlined. The huP2D10 heavy chain is a straight
CDR graft (i.e., there are no back mutations in the framework).
[0163] Shown below is the alignment of the K107 human kappa
acceptor (top) and the huP2D10 L1 light chain (bottom) variable
domain:
##STR00004##
[0164] CDRs are underlined. The huP2D10 .mu.l light chain has two
back mutations that are indicated in lower case in the framework
shown above: L46F in FR2 and Y87F in FR3.
[0165] Shown below is the alignment of the K107 human acceptor
(top) and the huP2D10 L2 light chain (bottom) variable domain:
##STR00005##
[0166] CDRs are underlined. The huP2D10 L2 light chain is a
straight CDR graft (without back mutations in the framework).
[0167] This is an exemplary amino acid sequence of the mature
huP2D10 H1 IgG1 heavy chain:
TABLE-US-00009 (SEQ ID NO: 64) 1 EVQLVESGGG LVQPGGSLRL SCAASGFTFS
RYAMSWVRQA PGKGLEWVAE 51 ISSGGSYPYY PDTVTGRFTI SRDNAKNSLY
LQMNSLRAED TAVYYCARVL 101 YYDYDGDRIE VMDYWGQGTL VTVSSASTKG
PSVFPLAPSS KSTSGGTAAL 151 GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA
VLQSSGLYSL SSVVTVPSSS 201 LGTQTYICNV NHKPSNTKVD KKVEPKSCDK
THTCPPCPAP ELLGGPSVFL 251 FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE
VKFNWYVDGV EVHNAKTKPR 301 EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK
VSNKALPAPI EKTISKAKGQ 351 PREPQVYTLP PSRDELTKNQ VSLTCLVKGF
YPSDIAVEWE SNGQPENNYK 401 TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV
FSCSVMHEAL HNHYTQKSLS 451 LSPG
[0168] Kabat numbering for the V.sub.H segment of the heavy chain
variable domain (SEQ ID NO:65) is shown below:
TABLE-US-00010 Kabat No. 1234567890 1234567890 1234567890 hP2D10
EVQLVESGGG LVQPGGSLRL SCAASGFTFS 1234567890 1234567890 RYAMSWVRQA
PGKGLEWVAE Kabat No. 12a3456789 0123456789 0123456789 hP2D10
ISSGGSYPYY PDTVTGRFTI SRDNAKNSLY 012abc3456 78901234 LQMNSLRAED
TAVYYCAR
[0169] This is an exemplary amino acid sequence of the mature
huP2D10 L1 light chain:
TABLE-US-00011 (SEQ ID NO: 66) 1 DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV
SSKGNTYLHW YLQKPGQSPQ 51 FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI
SRVEAEDVGV YFCSQSTHFP 101 RTFGGGTKVE IKRTVAAPSV FIFPPSDEQL
KSGTASVVCL LNNFYPREAK 151 VQWKVDNALQ SGNSQESVTE QDSKDSTYSL
SSTLTLSKAD YEKHKVYACE 201 VTHQGLSSPV TKSFNRGEC
[0170] Kabat numbering for this V.sub.L segment is shown below (SEQ
ID NO:67):
TABLE-US-00012 Kabat No. 1234567890 1234567890 1234567abc hP2D10
DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV de89012345 6789012345 SSKGNTYLHW
YLQKPGQSPQ Kabat No. 6789012345 6789012345 6789012345 hP2D10
FLIYKVSNRF SGVPDRFSGS GSGTDFTLKI 6789012345 6789012345 SRVEAEDVGV
YFCSQSTHFP
[0171] This is an exemplary amino acid sequence of the mature
huP2D10 L2 light chain:
TABLE-US-00013 (SEQ ID NO: 68) 1 DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV
SSKGNTYLHW YLQKPGQSPQ 51 LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI
SRVEAEDVGV YYCSQSTHFP 101 RTFGGGTKVE IKRTVAAPSV FIFPPSDEQL
KSGTASVVCL LNNFYPREAK 151 VQWKVDNALQ SGNSQESVTE QDSKDSTYSL
SSTLTLSKAD YEKHKVYACE 201 VTHQGLSSPV TKSFNRGEC
[0172] Kabat numbering for this V.sub.L segment is shown below (SEQ
ID NO:69):
TABLE-US-00014 Kabat No. 1234567890 1234567890 1234567abc hP2D10
DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV de89012345 6789012345 SSKGNTYLHW
YLQKPGQSPQ Kabat No. 6789012345 6789012345 6789012345 hP2D10
LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI 6789012345 6789012345 SRVEAEDVGV
YFCSQSTHFP
Example 2
[0173] The blocking monoclonal antibody to TWEAK, mP2D10,
significantly reduced clinical severity in models of multiple
sclerosis, stroke, and rheumatic arthritis. The pharmacokinetics
(PK) of anti-TWEAK monoclonal antibody, mP2D10, following
intravenous (IV) administration were modeled.
[0174] mP2D10 was administered to mice at 1, 10 or 100 mg/kg via IV
injection. Serum concentrations of mP2D10 was determined using
ELISA. The concentration-time PK profile was analyzed using a
two-compartment model with first-order elimination or
Michaelis-Menten elimination from the central compartment with a
volume of V1. The rate constants between the two compartments were
K12 (exiting compartment 1 to 2) and K21 (exiting compartment 2 to
1). For the first-order elimination model, the elimination rate
constant was K10. For the Michaelis-Menten elimination model, the
drug was cleared at the rate of Vm*C1/(Km+C1), in which, C1 was
mP2D10 in the concentration in the central compartment, Vm and Km
were constants. The data were fitted with ADAPT II software
(D'Argenio, D. Z. and A. Schumitzky. ADAPT II User's Guide:
Pharmacokinetic/Pharmacodynamic Systems Analysis Software.
Biomedical Simulations Resource, Los Angeles, 1997.) using the
Maximum Likelihood estimation procedure.
[0175] For the two-compartment linear elimination model, V1 was
23.2 mL/kg, K10 was 0.0096 h.sup.-1, the K12 was 2.501 and K21 was
1.053. The area under curve (AIC) value was 298 and the Schwarz
value was 304.2. For the two-compartment non-linear elimination
model, the V1 was 0.0235. The Vm was 9.22 mg/kg/hr, the Km was
484.2 .mu.g/mL. The K12 was 2.348 h.sup.-1, and the K21 was 0.966
h.sup.-1. The AIC value was 269 and the Schwarz value was 276. The
PK of mP2D10 was better predicted by a non-linear model than a
linear model.
[0176] The concentration-time profiles of mP2D10 were better
predicted by a two-compartment model with Michaelis-Menten
elimination than with first order elimination.
[0177] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments described herein described
herein.
Sequence CWU 1
1
69110PRTMus musculus 1Gly Phe Thr Phe Ser Arg Tyr Ala Met Ser1 5
10217PRTMus musculus 2Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr
Pro Asp Thr Val Thr1 5 10 15Gly316PRTMus musculus 3Val Leu Tyr Tyr
Asp Tyr Asp Gly Asp Arg Ile Glu Val Met Asp Tyr1 5 10
15410PRTArtificial SequenceSynthetically generated peptide 4Gly Xaa
Xaa Phe Xaa Xaa Tyr Ala Xaa Xaa1 5 1058PRTArtificial
SequenceSynthetically generated peptide 5Tyr Tyr Xaa Asp Xaa Val
Xaa Gly1 567PRTArtificial SequenceSynthetically generated peptide
6Xaa Xaa Xaa Xaa Asp Xaa Asp1 577PRTArtificial
SequenceSynthetically generated peptide 7Xaa Xaa Xaa Xaa Xaa Met
Xaa1 5816PRTMus musculus 8Arg Ser Ser Gln Ser Leu Val Ser Ser Lys
Gly Asn Thr Tyr Leu His1 5 10 1597PRTMus musculus 9Lys Val Ser Asn
Arg Phe Ser1 5109PRTMus musculus 10Ser Gln Ser Thr His Phe Pro Arg
Thr1 51116PRTArtificial SequenceSynthetically generated peptide
11Xaa Ser Ser Gln Ser Xaa Xaa Ser Ser Xaa Gly Asn Xaa Tyr Leu Xaa1
5 10 151216PRTArtificial SequenceSynthetically generated peptide
12Xaa Ser Ser Gln Ser Xaa Val Ser Ser Xaa Gly Asn Xaa Tyr Leu His1
5 10 15137PRTArtificial SequenceSynthetically generated peptide
13Xaa Xaa Ser Xaa Xaa Xaa Ser1 5147PRTArtificial
SequenceSynthetically generated peptide 14Lys Xaa Ser Xaa Arg Xaa
Ser1 5157PRTArtificial SequenceSynthetically generated peptide
15Xaa Gln Xaa Xaa Xaa Xaa Pro1 5167PRTArtificial
SequenceSynthetically generated peptide 16Ser Gln Xaa Xaa Xaa Phe
Pro1 517102PRTArtificial SequenceSynthetically generated peptide
17Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser
Ser 20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10018102PRTArtificial SequenceSynthetically generated peptide 18Asp
Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10019102PRTArtificial SequenceSynthetically generated peptide 19Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10
15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln
Ser 35 40 45Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10020102PRTArtificial SequenceSynthetically generated peptide 20Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly1 5 10
15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Phe Gln Gln Arg Pro Gly Gln
Ser 35 40 45Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10021102PRTArtificial SequenceSynthetically generated peptide 21Asp
Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10
15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10022102PRTArtificial SequenceSynthetically generated peptide 22Asp
Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly1 5 10
15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Pro 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10023102PRTArtificial SequenceSynthetically generated peptide 23Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10024102PRTArtificial SequenceSynthetically generated peptide 24Asp
Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10025102PRTArtificial SequenceSynthetically generated peptide 25Asp
Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly1 5 10
15Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Leu Gln Gln Arg Pro Gly Gln
Pro 35 40 45Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10026102PRTArtificial SequenceSynthetically generated peptide 26Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala 35 40 45Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile65 70 75 80Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr
10027110PRTArtificial SequenceSynthetically generated peptide 27Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45Gly Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp
Thr Val 50 55 60Thr Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp Gly
Asp Arg Ile Glu 100 105 11028110PRTArtificial SequenceSynthetically
generated peptide 28Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe
Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Arg Leu Glu Trp Met 35 40 45Gly Glu Ile Ser Ser Gly Gly Ser Tyr
Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Val Thr Ile Thr Arg
Asp Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr
Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100 105 11029110PRTArtificial
SequenceSynthetically generated peptide 29Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100
105 11030110PRTArtificial SequenceSynthetically generated peptide
30Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro
Asp Thr Val 50 55 60Thr Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp
Gly Asp Arg Ile Glu 100 105 11031110PRTArtificial
SequenceSynthetically generated peptide 31Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Val Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Thr Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100
105 11032110PRTArtificial SequenceSynthetically generated peptide
32Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Thr Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Gln Ala Leu Glu
Trp Met 35 40 45Gly Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro
Asp Thr Val 50 55 60Thr Gly Arg Val Thr Ile Thr Arg Asp Arg Ser Met
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp
Gly Asp Arg Ile Glu 100 105 11033110PRTArtificial
SequenceSynthetically generated peptide 33Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100
105 11034108PRTArtificial SequenceSynthetically generated peptide
34Gln Met Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Thr1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Arg Gln Arg Leu Glu Trp Ile
Gly Glu 35 40 45Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr
Val Thr Gly 50 55 60Arg Val Thr Ile Thr Arg Asp Met Ser Thr Ser Thr
Ala Tyr Met Glu65 70 75 80Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Ala 85 90 95Val Leu Tyr Tyr Asp Tyr Asp Gly Asp
Arg Ile Glu 100 10535110PRTArtificial SequenceSynthetically
generated peptide 35Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Ser Gly Gly Ser Tyr
Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr
Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100 105 11036111PRTArtificial
SequenceSynthetically generated peptide 36Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55
60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr
Cys 85 90 95Ala Lys Asp Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile
Glu 100 105 11037110PRTArtificial SequenceSynthetically generated
peptide 37Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Arg Tyr 20 25 30Ala Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr
Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp
Tyr Asp Gly Asp Arg Ile Glu 100 105 11038110PRTArtificial
SequenceSynthetically generated peptide 38Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Thr Thr Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100
105 11039110PRTArtificial SequenceSynthetically generated peptide
39Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro
Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Leu Tyr His Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp
Gly Asp Arg Ile Glu 100 105 11040110PRTArtificial
SequenceSynthetically generated peptide 40Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100
105 11041110PRTArtificial SequenceSynthetically generated peptide
41Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro
Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Leu Tyr Tyr Asp Tyr Asp
Gly Asp Arg Ile Glu 100 105 11042110PRTArtificial
SequenceSynthetically generated peptide 42Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100
105 11043110PRTArtificial SequenceSynthetically generated peptide
43Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro
Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp
Gly Asp Arg Ile Glu 100 105 11044110PRTArtificial
SequenceSynthetically generated peptide 44Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100
105 11045110PRTArtificial SequenceSynthetically generated peptide
45Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro
Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp
Gly Asp Arg Ile Glu 100 105 11046111PRTArtificial
SequenceSynthetically generated peptide 46Glu Val Gln Leu Val Glu
Ser Gly Gly Val Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95Ala Lys Asp Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu
100 105 11047110PRTArtificial SequenceSynthetically generated
peptide 47Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Arg Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr
Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Asp
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp
Tyr Asp Gly Asp Arg Ile Glu 100 105 11048110PRTArtificial
SequenceSynthetically generated peptide 48Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Thr Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Gly Ser Lys Ser Ile Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Thr Arg Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu 100
105 11049110PRTArtificial SequenceSynthetically generated peptide
49Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Tyr Val 35 40 45Ser Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro
Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Gly Ser Leu Arg Ala Glu Asp
Met Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp
Gly Asp Arg Ile Glu 100 105 11050125PRTMus musculus 50Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly1 5 10 15Ser Leu
Lys Leu Phe Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala
Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val 35 40
45Ala Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val
50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80Leu Glu Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg
Ile Glu Val Met 100 105 110Asp Tyr Trp Gly Gln Gly Thr Ala Val Ile
Val Ser Ser 115 120 12551112PRTMus musculus 51Asp Val Val Met Thr
Gln Ser Pro Leu Ser Leu Ser Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser 20 25 30Lys Gly Asn
Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys
Phe Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Ala Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser
85 90 95Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Thr Leu Glu Ile
Lys 100 105 11052125PRTHomo sapiens 52Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys
Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Asp Pro Met Thr Thr Val Val Lys Pro Ser Leu Ala Thr Asn
100 105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 12553112PRTHomo sapiens 53Asp Val Val Met Thr Gln Ser Pro Leu
Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr
Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln
Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11054125PRTMus musculus 54Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Arg Pro Gly Gly1 5 10 15Ser Leu Lys Leu Phe Cys Ala Ala Ser
Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ser
Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Glu Ile Ser Ser Gly Gly
Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Glu Met Ser
Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Val
Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu Val Met 100 105 110Asp
Tyr Trp Gly Gln Gly Thr Ala Val Ile Val Ser Ser 115 120
12555125PRTHomo sapiens 55Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Trp Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Asn Ile Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Pro Met Thr Thr Val Val Lys Pro Ser Leu Ala Thr Asn 100 105 110Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
12556112PRTMus musculus 56Asp Val Val Met Thr Gln Ser Pro Leu Ser
Leu Ser Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Val Ser Ser 20 25 30Lys Gly Asn Thr Tyr Leu His Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Lys Phe Leu Ile Tyr Lys
Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Ala
Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Phe
Pro Arg Thr Phe Gly Gly Gly Thr Thr Leu Glu Ile Lys 100 105
11057112PRTHomo sapiens 57Asp Val Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1
5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 11058125PRTHomo sapiens 58Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Pro Met Thr Thr Val Val Lys
Pro Ser Leu Ala Thr Asn 100 105 110Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 12559125PRTArtificial
SequenceSynthetically generated peptide 59Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp Gly Asp Arg Ile Glu Val
Met 100 105 110Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 12560112PRTHomo sapiens 60Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu
Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile
Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu
Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11061111PRTArtificial SequenceSynthetically generated peptide 61Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Phe Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Cys Ser Gln Ser Thr 85 90 95His Phe Pro Arg Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 11062112PRTHomo sapiens 62Asp Val Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30Asn
Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Met Gln Ala 85 90 95Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 11063112PRTArtificial SequenceSynthetically
generated peptide 63Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Val Ser Ser 20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu
Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
11064454PRTArtificial SequenceSynthetically generated peptide 64Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Glu Ile Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp
Thr Val 50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Leu Tyr Tyr Asp Tyr Asp Gly
Asp Arg Ile Glu Val Met 100 105 110Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr 115 120 125Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu145 150 155 160Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170
175Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys 195 200 205Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu 210 215 220Pro Lys Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro225 230 235 240Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295
300Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp305 310 315 320Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 325 330 335Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 340 345 350Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys385 390 395 400Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410
415Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser 435 440 445Leu Ser Leu Ser Pro Gly 4506598PRTArtificial
SequenceSynthetically generated peptide 65Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Glu Ile
Ser Ser Gly Gly Ser Tyr Pro Tyr Tyr Pro Asp Thr Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg 66219PRTArtificial SequenceSynthetically generated
peptide 66Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Val Ser Ser 20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45Pro Gln Phe Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150
155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 165 170 175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu 180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly
Glu Cys 210 21567100PRTArtificial SequenceSynthetically generated
peptide 67Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Val Ser Ser 20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys
Pro Gly Gln Ser 35 40 45Pro Gln Phe Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95Thr His Phe Pro
10068219PRTArtificial SequenceSynthetically generated peptide 68Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ser Gln Ser 85 90 95Thr His Phe Pro Arg Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 110Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150 155 160Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170
175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 195 200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
21569100PRTArtificial SequenceSynthetically generated peptide 69Asp
Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10
15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Ser Ser
20 25 30Lys Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Phe Cys Ser Gln Ser 85 90 95Thr His Phe Pro 100
* * * * *