U.S. patent application number 17/509351 was filed with the patent office on 2022-03-31 for anti-neuropilin antigen-binding proteins and methods of use thereof.
The applicant listed for this patent is Potenza Therapeutics, Inc.. Invention is credited to Heather Brodkin, Daniel Hicklin, Nels P. Nielson, Jose-Andres Salmeron-Garcia, Cynthia Seidel-Dugan, William Winston.
Application Number | 20220098312 17/509351 |
Document ID | / |
Family ID | 1000006025804 |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220098312 |
Kind Code |
A1 |
Hicklin; Daniel ; et
al. |
March 31, 2022 |
ANTI-NEUROPILIN ANTIGEN-BINDING PROTEINS AND METHODS OF USE
THEREOF
Abstract
Provided herein are antigen-binding proteins (ABPs) that
selectively bind to NRP-1 and its isoforms and homologs, and
compositions comprising the ABPs. Also provided are methods of
using the ABPs, such as therapeutic and diagnostic methods.
Inventors: |
Hicklin; Daniel; (Montclair,
NJ) ; Seidel-Dugan; Cynthia; (Belmont, MA) ;
Winston; William; (Marlborough, MA) ;
Salmeron-Garcia; Jose-Andres; (Westminster, MA) ;
Nielson; Nels P.; (Lebanon, NH) ; Brodkin;
Heather; (West Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Potenza Therapeutics, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
1000006025804 |
Appl. No.: |
17/509351 |
Filed: |
October 25, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16239234 |
Jan 3, 2019 |
11186644 |
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17509351 |
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15900158 |
Feb 20, 2018 |
10227413 |
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16239234 |
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PCT/US2017/067782 |
Dec 21, 2017 |
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15900158 |
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62438733 |
Dec 23, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/565 20130101;
A61K 2039/505 20130101; C07K 2317/92 20130101; C07K 2317/52
20130101; C07K 16/2818 20130101; C07K 2317/524 20130101; A61P 35/00
20180101; Y02A 50/30 20180101; C07K 2317/21 20130101; C07K 2317/55
20130101; C07K 16/2863 20130101; C07K 2317/34 20130101; C07K
16/2827 20130101; A61K 2039/507 20130101; C07K 2317/71 20130101;
C07K 2317/526 20130101; C07K 2317/622 20130101; C07K 2317/30
20130101; C07K 2317/76 20130101; C07K 2317/31 20130101; C07K
2317/33 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Claims
1-21. (canceled)
22. A method of preventing or treating cancer in a subject in need
thereof, the method comprising administering to the subject an
effective amount of an antigen binding protein (ABP) that
specifically binds human NRP-1 (hNRP-1; SEQ ID NO:130), wherein the
ABP comprises the following six CDR sequences: (a) a CDR-H3 having
the sequence set forth in SEQ ID NO:47; (b) a CDR-H2 having the
sequence X.sub.1ISGSGGX.sub.2TYYADSVX.sub.3G, wherein X.sub.1 is I
or A, X.sub.2 is S or A, and X.sub.3 is K or E, as set forth in SEQ
ID NO:136; (c) a CDR-H1 having the sequence FTFX.sub.1SX.sub.2AMV,
wherein X.sub.1 is A, K, or S and X.sub.2 is Y or V, as set forth
in SEQ ID NO:137; (d) a CDR-L3 having the sequence set forth in SEQ
ID NO:81; (e) a CDR-L2 having the sequence set forth in SEQ ID
NO:71; and (f) a CDR-L1 having the sequence set forth in SEQ ID
NO:63, thereby preventing or treating cancer in the subject.
23. The method of claim 22, wherein the ABP comprises: (a) a CDR-H3
of SEQ ID NO:47, a CDR-H2 of SEQ ID NO:27, a CDR-H1 of SEQ ID
NO:12, a CDR-L3 of SEQ ID NO:81, a CDR-L2 of SEQ ID NO:71, and a
CDR-L1 of SEQ ID NO:63; (b) a CDR-H3 of SEQ ID NO:47, a CDR-H2 of
SEQ ID NO:28, a CDR-H1 of SEQ ID NO:13, a CDR-L3 of SEQ ID NO:81, a
CDR-L2 of SEQ ID NO:71, and a CDR-L1 of SEQ ID NO:63; (c) a CDR-H3
of SEQ ID NO:47, a CDR-H2 of SEQ ID NO:29, a CDR-H1 of SEQ ID
NO:14, a CDR-L3 of SEQ ID NO:81, a CDR-L2 of SEQ ID NO:71, and a
CDR-L1 of SEQ ID NO:63; or (d) a CDR-H3 of SEQ ID NO:47, a CDR-H2
of SEQ ID NO:30, a CDR-H1 of SEQ ID NO:14, a CDR-L3 of SEQ ID
NO:81, a CDR-L2 of SEQ ID NO:71, and a CDR-L1 of SEQ ID NO:63.
24. The method of claim 23, wherein: (a) the ABP of claim 2(a)
comprises a VH sequence of SEQ ID NO:92 and a VL sequence of SEQ ID
NO:104; (b) the ABP of claim 2(b) comprises a VH sequence of SEQ ID
NO:93 and a VL sequence of SEQ ID NO:104; (c) the ABP of claim 2(c)
comprises a VH sequence of SEQ ID NO:94 and a VL sequence of SEQ ID
NO:104; (d) the ABP of claim 2(d) comprises a VH sequence of SEQ ID
NO:95 and a VL sequence of SEQ ID NO:104; or (e) the ABP of claim
2(d) comprises a VH sequence of SEQ ID NO:96 and a VL sequence of
SEQ ID NO:104.
25. The method of claim 24, wherein: (a) the ABP of claim 2 (a)
comprises a heavy chain of SEQ ID NO:114 and a light chain of SEQ
ID NO:126; (b) the ABP of claim 2(b) comprises a heavy chain of SEQ
ID NO:115 and a light chain of SEQ ID NO:126; (c) the ABP of claim
2(c) comprises a heavy chain of SEQ ID NO:116 and a light chain of
SEQ ID NO:126; (d) the ABP of claim 2(d) comprises a heavy chain of
SEQ ID NO:117 and a light chain of SEQ ID NO:126; or (e) the ABP of
claim 2(d) comprises a heavy chain of SEQ ID NO:118 and a light
chain of SEQ ID NO:126.
26. The method of claim 22, wherein the ABP specifically
antagonizes hNRP-1 binding to a neuropilin-1 ligand.
27. The method of claim 22, wherein the ABP specifically binds one
or more residues on hNRP-1 (SEQ ID NO:130) selected from the group
consisting of Y297, T316, D320, E348, T349, K350, K351, K352, Y353,
Y354, E412, T413, G414, and 1415.
28. The method of claim 22, wherein the ABP specifically binds to
NRP-1 from humans, mice, and cynomolgus monkeys.
29. The method of claim 22, wherein the ABP binds to a different
epitope on hNRP-1 than the epitope on hNRP-1 to which SEC10 binds,
wherein the SEC10 comprises a heavy chain of SEQ ID NO: 141 and a
light chain of SEQ ID NO: 142.
30. The method of claim 22, wherein the ABP specifically binds to
the b1 domain of hNRP-1.
31. The method of claim 22, wherein the ABP antagonizes the
interaction between a hNRP-1 polypeptide and one or both of a
vascular endothelial cell growth factor (VEGF) polypeptide and a
semaphorin (SEMA) polypeptide.
32. The method of claim 22, wherein the method induces or enhances
an immune response to a cancer-associated antigen.
33. The method of claim 22, wherein the ABP is capable of
decreasing Treg survival in the subject, binding to an
extracellular domain of the NRP, or a combination thereof.
34. The method of claim 22, further comprising administering one or
more additional therapeutic agents to the subject.
35. The method of claim 34, wherein the additional therapeutic
agent is selected from the group consisting of radiation, a
cytotoxic agent, a chemotherapeutic agent, a cytostatic agent, an
anti-hormonal agent, a VEGF inhibitor, an immunostimulatory agent,
an anti-angiogenic agent, and combinations thereof.
36. The method of claim 35, wherein the additional therapeutic
agent is an immunostimulatory agent.
37. The method of claim 36, wherein the immunostimulatory agent
comprises: (i) an agent that blocks signaling of an inhibitory
receptor expressed by an immune cell or a ligand thereof; (ii) an
agonist to a stimulatory receptor expressed by an immune cell;
(iii) a cytokine; or (iv) a vaccine to a cancer-associated
antigen.
38. The method of claim 36, wherein the immunostimulatory agent is
an anti-PD-1 antibody.
39. The method of claim 38, wherein the anti-PD-1 antibody is
pembrolizumab, nivolumab, or a combination thereof.
40. A method of modulating an immune response in a subject in need
thereof, the method comprising administering to the subject an
effective amount of an antigen binding protein (ABP) that
specifically binds human NRP-1 (hNRP-1; SEQ ID NO:130), wherein the
ABP comprises the following six CDR sequences: (a) a CDR-H3 having
the sequence set forth in SEQ ID NO:47; (b) a CDR-H2 having the
sequence X.sub.1ISGSGGX.sub.2TYYADSVX.sub.3G, wherein X.sub.1 is I
or A, X.sub.2 is S or A, and X.sub.3 is K or E, as set forth in SEQ
ID NO:136; (c) a CDR-H1 having the sequence FTFX.sub.1SX.sub.2AMV,
wherein X.sub.1 is A, K, or S and X.sub.2 is Y or V, as set forth
in SEQ ID NO:137; (d) a CDR-L3 having the sequence set forth in SEQ
ID NO:81; (e) a CDR-L2 having the sequence set forth in SEQ ID
NO:71; and (f) a CDR-L1 having the sequence set forth in SEQ ID
NO:63, thereby modulating an immune response in the subject.
41. A method of increasing T effector cell (Teff) function in a
subject in need thereof, the method comprising administering to the
subject an effective amount of an antigen binding protein (ABP)
that specifically binds human NRP-1 (hNRP-1; SEQ ID NO:130),
wherein the ABP comprises the following six CDR sequences: (a) a
CDR-H3 having the sequence set forth in SEQ ID NO:47; (b) a CDR-H2
having the sequence X.sub.1ISGSGGX.sub.2TYYADSVX.sub.3G, wherein
X.sub.1 is I or A, X.sub.2 is S or A, and X.sub.3 is K or E, as set
forth in SEQ ID NO:136; (c) a CDR-H1 having the sequence
FTFX.sub.1SX.sub.2AMV, wherein X.sub.1 is A, K, or S and X.sub.2 is
Y or V, as set forth in SEQ ID NO:137; (d) a CDR-L3 having the
sequence set forth in SEQ ID NO:81; (e) a CDR-L2 having the
sequence set forth in SEQ ID NO:71; and (f) a CDR-L1 having the
sequence set forth in SEQ ID NO:63, thereby increasing T effector
cell (Teff) function in the subject.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 16/239,234, filed on Jan. 3, 2019;
which is a continuation application of U.S. patent application Ser.
No. 15/900,158, filed on Feb. 20, 2018, issued as U.S. Pat. No.
10,227,413 on Mar. 12, 2019; which claims the benefit of PCT
International Application Serial No. PCT/US2017/067782, filed on
Dec. 21, 2017; which claims the benefit of U.S. Provisional
Application Ser. No. 62/438,733, filed on Dec. 23, 2016. The entire
contents of each of the foregoing applications are expressly
incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 3, 2019, is named 127206-03604_sequencelisting.txt and is
192,455 bytes in size.
FIELD
[0003] Provided herein are antigen-binding proteins (ABPs) with
binding specificity for NRP-1 and compositions comprising such
ABPs, including pharmaceutical compositions, diagnostic
compositions, and kits. Also provided are methods of making NRP-1
ABPs, and methods of using NRP-1 ABPs, for example, for therapeutic
purposes, diagnostic purposes, and research purposes.
BACKGROUND
[0004] Multiple studies have demonstrated that tumors are able to
establish an immunosuppressive microenvironment to escape immune
surveillance and promote tumor development. Regulatory T cells
(Tregs) are an important component of the immunosuppressive milieu
in the tumor environment and work by dampening T cell immunity to
tumor associated antigens. Tregs are therefore a major obstacle in
mounting an effective anti-tumor immune response. Depletion of
Tregs in murine models of cancer inhibits tumor growth; however,
the accompanying autoimmune and inflammatory disorders associated
with a complete depletion of Tregs may limit the clinical utility
of this approach. Strategies which specifically target Tregs, in
the inflammatory tumor microenvironment, may be a viable
alternative. Recent studies in several laboratories have identified
Neuropilin 1 (NRP-1) as a candidate target for modulating Treg
activity in tumors without impacting Tregs in the periphery (see,
e.g., Chaudhary and Elkord, Vaccines (2016) September; 4(3): 28;
Bos et al., J Exp Med (2013) 210 (11):2435-66; Teng et al., Cancer
Res. (2010) 70 (20):7800.
[0005] NRP-1 is a multifunctional 130-kDa transmembrane protein
with a large extracellular domain containing two N-terminal CUB
domains (a1 and a2), two coagulation factor V/VIII homology domains
(b1 and b2) and a single MAM domain (c). The cytoplasmic tail is
short and does not display any catalytic activity on its own. NRP-1
is a receptor with multiple known ligands and co-receptors,
including semaphorins, VEGF, P1GF and plexins, among others
(Appleton et al., Embo J. (2007) Nov. 28; 26(23): 4902-4912).
[0006] NRP-1 is expressed on human and murine Tregs, and this
expression identifies a highly-suppressive Treg subset. Within the
tumor microenvironment, NRP-1 expression is required for Treg
stability and function but does not impact Tregs outside the
inflammatory environment of tumors. Recent studies have identified
the immune cell-expressed ligand semaphorin 4A (Sema4a) as an
additional ligand for NRP-1, and demonstrated that the sema4a/NRP-1
interaction is an important mediator of Treg stability in vitro and
in inflammatory sites in vivo. These data suggest that NRP-1 is
required for Treg lineage stability and function (see, e.g.,
Delgoffe et al., Nature (2013) Sep. 12; 501(7466):252-6.).
[0007] Several lines of evidence support the utility of targeting
the interaction of NRP-1 and its associated proteins, in particular
targeting the NRP-1/Sema axis, on Tregs as a strategy for
modulating the immunosuppressive microenvironment found in tumors.
For instance, mice with Treg targeted NRP-1 knock-out exhibit
reduced tumor growth in several murine tumor models, without any
other autoimmune phenotypes. Additionally, antagonists to NRP-1 or
Sema reverse Treg suppressive activity and demonstrate anti-tumor
efficacy again in the absence of autoimmune adverse events.
Furthermore, the NRP-1-VEGFA axis has been proposed as an important
pathway regulating the chemotaxis of Tregs into the tumor
micro-environment, and an antagonistic Ab that blocks this
interaction on Tregs could inhibit the influx of these suppressive
cells into the tumor.
[0008] There is emerging evidence suggesting the NRP-1 is expressed
on the surface of immune cells in human tumors. NRP-1+ Tregs are
found in the draining lymph nodes (DLN) from cervical cancer
patents, and there was a significant drop in the percentage of
Tregs in DLN in patients with a pathological response to
preoperative chemoradiation. In addition, NRP-1+ Tregs have been
observed in tumor infiltrating lymphocytes (TILs) in patients with
melanoma and head and neck squamous cell carcinoma.
[0009] Thus, there is a need for therapeutics that can antagonize
NRP-1 without inducing autoimmune disease. Provided herein are ABPs
that fulfill this need.
SUMMARY
[0010] Provided herein are ABPs that specifically bind NRP-1 and
methods of using such ABPs.
[0011] In one aspect, provided herein are is an isolated
multivalent antigen binding protein (ABP) that specifically binds
human NRP-1 (hNRP-1; SEQ ID NO:130), wherein the ABP comprises the
following six CDR sequences: [0012] (a) a CDR-H3 having the
sequence set forth in SEQ ID NO:47; [0013] (b) a CDR-H2 having the
sequence X.sub.1ISGSGGX.sub.2TYYADSVX.sub.3G, wherein X.sub.1 is I
or A, X.sub.2 is S or A, and X.sub.3 is K or E, as set forth in SEQ
ID NO:136; [0014] (c) a CDR-H1 having the sequence
FTFX.sub.1SX.sub.2AMV, wherein X.sub.1 is A, K, or S and X.sub.2 is
Y or V, as set forth in SEQ ID NO:137; [0015] (d) a CDR-L3 having
the sequence set forth in SEQ ID NO:81; [0016] (e) a CDR-L2 having
the sequence set forth in SEQ ID NO:71; and [0017] (f) a CDR-L1
having the sequence set forth in SEQ ID NO:63.
[0018] In one embodiment, the ABP comprises a CDR-H3 of SEQ ID
NO:47, a CDR-H2 of SEQ ID NO:27, a CDR-H1 of SEQ ID NO:12, a CDR-L3
of SEQ ID NO:81, a CDR-L2 of SEQ ID NO:71, and a CDR-L1 of SEQ ID
NO:63; or a CDR-H3 of SEQ ID NO:47, a CDR-H2 of SEQ ID NO:28, a
CDR-H1 of SEQ ID NO:13, a CDR-L3 of SEQ ID NO:81, a CDR-L2 of SEQ
ID NO:71, and a CDR-L1 of SEQ ID NO:63; or a CDR-H3 of SEQ ID
NO:47, a CDR-H2 of SEQ ID NO:29, a CDR-H1 of SEQ ID NO:14, a CDR-L3
of SEQ ID NO:81, a CDR-L2 of SEQ ID NO:71, and a CDR-L1 of SEQ ID
NO:63; or a CDR-H3 of SEQ ID NO:47, a CDR-H2 of SEQ ID NO:30, a
CDR-H1 of SEQ ID NO:14, a CDR-L3 of SEQ ID NO:81, a CDR-L2 of SEQ
ID NO:71, and a CDR-L1 of SEQ ID NO:63.
[0019] In another embodiment, the ABP comprises a V.sub.H sequence
of SEQ ID NO:92 and a V.sub.L sequence of SEQ ID NO:104; a V.sub.H
sequence of SEQ ID NO:93 and a V.sub.L sequence of SEQ ID NO:104; a
V.sub.H sequence of SEQ ID NO:94 and a V.sub.L sequence of SEQ ID
NO:104; a V.sub.H sequence of SEQ ID NO:95 and a V.sub.L sequence
of SEQ ID NO:104; or a V.sub.H sequence of SEQ ID NO:96 and a
V.sub.L sequence of SEQ ID NO:104.
[0020] In another embodiment, the ABP comprises a heavy chain of
SEQ ID NO:114 and a light chain of SEQ ID NO:126; a heavy chain of
SEQ ID NO:115 and a light chain of SEQ ID NO:126; a heavy chain of
SEQ ID NO:116 and a light chain of SEQ ID NO:126; a heavy chain of
SEQ ID NO:117 and a light chain of SEQ ID NO:126; or a heavy chain
of SEQ ID NO:118 and a light chain of SEQ ID NO:126.
[0021] In another aspect is provided an isolated multivalent
antigen binding protein (ABP) that specifically binds human NRP-1
(hNRP-1; SEQ ID NO:130), wherein the ABP comprises the following
six CDR sequences: [0022] (a) a CDR-H3 having the sequence set
forth in SEQ ID NO:41; [0023] (b) a CDR-H2 having the sequence set
forth in SEQ ID NO:23; [0024] (c) a CDR-H1 having the sequence set
forth in SEQ ID NO:8; [0025] (d) a CDR-L3 having the sequence set
forth in SEQ ID NO:77; [0026] (e) a CDR-L2 having the sequence set
forth in SEQ ID NO:67, and [0027] (f) a CDR-L1 having the sequence
set forth in SEQ ID NO:59.
[0028] In one embodiment, the ABP comprises a V.sub.H sequence of
SEQ ID NO:85 and a V.sub.L sequence of SEQ ID NO:100; or a V.sub.H
sequence of SEQ ID NO:86 and a V.sub.L sequence of SEQ ID NO:100.
In another embodiment, the ABP comprises a heavy chain of SEQ ID
NO:107 and a kappa light chain of SEQ ID NO:122; and a heavy chain
of SEQ ID NO:108 and a kappa light chain of SEQ ID NO:122.
[0029] In another aspect is provided an isolated multivalent
antigen binding protein (ABP) that specifically binds human NRP-1
(hNRP-1; SEQ ID NO:130), wherein the ABP comprises the following
six CDR sequences: [0030] (a) a CDR-H3 having the sequence
ARDLGYYGSGMHX, wherein X is A or V, as set forth in SEQ ID NO:138;
[0031] (a) a CDR-H2 having the sequence set forth in SEQ ID NO:24;
[0032] (b) a CDR-H1 having the sequence set forth in SEQ ID NO:9;
[0033] (c) a CDR-L3 having the sequence set forth in SEQ ID NO:78;
[0034] (d) a CDR-L2 having the sequence set forth in SEQ ID NO:68;
and [0035] (e) a CDR-L1 having the sequence set forth in SEQ ID
NO:60.
[0036] In one embodiment, the ABP comprises: a CDR-H3 of SEQ ID
NO:42, a CDR-H2 of SEQ ID NO:24, a CDR-H1 of SEQ ID NO:9, a CDR-L3
of SEQ ID NO:78, a CDR-L2 of SEQ ID NO:68, and a CDR-L1 of SEQ ID
NO:60; or a CDR-H3 of SEQ ID NO:43, a CDR-H2 of SEQ ID NO:24, a
CDR-H1 of SEQ ID NO:9, a CDR-L3 of SEQ ID NO:78, a CDR-L2 of SEQ ID
NO:68, and a CDR-L1 of SEQ ID NO:60. In another embodiment, the ABP
comprises a V.sub.H sequence of SEQ ID NO:87 and a V.sub.L sequence
of SEQ ID NO:101; or the ABP comprises a V.sub.H sequence of SEQ ID
NO:88 and a V.sub.L sequence of SEQ ID NO:101. In another
embodiment, the ABP comprises a heavy chain of SEQ ID NO:109 and a
kappa light chain of SEQ ID NO:123; or the ABP comprises a heavy
chain of SEQ ID NO:110 and a kappa light chain of SEQ ID
NO:123.
[0037] In another aspect is provided an isolated multivalent
antigen binding protein (ABP) that specifically binds human NRP-1
(hNRP-1; SEQ ID NO:130), wherein the ABP comprises the following
six CDR sequences: [0038] (a) a CDR-H3 having the sequence,
ARDRGMYYASGFXP, wherein X is G or N, as set forth in (SEQ ID
NO:139); [0039] (b) a CDR-H2 having the sequence set forth in SEQ
ID NO:25; [0040] (c) a CDR-H1 having the sequence set forth in SEQ
ID NO:10; [0041] (d) a CDR-L3 having the sequence set forth in SEQ
ID NO:79; [0042] (e) a CDR-L2 having the sequence set forth in SEQ
ID NO:69; and [0043] (f) a CDR-L1 having the sequence set forth in
SEQ ID NO:61.
[0044] In one embodiment the ABP comprises a CDR-H3 of SEQ ID
NO:44, a CDR-H2 of SEQ ID NO:25, a CDR-H1 of SEQ ID NO:10, a CDR-L3
of SEQ ID NO:79, a CDR-L2 of SEQ ID NO:69, and a CDR-L1 of SEQ ID
NO:61; or a CDR-H3 of SEQ ID NO:45, a CDR-H2 of SEQ ID NO:25, a
CDR-H1 of SEQ ID NO:10, a CDR-L3 of SEQ ID NO:79, a CDR-L2 of SEQ
ID NO:69, and a CDR-L1 of SEQ ID NO:61. In another embodiment, the
ABP comprises a V.sub.H sequence of SEQ ID NO:89 and a V.sub.L
sequence of SEQ ID NO:102; or a V.sub.H sequence of SEQ ID NO:90
and a V.sub.L sequence of SEQ ID NO:102. In another embodiment, the
ABP comprises a heavy chain of SEQ ID NO:111 and a kappa light
chain of SEQ ID NO:124; or the ABP comprises a heavy chain of SEQ
ID NO:112 and a kappa light chain of SEQ ID NO:124.
[0045] In another aspect is provided an isolated multivalent
antigen binding protein (ABP) that specifically binds human NRP-1
(hNRP-1; SEQ ID NO:130), comprising the following six CDR
sequences: [0046] (a) a CDR-H3 having the sequence set forth in SEQ
ID NO:46; [0047] (b) a CDR-H2 having the sequence set forth in SEQ
ID NO:26; [0048] (c) a CDR-H1 having the sequence set forth in SEQ
ID NO:11; [0049] (d) a CDR-L3 having the sequence set forth in SEQ
ID NO:80; [0050] (e) a CDR-L2 having the sequence set forth in SEQ
ID NO:70; and [0051] (f) a CDR-L1 having the sequence set forth in
SEQ ID NO:62.
[0052] In one embodiment, the ABP comprises a V.sub.H sequence of
SEQ ID NO:91 and a V.sub.L sequence of SEQ ID NO:103. In another
embodiment, the ABP comprises a heavy chain of SEQ ID NO:113 and a
kappa light chain of SEQ ID NO:125.
[0053] In another aspect is provided an isolated multivalent
antigen binding protein (ABP) that specifically binds human NRP-1
(hNRP-1; SEQ ID NO:130), comprising the following six CDR
sequences: [0054] (a) a CDR-H3 having the sequence set forth in SEQ
ID NO:48; [0055] (b) a CDR-H2 having the sequence set forth in SEQ
ID NO:31; [0056] (c) a CDR-H1 having the sequence set forth in SEQ
ID NO:15; [0057] (d) a CDR-L3 having the sequence set forth in SEQ
ID NO:82; [0058] (e) a CDR-L2 having the sequence set forth in SEQ
ID NO:68; and [0059] (f) a CDR-L1 having the sequence set forth in
SEQ ID NO:64.
[0060] In one embodiment, the ABP comprises a V.sub.H sequence of
SEQ ID NO:97 and a V.sub.L sequence of SEQ ID NO:105, or a V.sub.H
sequence of SEQ ID NO:98 and a V.sub.L sequence of SEQ ID NO:105.
In another embodiment, the ABP comprises: a heavy chain of SEQ ID
NO:119 and a kappa light chain of SEQ ID NO:127; or a heavy chain
of SEQ ID NO:120 and a kappa light chain of SEQ ID NO:127.
[0061] In another aspect is provided an isolated multivalent
antigen binding protein (ABP) that specifically binds human NRP-1
(hNRP-1; SEQ ID NO:130), comprising the following six CDR
sequences: [0062] (a) a CDR-H3 having the sequence set forth in SEQ
ID NO:49; [0063] (b) a CDR-H2 having the sequence set forth in SEQ
ID NO:32; [0064] (c) a CDR-H1 having the sequence set forth in SEQ
ID NO:16; [0065] (d) a CDR-L3 having the sequence set forth in SEQ
ID NO:83; [0066] (e) a CDR-L2 having the sequence set forth in SEQ
ID NO:72; and [0067] (f) a CDR-L1 having the sequence set forth in
SEQ ID NO:65.
[0068] In one embodiment, the ABP comprises a V.sub.H sequence of
SEQ ID NO:99 and a V.sub.L sequence of SEQ ID NO:106. In another
embodiment, the ABP comprises a heavy chain of SEQ ID NO:121 and a
kappa light chain of SEQ ID NO:128.
[0069] In another aspect is provided an isolated antigen binding
protein (ABP) that specifically binds human NRP-1 (hNRP-1; SEQ ID
NO:130), comprising a CDR-H3 having at least about 80% identity to
a CDR-H3 of a V.sub.H region selected from SEQ ID NOs:41-49; a
CDR-H2 having at least about 80% identity to a CDR-H2 of a V.sub.H
region selected from SEQ ID NOs:23-32; a CDR-H1 having at least
about 80% identity to a CDR-H1 of a V.sub.H region selected from
SEQ ID NOs:8-16; a CDR-L3 having at least about 80% identity to a
CDR-L3 of a V.sub.L region selected from SEQ ID NOs:77-83; a CDR-L2
having at least about 80% identity to a CDR-L2 of a V.sub.L region
selected from SEQ ID NOs:67-72; and a CDR-L1 having at least about
80% identity to a CDR-L1 of a V.sub.L region selected from SEQ ID
NOs:59-65. In one embodiment, the CDR-H3, CDR-H2, CDR-H1, CDR-L3,
CDR-L2, and CDR-L1 are each identified according to a numbering
scheme selected from the Kabat numbering scheme, the Chothia
numbering scheme, or the IMGT numbering scheme. In another
embodiment, the CDR-H1 is identified as defined by both the Chothia
and Kabat numbering schemes, inclusive of the boundaries of both
numbering schemes. In one embodiment, the CDR-H3 comprises a CDR-H3
selected from SEQ ID NOs:41-49, or a variant thereof having 1, 2,
or 3 amino acid substitutions; the CDR-H2 comprises a CDR-H3
selected from SEQ ID NOs:23-32, or a variant thereof having 1, 2,
or 3 amino acid substitutions; the CDR-H1 comprises a CDR-H1
selected from SEQ ID NOs:8-16, or a variant thereof having 1 or 2
amino acid substitutions; the CDR-L3 comprises a CDR-L3 selected
from SEQ ID NOs:77-83, or a variant thereof having 1 or 2 amino
acid substitutions; the CDR-L2 comprises a CDR-L2 selected from SEQ
ID NOs:67-72, or a variant thereof having 1 amino acid
substitution; and the CDR-L1 comprises a CDR-L1 selected from SEQ
ID NOs:59-65, or a variant thereof having 1 or 2 amino acid
substitutions. In one embodiment, the amino acid substitutions are
conservative amino acid substitutions.
[0070] In another aspect is provided an ABP that specifically binds
human NRP-1, wherein the ABP: [0071] (a) competes or cross-competes
for binding to NRP-1 with an antibody selected from MAB1, MAB2,
MAB3, MAB4, MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, MAB12,
MAB13, MAB14, or MAB15, each as provided in Appendix A of this
disclosure; [0072] (b) is specific for cell surface NRP-1; [0073]
(c) specifically blocks NRP-1 binding to a transmembrane semaphorin
polypeptide; [0074] (d) blocks the interaction between a NRP-1
polypeptide and a vascular endothelial cell growth factor (VEGF)
polypeptide; [0075] (e) is capable of inhibiting Treg suppression
in a human subject; [0076] (f) co-stimulates an effector T cell in
combination with antigen presentation from an antigen-presenting
cell; [0077] (g) inhibits the suppression of an effector T cell by
a regulatory T cell; [0078] (h) reduces the number of effector T
cells in a tissue or in systemic circulation; [0079] (i) does not
substantially bind platelets; [0080] (j) does not substantially
cause thrombocytopenia when administered to a patient; [0081] (k)
blocks SEMA3 binding to NRP-1; [0082] (l) does not bind to
NRP-1-negative cells; or [0083] (m) is capable of any combination
of (a)-(l).
[0084] In one embodiment, the ABP antibody does not compete or
cross-compete for binding with an antibody selected from MAB1,
MAB2, MAB3, MAB4, MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11,
MAB12, MAB13, MAB14, or MAB15, each as provided in Appendix A of
this disclosure. In one embodiment, the ABP is an ABP selected from
MAB1, MAB2, MAB3, MAB4, MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11,
MAB12, MAB13, MAB14, or MAB15, each as provided in Appendix A of
this disclosure. In one embodiment, the NRP-1 is selected from
hNRP-1 (SEQ ID NO:130), cNRP-1 (SEQ ID NO:132), mNRP-1 (SEQ ID
NO:134), rNRP-1 (SEQ ID NO:135), and combinations thereof.
[0085] In one embodiment, the ABP comprises an antibody. In one
embodiment, the antibody is a monoclonal antibody. In another
embodiment, the antibody is selected from a human antibody, a
humanized antibody or a chimeric antibody. In one embodiment, the
ABP is multivalent. In another embodiment, the ABP comprises an
antibody fragment. In another embodiment, the ABP comprises an
alternative scaffold. In another embodiment, the ABP comprises an
immunoglobulin constant region. In another embodiment, the ABP
comprises heavy chain constant region of a class selected from IgA,
IgD, IgE, IgG, or IgM. In another embodiment, ABP comprises a heavy
chain constant region of the class IgG and a subclass selected from
IgG4, IgG1, IgG2, or IgG3. In another embodiment, the IgG is an
IgG4. In another embodiment, the IgG is an IgG1.
[0086] In one embodiment, the ABP comprises a common light chain
antibody, an antibody with a knobs-into-holes modification, an scFv
attached to an IgG, a Fab attached to an IgG, a diabody, a
tetravalent bispecific antibody, a DVD-IgMAB, a DARTT M, a
DuoBody.RTM., a CovX-Body, an Fcab antibody, a TandAb.RTM., a
tandem Fab, a Zybody.sup.MAB, or combinations thereof.
[0087] In one embodiment, the ABP blocks binding of semaphorin 3A
(SEMA3A) to NRP-1 by at least about 10%, at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, or at least
about 90%. In on embodiment, the ABP reduces binding of semaphorin
3A to NRP-1 by at least about 50%. In one embodiment, the tissue is
a tumor. In another embodiment, the NRP-1 is expressed on the
surface of a target cell.
[0088] In one embodiment, the ABP comprises a polypeptide sequence
having a pyroglutamate (pE) residue at its N-terminus. In another
embodiment, the ABP comprises a V.sub.H sequence in which an
N-terminal Q is substituted with pE. In another embodiment, the ABP
comprises a V.sub.H sequence in which an N-terminal E is
substituted with pE. In another embodiment, the ABP comprises a
V.sub.L sequence in which an N-terminal E is substituted with pE.
In another embodiment, the ABP comprises a heavy chain sequence in
which an N-terminal Q is substituted with pE. In another
embodiment, the ABP comprises a heavy chain sequence in which an
N-terminal E is substituted with pE. In another embodiment, the ABP
comprises a light chain sequence in which an N-terminal E is
substituted with pE.
[0089] In one embodiment, the ABP specifically binds to human NRP-1
with a kD of less than 20 nM, less than 10 nM, less than 5 nM, less
than 2 nM, less than 1 nM, less than 0.5 nM, or less than 0.2 nM.
In another embodiment, the ABP specifically binds to NRP-1 from
humans, mice, and cynomolgus monkeys. In one embodiment, the ABP
binds to a different epitope on NRP-1 than the epitope on NRP-1 to
which SEC10 binds. In one embodiment, the ABP binds to the a1, a2,
b1, or b2 domain of NRP-1. In another embodiment, the ABP binds to
more than one domain of NRP-1. In another embodiment, the ABP binds
to the b2 domain of NRP-1. In another embodiment, the ABP binds to
the b1 domain of NRP-1.
[0090] In another aspect is provided any of the ABPs disclosed
herein for use as a medicament. In another embodiment, the ABP is
provided for use in the treatment of a cancer or viral infection.
In one embodiment, the cancer is selected from a solid tumor and a
hematological tumor.
[0091] In another aspect is provided a kit comprising any of the
ABPs disclosed herein, and instructions for use of the ABP. In one
embodiment, the kit comprises a lyophilized ABP. In another
embodiment, the kit comprises a fluid for reconstitution of the
lyophilized ABP.
[0092] In another aspect is provided an isolated polynucleotide
encoding an ABP disclosed herein, a V.sub.H thereof, a V.sub.L
thereof, a light chain thereof, a heavy chain thereof or an
antigen-binding portion thereof.
[0093] In another aspect is provided a vector comprising the
isolated polynucleotide encoding an ABP disclosed herein, a V.sub.H
thereof, a V.sub.L thereof, a light chain thereof, a heavy chain
thereof or an antigen-binding portion thereof.
[0094] In another aspect is provided a host cell comprising any of
the vectors or polynucleotides disclosed herein. In one embodiment,
the host cell is selected from a bacterial cell, a fungal cell, and
a mammalian cell. In another embodiment, the host cell is selected
from an E. coli cell, a Saccharomyces cerevisiae cell, and a CHO
cell.
[0095] In another aspect is provided a cell-free expression
reaction comprising any of the vectors or polynucleotides disclosed
herein.
[0096] In another aspect is provided a method of producing an ABP
as disclosed herein, comprising expressing the ABP in the host cell
disclosed herein and isolating the expressed ABP.
[0097] In another aspect is provided a pharmaceutical composition
comprising any of the ABPs disclosed herein and a pharmaceutically
acceptable excipient. In one embodiment, the ABP is present in the
composition in an amount effective to locally inhibit the
NRP-1:semaphorin-4 interaction in a tumor. In one embodiment, the
anti-NRP-1 antibody is present in the composition in an amount
effective to inhibit an interaction between NRP-1 and a
transmembrane semaphorin polypeptide when administered to a human
subject. In another embodiment, the anti-NRP-1 antibody
specifically blocks NRP-1 binding to a transmembrane semaphorin
polypeptide. In another embodiment, the anti-NRP-1 antibody blocks
the interaction between a NRP-1 polypeptide and a vascular
endothelial cell growth factor (VEGF) polypeptide. In another
embodiment, the anti-NRP-1 antibody blocks binding of a semaphorin
polypeptide. In one embodiment, the anti-NRP1 antibody blocks SEMA3
binding. In another embodiment, the anti-NRP-1 antibody blocks
SEMA4 binding. In another embodiment, the antibody blocks
interaction between a NRP-1 polypeptide and SEMA3. In another
embodiment, the antibody blocks interaction between a NRP-1
polypeptide and VEGF. In one embodiment, the antibody blocks a
semaphorin polypeptide binding but does not block VEGF binding. In
another embodiment, the anti-NRP-1 antibody is capable of
inhibiting Treg suppression in the human subject. In another
embodiment, the anti-NRP-1 antibody is capable of decreasing Treg
survival and/or stability in the human subject. In one embodiment,
the anti-NRP-1 antibody is present in the composition in an amount
effective to locally inhibit the NRP-1:semaphorin-4 interaction in
a tumor. In another embodiment, the anti-NRP-1 antibody is present
in the composition in an amount effective to prevent development of
an undesired autoimmune and/or inflammatory manifestation. In one
embodiment, human subject is suffering from a cancer. In one
embodiment, the amount of the ABP in the pharmaceutical composition
is sufficient to (a) reduce the suppression of effector T cells by
regulatory T cells; (b) activate effector T cells; (c) reduce the
number of regulatory T cells in a tissue or systemically; (d)
induce or enhance proliferation of effector T cells; (e) inhibit
the rate of tumor growth; (f) induce tumor regression; or (g)
combinations thereof, in a subject.
[0098] In one embodiment, the pharmaceutical composition is for use
as a medicament. In one embodiment, the pharmaceutical composition
is for use in the treatment of a cancer or a viral infection. In
one embodiment, the pharmaceutical composition is for use in the
treatment of a cancer, wherein the cancer is selected from brain,
prostate, breast, colon, skin, and lung cancer. In one embodiment,
the pharmaceutical composition comprises a pharmaceutically
acceptable excipient. In one embodiment, the ABP in the
pharmaceutical composition is sufficient to (a) reduce the
suppression of effector T cells by regulatory T cells; (b) activate
effector T cells; (c) reduce the number of regulatory T cells in a
tissue or systemically; (d) induce or enhance proliferation of
effector T cells; (e) inhibit the rate of tumor growth; (f) induce
tumor regression; or (g) combinations thereof, in a subject.
[0099] In another aspect is provided a method of inhibiting a
function or decreasing stability of a regulatory T cell (Treg) in a
subject, comprising exposing the Treg in vivo to an inhibitor of
neuropilin-1 (NRP-1):semaphorin-4A axis in the Treg, wherein an
effective amount of an ABP provided herein or a pharmaceutical
composition provided herein is administered to the subject. In one
embodiment, the method comprises increasing T effector cell
(T.sub.eff) function or exposing the T.sub.eff in vivo to an ABP
provided herein, comprising administering to a subject an effective
amount of a pharmaceutical composition provided herein. In one
embodiment, the subject has a cancer. In one embodiment, the method
induces or enhances an immune response to a cancer-associated
antigen. In one embodiment, the ABP is capable of (a) decreasing
Treg survival and/or stability in the human subject; (b) binding to
an extracellular domain of the NRP-1 polypeptide; or (c) a
combination thereof.
[0100] In one embodiment, the method further comprises
administering one or more additional therapeutic agents. In one
embodiment, the additional therapeutic agent is selected from
radiation, a cytotoxic agent, a chemotherapeutic agent, a
cytostatic agent, an anti-hormonal agent, a VEGF inhibitor, an
immunostimulatory agent, an anti-angiogenic agent, and combinations
thereof. In one embodiment, the additional therapeutic agent is an
immunostimulatory agent. In one embodiment, the immunostimulatory
agent comprises an agent that blocks signaling of an inhibitory
receptor expressed by an immune cell or a ligand thereof. In one
embodiment, the inhibitory receptor expressed by an immune cell or
ligand thereof is selected from PVRIG, VISTA, CCR4, CD27, CTLA-4,
PD-1, PD-L1, LAG-3, Tim3, TIGIT, neuritin, BTLA, KIR, and
combinations thereof. In one embodiment, the immunostimulatory
agent comprises an agonist to a stimulatory receptor expressed by
an immune cell. In one embodiment, the stimulatory receptor
expressed by an immune cell is selected from OX40, GITR, ICOS,
CD28, CD37, CD40, 4-1BB, and combinations thereof. In one
embodiment, the immunostimulatory agent comprises a cytokine. In
another embodiment, the immunostimulatory agent comprises a vaccine
to a cancer-associated antigen.
[0101] In another aspect is provided a method of modulating an
immune response in a subject in need thereof, comprising
administering to the subject an effective amount of an ABP provided
herein. In one embodiment, the method further comprises
administering one or more additional therapeutic agents to the
subject. In one embodiment, the additional therapeutic agent is (i)
an agonist to a stimulatory receptor of an immune cell or (ii) an
antagonist of an inhibitory receptor of an immune cell, wherein the
receptor of an immune cell is selected from OX40, CD2, CD27, CDS,
ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), CD28,
CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, GITR, SLAMF7, NKp80,
CD160, B7-H3, CD83 ligand, and combinations thereof. In another
embodiment, the additional therapeutic agent is an oncolytic virus
selected from herpes simplex virus, vesicular stomatitis virus,
adenovirus, Newcastle disease virus, vaccinia virus, a maraba
virus, and combinations thereof. In one embodiment, the additional
therapeutic agent is formulated in the same pharmaceutical
composition as the ABP. In another embodiment, the additional
therapeutic agent is formulated in a different pharmaceutical
composition from the ABP.
[0102] In one embodiment, the additional therapeutic agent is
administered prior to administering the ABP. In another embodiment,
the additional therapeutic agent is administered after
administering the ABP. In another embodiment, the additional
therapeutic agent is administered contemporaneously with the ABP.
In one embodiment, the method does not substantially cause
thrombocytopenia in the subject.
[0103] In another aspect is provided an anti-human NRP-1 antibody
or an antigen-binding fragment thereof, comprising a heavy chain
variable region comprising a CDR-H3 consisting of SEQ ID NO:47, a
CDR-H2 consisting of SEQ ID NO:30, and a CDR-H1 consisting of SEQ
ID NO:14; and a light chain variable region comprising a CDR-L3
consisting of SEQ ID NO:81, a CDR-L2 consisting of SEQ ID NO:71,
and a CDR-L1 consisting of SEQ ID NO:63. In one embodiment, the
antibody or antigen-binding fragment is selected from any one of
the following (1) and (2): [0104] (1) an anti-human NRP-1 antibody
or an antigen-binding fragment thereof, comprising a heavy chain
variable region consisting of SEQ ID NO:96, and a light chain
variable region consisting of SEQ ID NO:104; and [0105] (2) an
anti-human NRP-1 antibody or the antigen-binding fragment thereof,
comprising a heavy chain variable region consisting of SEQ ID NO:96
in which E of the amino acid number 1 is modified to pyroglutamate,
and a light chain variable region consisting of SEQ ID NO:104.
[0106] In one embodiment is a method for producing an anti-human
NRP-1 antibody or an antigen-binding fragment thereof, comprising
culturing host cell(s) selected from the group consisting of (a) to
(c) below to express a tetravalent anti-human NRP-1 antibody or an
antigen-binding fragment thereof: [0107] (a) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the heavy chain variable region
of the anti-human NRP-1 antibody or the antigen-binding fragment
thereof of the above embodiment (1) and a polynucleotide comprising
a base sequence encoding the light chain variable region of the
antibody or the antigen-binding fragment thereof; [0108] (b) a host
cell transformed with an expression vector comprising a
polynucleotide comprising a base sequence encoding the heavy chain
variable region of the anti-human NRP-1 antibody or the
antigen-binding fragment thereof of the above embodiment (1) and an
expression vector comprising a polynucleotide comprising a base
sequence encoding the light chain variable region of the antibody
or the antigen-binding fragment thereof; and [0109] (c) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the heavy chain variable region
of the anti-human NRP-1 antibody or the antigen-binding fragment
thereof of claim the above embodiment (1) and a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the light chain variable region
of the antibody or the antigen-binding fragment thereof.
[0110] In another embodiment is provided (1) a polynucleotide
comprising a base sequence encoding the heavy chain variable region
of the anti-human NRP-1 antibody or the antigen-binding fragment
thereof of the above aspect, and (2) a polynucleotide comprising a
base sequence encoding the light chain variable region of the
anti-human NRP-1 antibody or the antigen-binding fragment thereof
of the above aspect.
[0111] In another embodiment is provided an expression vector
comprising: (a) a polynucleotide comprising a base sequence
encoding the heavy chain variable region of the anti-human NRP-1
antibody or the antigen-binding fragment thereof of the above
aspect, and/or (b) a polynucleotide comprising a base sequence
encoding the light chain variable region of the anti-human NRP-1
antibody or the antigen-binding fragment thereof of the above
aspect.
[0112] In another embodiment is provided a host cell transformed
with an expression vector selected from the group consisting of (a)
to (d): [0113] (a) a host cell transformed with an expression
vector comprising a polynucleotide comprising a base sequence
encoding the heavy chain variable region of the anti-human NRP-1
antibody or the antigen-binding fragment thereof of the above
aspect, and a polynucleotide comprising a base sequence encoding
the light chain variable region of the antibody or the
antigen-binding fragment thereof; [0114] (b) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the heavy chain variable region
of the anti-human NRP-1 antibody or the antigen-binding fragment
thereof of the above aspect and an expression vector comprising a
polynucleotide comprising a base sequence encoding the light chain
variable region of the antibody or the antigen-binding fragment
thereof of the above aspect; [0115] (c) a host cell transformed
with an expression vector comprising a polynucleotide comprising a
base sequence encoding the heavy chain variable region of the
anti-human NRP-1 antibody or the antigen-binding fragment thereof
of the above aspect; and [0116] (d) a host cell transformed with an
expression vector comprising a polynucleotide comprising a base
sequence encoding the light chain variable region of the anti-human
NRP-1 antibody or the antigen-binding fragment thereof of the above
aspect.
[0117] In another embodiment is provided an anti-human NRP-1
antibody or an antigen-binding fragment thereof according to the
above aspect, which is selected from the group consisting of (1) to
(4): [0118] (1) an anti-human NRP-1 antibody comprising a heavy
chain consisting of SEQ ID NO:118, and a light chain consisting of
SEQ ID NO:126; [0119] (2) an anti-human NRP-1 antibody comprising a
heavy chain consisting of SEQ ID NO:118 in which E of the amino
acid number 1 is modified to pyroglutamate, and a light chain
consisting of SEQ ID NO:126; [0120] (3) an anti-human NRP-1
antibody comprising a heavy chain consisting of the amino acid
sequence of amino acid numbers 1 to 453 of SEQ ID NO:118, and a
light chain consisting of SEQ ID NO:126; and [0121] (4) an
anti-human NRP-1 antibody comprising a heavy chain consisting of
the amino acid sequence of amino acid numbers 1 to 453 of SEQ ID
NO:118 in which E of the amino acid number 1 is modified to
pyroglutamate, and a light chain consisting of SEQ ID NO:126.
[0122] In one embodiment, the anti-human NRP-1 antibody is for use
in preventing or treating cancer. In another embodiment, the
anti-human NRP-1 antibody is for manufacture of a pharmaceutical
composition for preventing or treating cancer.
[0123] A polynucleotide, which is selected from the group
consisting of (1) and (2): [0124] (1) a polynucleotide comprising a
base sequence encoding the heavy chain of the anti-human NRP-1
antibody according to the above embodiment (1), and [0125] (2) a
polynucleotide comprising a base sequence encoding the light chain
of the anti-human NRP-1 antibody according to the above embodiment
(1).
[0126] An expression vector comprising: [0127] (a) a polynucleotide
comprising a base sequence encoding the heavy chain of the
anti-human NRP-1 antibody of the above embodiment (1), and/or
[0128] (b) a polynucleotide comprising a base sequence encoding the
light chain of the anti-human NRP-1 antibody of the above
embodiment (1).
[0129] A host cell transformed with an expression vector selected
from the group consisting of (a) to (d): [0130] (a) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the heavy chain of the
anti-human NRP-1 antibody of the above embodiment (1) and a
polynucleotide comprising a base sequence encoding the light chain
of the antibody; [0131] (b) a host cell transformed with an
expression vector comprising a polynucleotide comprising a base
sequence encoding the heavy chain of the anti-human NRP-1 antibody
of the above embodiment (1) and an expression vector comprising a
polynucleotide comprising a base sequence encoding the light chain
of the antibody; [0132] (c) a host cell transformed with an
expression vector comprising a polynucleotide comprising a base
sequence encoding the heavy chain of the anti-human NRP-1 antibody
of the above embodiment (1); and [0133] (d) a host cell transformed
with an expression vector comprising a polynucleotide comprising a
base sequence encoding the light chain of the anti-human NRP-1
antibody of the above embodiment (1).
[0134] A method for producing an anti-human NRP-1 antibody,
comprising culturing host cell(s) selected from the group
consisting of (a) to (c) below to express an anti-human NRP-1
antibody: [0135] (a) a host cell transformed with an expression
vector comprising a polynucleotide comprising a base sequence
encoding the heavy chain of the anti-human NRP-1 antibody of the
above embodiment (1) and a polynucleotide comprising a base
sequence encoding the light chain of the antibody; [0136] (b) a
host cell transformed with an expression vector comprising a
polynucleotide comprising a base sequence encoding the heavy chain
of the anti-human NRP-1 antibody of the above embodiment (1) and an
expression vector comprising a polynucleotide comprising a base
sequence encoding the light chain of the antibody; and [0137] (c) a
host cell transformed with an expression vector comprising a
polynucleotide comprising a base sequence encoding the heavy chain
of the anti-human NRP-1 antibody of the above embodiment (1) and a
host cell transformed with an expression vector comprising a
polynucleotide comprising a base sequence encoding the light chain
of the antibody.
[0138] In one embodiment is provided a pharmaceutical composition
comprising the anti-human NRP-1 antibody of the above embodiment
and a pharmaceutically acceptable excipient. In another embodiment
is provided a pharmaceutical composition comprising the anti-human
NRP-1 antibody of the above embodiment (1), the anti-human NRP-1
antibody of the above embodiment (2), the anti-human NRP-1 antibody
of the above embodiment (3), and/or the anti-human NRP-1 antibody
of the above embodiment (4), and a pharmaceutically acceptable
excipient. In one embodiment the pharmaceutical composition is a
pharmaceutical composition for treating cancer. In another
embodiment, the composition is administered in combination with
radiation, a cytotoxic agent, a chemotherapeutic agent, a
cytostatic agent, an anti-hormonal agent, a VEGF inhibitor, an
immunostimulatory agent, an anti-angiogenic agent, or combinations
thereof.
[0139] In another embodiment is provided a method for preventing or
treating cancer, comprising administering a therapeutically
effective amount of the anti-human NRP-1 antibody of the above
aspect. In one embodiment, the method further comprises
administering one or more additional therapeutic agents. In one
embodiment, the additional therapeutic agent is selected from the
group consisting of radiation, a cytotoxic agent, a
chemotherapeutic agent, a cytostatic agent, an anti-hormonal agent,
a VEGF inhibitor, an immunostimulatory agent, an anti-angiogenic
agent, and combinations thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0140] FIG. 1A is graphs showing tumor growth inhibition in CT26
tumor-bearing mice treated with a murine version of MABs 2, 3, 4,
5, 7, 12, 13, 14, and 15, as well as an IgG control and the
anti-NRP-1 antibody SEC10 as a comparator. Mice were treated with
MAB monotherapy (FIG. 1A). Antibody treatment times (days) are
shown by arrows.
[0141] FIG. 1B is graphs showing tumor growth inhibition in CT26
tumor-bearing mice treated with a murine version of MABs 2, 3, 4,
5, 7, 12, 13, 14, and 15, as well as an IgG control and the
anti-NRP-1 antibody SEC10 as a comparator. Mice were treated with
MAB in combination with a PD-1 antibody (FIG. 1B). Antibody
treatment times (days) are shown by arrows.
[0142] FIG. 1C is a graph showing tumor growth inhibition in CT26
tumor-bearing mice treated with monotherapy and combination therapy
as described herein. Provided are: i) a murine version of MAB12,
ii) a PD-1 inhibitor, and iii) a combination of mMAB12 and the PD-1
inhibitor. Antibody treatment times (days) are shown by arrows.
[0143] FIG. 2A is graphs showing tumor growth inhibition in MC38
tumor-bearing mice treated with a murine version of MABs 2, 3, 4,
5, 7, 12, 13, 14, and 15, as well as an IgG control and SEC10 as a
comparator. Mice were treated with MAB monotherapy (FIG. 2A).
Antibody treatment times (days) are shown by arrows.
[0144] FIG. 2B is graphs showing tumor growth inhibition in MC38
tumor-bearing mice treated with a murine version of MABs 2, 3, 4,
5, 7, 12, 13, 14, and 15, as well as an IgG control and SEC10 as a
comparator. Mice were treated with MAB in combination with a PD-L1
antibody (FIG. 2B). Antibody treatment times (days) are shown by
arrows.
[0145] FIG. 2C is graphs showing tumor growth inhibition in MC38
tumor-bearing mice treated with a murine version of MABs 2, 3, 4,
5, 7, 12, 13, 14, and 15, as well as an IgG control and SEC10 as a
comparator. Antibody treatment times (days) are shown by arrows.
The anti-tumor efficacy of mMAB12 alone or in combination with
PD-L1 antibody in the MC38 syngeneic colon mouse tumor model is
shown in FIG. 2C.
[0146] FIG. 3 is two graphs showing epitope binning data for the
anti-NRP-1 antibodies MAB12 and SEC10. The top panel shows binning
data for MAB12 and SEC10 with 5 .mu.g/mL MAB12 immobilized on
anti-human Fc AHC sensors. The bottom panel shows binning data for
MAB12 and SEC10 with 5 .mu.g/mL SEC10 immobilized on the sensors.
NRP1 protein is bound to the immobilized antibody and binding of
the second antibody is evaluated. The traces show that MAB12 and
SEC10 are able to simultaneously bind NRP1.
DETAILED DESCRIPTION
1. Definitions
[0147] Unless otherwise defined, all terms of art, notations and
other scientific terminology used herein are intended to have the
meanings commonly understood by those of skill in the art to which
this invention pertains. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not
necessarily be construed to represent a difference over what is
generally understood in the art. The techniques and procedures
described or referenced herein are generally well understood and
commonly employed using conventional methodologies by those skilled
in the art, such as, for example, the widely utilized molecular
cloning methodologies described in Sambrook et al., Molecular
Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,
procedures involving the use of commercially available kits and
reagents are generally carried out in accordance with
manufacturer-defined protocols and conditions unless otherwise
noted.
[0148] As used herein, the singular forms "a," "an," and "the"
include the plural referents unless the context clearly indicates
otherwise. The terms "include," "such as," and the like are
intended to convey inclusion without limitation, unless otherwise
specifically indicated.
[0149] As used herein, the term "comprising" also specifically
includes embodiments "consisting of" and "consisting essentially
of" the recited elements, unless specifically indicated
otherwise
[0150] The term "about" indicates and encompasses an indicated
value and a range above and below that value. In certain
embodiments, the term "about" indicates the designated
value.+-.10%, .+-.5%, or .+-.1%. In certain embodiments, where
applicable, the term "about" indicates the designated
value(s).+-.one standard deviation of that value(s).
[0151] The term "immunoglobulin" refers to a class of structurally
related proteins generally comprising two pairs of polypeptide
chains: one pair of light (L) chains and one pair of heavy (H)
chains. In an "intact immunoglobulin," all four of these chains are
interconnected by disulfide bonds. The structure of immunoglobulins
has been well characterized. See, e.g., Paul, Fundamental
Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins,
Philadelphia, Pa. Briefly, each heavy chain typically comprises a
heavy chain variable region (V.sub.H) and a heavy chain constant
region (C.sub.H). The heavy chain constant region typically
comprises three domains, abbreviated C.sub.H1, C.sub.H2, and
C.sub.H3. Each light chain typically comprises a light chain
variable region (V.sub.L) and a light chain constant region. The
light chain constant region typically comprises one domain,
abbreviated C.sub.L.
[0152] The term "antigen-binding protein" (ABP) refers to a protein
comprising one or more antigen-binding domains that specifically
bind to an antigen or epitope. In some embodiments, the
antigen-binding domain binds the antigen or epitope with
specificity and affinity similar to that of naturally occurring
antibodies. In some embodiments, the ABP comprises an antibody. In
some embodiments, the ABP consists of an antibody. In some
embodiments, the ABP consists essentially of an antibody. In some
embodiments, the ABP comprises an alternative scaffold. In some
embodiments, the ABP consists of an alternative scaffold. In some
embodiments, the ABP consists essentially of an alternative
scaffold. In some embodiments, the ABP comprises an antibody
fragment. In some embodiments, the ABP consists of an antibody
fragment. In some embodiments, the ABP consists essentially of an
antibody fragment. A "NRP-1 ABP," "anti-NRP-1 ABP," or
"NRP-1-specific ABP" is an ABP, as provided herein, which
specifically binds to the antigen NRP-1. In some embodiments, the
ABP binds the extracellular domain of NRP-1. In certain
embodiments, a NRP-1 ABP provided herein binds to an epitope of
NRP-1 that is conserved between or among NRP-1 proteins from
different species.
[0153] The term "antibody" is used herein in its broadest sense and
includes certain types of immunoglobulin molecules comprising one
or more antigen-binding domains that specifically bind to an
antigen or epitope. An antibody specifically includes intact
antibodies (e.g., intact immunoglobulins), antibody fragments, and
multi-specific antibodies. An antibody is one type of ABP.
[0154] The term "antigen-binding domain" means the portion of an
ABP that is capable of specifically binding to an antigen or
epitope. One example of an antigen-binding domain is an
antigen-binding domain formed by a V.sub.H-V.sub.L dimer of an
antibody. Another example of an antigen-binding domain is an
antigen-binding domain formed by diversification of certain loops
from the tenth fibronectin type III domain of an adnectin.
[0155] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a naturally
occurring antibody structure and having heavy chains that comprise
an Fc region. For example, when used to refer to an IgG molecule, a
"full length antibody" is an antibody that comprises two heavy
chains and two light chains. An "anti-human NRP-1 antibody" is the
intact antibody, as provided herein, which specifically binds to
the human NRP-1.
[0156] The term "Fc region" means the C-terminal region of an
immunoglobulin heavy chain that, in naturally occurring antibodies,
interacts with Fc receptors and certain proteins of the complement
system. The structures of the Fc regions of various
immunoglobulins, and the glycosylation sites contained therein, are
known in the art. See Schroeder and Cavacini, J. Allergy Clin.
Immunol., 2010, 125:S41-52, incorporated by reference in its
entirety. The Fc region may be a naturally occurring Fc region, or
an Fc region modified as described in the art or elsewhere in this
disclosure.
[0157] The V.sub.H and V.sub.L regions may be further subdivided
into regions of hypervariability ("hypervariable regions (HVRs);"
also called "complementarity determining regions" (CDRs))
interspersed with regions that are more conserved. The more
conserved regions are called framework regions (FRs). Each V.sub.H
and V.sub.L generally comprises three CDRs and four FRs, arranged
in the following order (from N-terminus to C-terminus):
FR1--CDR1--FR2--CDR2--FR3--CDR3--FR4. The CDRs are involved in
antigen binding, and influence antigen specificity and binding
affinity of the antibody. See Kabat et al., Sequences of Proteins
of Immunological Interest 5th ed. (1991) Public Health Service,
National Institutes of Health, Bethesda, Md., incorporated by
reference in its entirety.
[0158] The light chain from any vertebrate species can be assigned
to one of two types, called kappa (.kappa.) and lambda (.lamda.),
based on the sequence of its constant domain
[0159] The heavy chain from any vertebrate species can be assigned
to one of five different classes (or isotypes): IgA, IgD, IgE, IgG,
and IgM. These classes are also designated .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively. The IgG and IgA classes
are further divided into subclasses on the basis of differences in
sequence and function. Humans express the following subclasses:
IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
[0160] The amino acid sequence boundaries of a CDR can be
determined by one of skill in the art using any of a number of
known numbering schemes, including those described by Kabat et al.,
supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol.
Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al.,
1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme);
Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT"
numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001,
309:657-70 ("AHo" numbering scheme); each of which is incorporated
by reference in its entirety.
[0161] Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3,
CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia
schemes. For CDR-H1, residue numbering is provided using both the
Kabat and Chothia numbering schemes.
[0162] CDRs may be assigned, for example, using antibody numbering
software, such as Abnum, available at www.bioinf.org.uk/abs/abnum/,
and described in Abhinandan and Martin, Immunology, 2008,
45:3832-3839, incorporated by reference in its entirety.
TABLE-US-00001 TABLE 1 Residues in CDRs according to Kabat and
Chothia numbering schemes. CDR Kabat Chothia L1 L24-L34 L24-L34 L2
L50-L56 L50-L56 L3 L89-L97 L89-L97 H1 (Kabat Numbering) H31-H35B
H26-H32 or H34* H1 (Chothia Numbering) H31-H35 H26-H32 H2 H50-H65
H52-H56 H3 H95-H102 H95-H102 *The C-terminus of CDR-H1, when
numbered using the Kabat numbering convention, varies between H32
and H34, depending on the length of the CDR.
[0163] The "EU numbering scheme" is generally used when referring
to a residue in an antibody heavy chain constant region (e.g., as
reported in Kabat et al., supra). Unless stated otherwise, the EU
numbering scheme is used to refer to residues in antibody heavy
chain constant regions described herein.
[0164] An "antibody fragment" or an "antigen-binding fragment"
comprises a portion of an intact antibody, such as the
antigen-binding or variable region of an intact antibody. Antibody
fragments include, for example, Fv fragments, Fab fragments,
F(ab').sub.2 fragments, Fab' fragments, scFv (sFv) fragments, and
scFv-Fc fragments.
[0165] "Fv" fragments comprise a non-covalently-linked dimer of one
heavy chain variable domain and one light chain variable
domain.
[0166] "Fab" fragments comprise, in addition to the heavy and light
chain variable domains, the constant domain of the light chain and
the first constant domain (Cm) of the heavy chain. Fab fragments
may be generated, for example, by recombinant methods or by papain
digestion of a full-length antibody.
[0167] "F(ab').sub.2" fragments contain two Fab' fragments joined,
near the hinge region, by disulfide bonds. F(ab').sub.2 fragments
may be generated, for example, by recombinant methods or by pepsin
digestion of an intact antibody. The F(ab') fragments can be
dissociated, for example, by treatment with
.beta.-mercaptoethanol.
[0168] "Single-chain Fv" or "sFv" or "scFv" antibody fragments
comprise a V.sub.H domain and a V.sub.L domain in a single
polypeptide chain. The V.sub.H and V.sub.L are generally linked by
a peptide linker. See Pluckthun A. (1994). Any suitable linker may
be used. In some embodiments, the linker is a (GGGGS).sub.n (SEQ ID
NO:140). In some embodiments, n=1, 2, 3, 4, 5, or 6. See Antibodies
from Escherichia coli. In Rosenberg M. & Moore G. P. (Eds.),
The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315).
Springer-Verlag, New York, incorporated by reference in its
entirety.
[0169] "scFv-Fc" fragments comprise an scFv attached to an Fc
domain. For example, an Fc domain may be attached to the C-terminal
of the scFv. The Fc domain may follow the V.sub.H or V.sub.L,
depending on the orientation of the variable domains in the scFv
(i.e., V.sub.H-V.sub.L or V.sub.L-V.sub.H). Any suitable Fc domain
known in the art or described herein may be used. In some cases,
the Fc domain comprises an IgG4 Fc domain
[0170] The term "single domain antibody" refers to a molecule in
which one variable domain of an antibody specifically binds to an
antigen without the presence of the other variable domain. Single
domain antibodies, and fragments thereof, are described in Arabi
Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans
et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is
incorporated by reference in its entirety. Single domain antibodies
are also known as sdAbs or nanobodies.
[0171] The term "monoclonal antibody" refers to an antibody from a
population of substantially homogeneous antibodies. A population of
substantially homogeneous antibodies comprises antibodies that are
substantially similar and that bind the same epitope(s), except for
variants that may normally arise during production of the
monoclonal antibody. Such variants are generally present in only
minor amounts. A monoclonal antibody is typically obtained by a
process that includes the selection of a single antibody from a
plurality of antibodies. For example, the selection process can be
the selection of a unique clone from a plurality of clones, such as
a pool of hybridoma clones, phage clones, yeast clones, bacterial
clones, or other recombinant DNA clones. The selected antibody can
be further altered, for example, to improve affinity for the target
("affinity maturation"), to humanize the antibody, to improve its
production in cell culture, and/or to reduce its immunogenicity in
a subject.
[0172] The term "chimeric antibody" refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0173] "Humanized" forms of non-human antibodies are chimeric
antibodies that contain minimal sequence derived from the non-human
antibody. A humanized antibody is generally a human antibody
(recipient antibody) in which residues from one or more CDRs are
replaced by residues from one or more CDRs of a non-human antibody
(donor antibody). The donor antibody can be any suitable non-human
antibody, such as a mouse, rat, rabbit, chicken, or non-human
primate antibody having a desired specificity, affinity, or
biological effect. In some instances, selected framework region
residues of the recipient antibody are replaced by the
corresponding framework region residues from the donor antibody.
Humanized antibodies may also comprise residues that are not found
in either the recipient antibody or the donor antibody. Such
modifications may be made to further refine antibody function. For
further details, see Jones et al., Nature, 1986, 321:522-525;
Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op.
Struct. Biol., 1992, 2:593-596, each of which is incorporated by
reference in its entirety.
[0174] A "human antibody" is one which possesses an amino acid
sequence corresponding to that of an antibody produced by a human
or a human cell, or derived from a non-human source that utilizes a
human antibody repertoire or human antibody-encoding sequences
(e.g., obtained from human sources or designed de novo). Human
antibodies specifically exclude humanized antibodies.
[0175] By "SEC10" is meant an anti-NRP-1 antibody previously in
clinical trials for treatment of solid tumors, with and without
bevacizumab. See, e.g., "A Study of MNRP1685A in Patients with
Locally Advanced or Metastatic Solid Tumors," clinicaltrials.gov
Identifier NCT00747734.
[0176] By "SEC3" is meant the pan-anti-NRP-1 antibody set forth in
SEQ ID NO:144, also described, e.g., in Appleton, et. al., The EMBO
Journal (2007) 26, 4902-4912.
[0177] By "MAB59941" is meant an anti-mouse Neuropilin-1 antibody
available from R&D Systems, Clone #761704.
[0178] An "isolated ABP" or "isolated nucleic acid" is an ABP or
nucleic acid that has been separated and/or recovered from a
component of its natural environment. Components of the natural
environment may include enzymes, hormones, and other proteinaceous
or nonproteinaceous materials. In some embodiments, an isolated ABP
is purified to a degree sufficient to obtain at least 15 residues
of N-terminal or internal amino acid sequence, for example by use
of a spinning cup sequenator. In some embodiments, an isolated ABP
is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE)
under reducing or nonreducing conditions, with detection by
Coomassie blue or silver stain. In some embodiments, an isolated
ABP may include an ABP in situ within recombinant cells, since at
least one component of the ABP's natural environment is not
present. In some aspects, an isolated ABP or isolated nucleic acid
is prepared by at least one purification step. In some embodiments,
an isolated ABP or isolated nucleic acid is purified to at least
80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, an
isolated ABP or isolated nucleic acid is purified to at least 80%,
85%, 90%, 95%, or 99% by volume. In some embodiments, an isolated
ABP or isolated nucleic acid is provided as a solution comprising
at least 85%, 90%, 95%, 98%, 99% to 100% ABP or nucleic acid by
weight. In some embodiments, an isolated ABP or isolated nucleic
acid is provided as a solution comprising at least 85%, 90%, 95%,
98%, 99% to 100% ABP or nucleic acid by volume.
[0179] "Affinity" refers to the strength of the sum total of
non-covalent interactions between a single binding site of a
molecule (e.g., an ABP) and its binding partner (e.g., an antigen
or epitope). Unless indicated otherwise, as used herein, "affinity"
refers to intrinsic binding affinity, which reflects a 1:1
interaction between members of a binding pair (e.g., ABP and
antigen or epitope). The affinity of a molecule X for its partner Y
can be represented by the dissociation equilibrium constant
(K.sub.D). The kinetic components that contribute to the
dissociation equilibrium constant are described in more detail
below. Affinity can be measured by common methods known in the art,
including those described herein, such as surface plasmon resonance
(SPR) technology (e.g., BIACORE.RTM.) or biolayer interferometry
(e.g., FORTEBIO.RTM.).
[0180] With regard to the binding of an ABP to a target molecule,
the terms "bind," "specific binding," "specifically binds to,"
"specific for," "selectively binds," and "selective for" a
particular antigen (e.g., a polypeptide target) or an epitope on a
particular antigen mean binding that is measurably different from a
non-specific or non-selective interaction (e.g., with a non-target
molecule). Specific binding can be measured, for example, by
measuring binding to a target molecule and comparing it to binding
to a non-target molecule. Specific binding can also be determined
by competition with a control molecule that mimics the epitope
recognized on the target molecule. In that case, specific binding
is indicated if the binding of the ABP to the target molecule is
competitively inhibited by the control molecule. In some aspects,
the affinity of a NRP-1 ABP for a non-target molecule is less than
about 50% of the affinity for NRP-1. In some aspects, the affinity
of a NRP-1 ABP for a non-target molecule is less than about 40% of
the affinity for NRP-1. In some aspects, the affinity of a NRP-1
ABP for a non-target molecule is less than about 30% of the
affinity for NRP-1. In some aspects, the affinity of a NRP-1 ABP
for a non-target molecule is less than about 20% of the affinity
for NRP-1. In some aspects, the affinity of a NRP-1 ABP for a
non-target molecule is less than about 10% of the affinity for
NRP-1. In some aspects, the affinity of a NRP-1 ABP for a
non-target molecule is less than about 1% of the affinity for
NRP-1. In some aspects, the affinity of a NRP-1 ABP for a
non-target molecule is less than about 0.1% of the affinity for
NRP-1.
[0181] The term "k.sub.d" (sec.sup.-1), as used herein, refers to
the dissociation rate constant of a particular ABP-antigen
interaction. This value is also referred to as the k.sub.off
value.
[0182] The term "k.sub.a" (M.sup.-1.times.sec.sup.-1), as used
herein, refers to the association rate constant of a particular
ABP-antigen interaction. This value is also referred to as the
k.sub.on value.
[0183] The term "K.sub.D" (M), as used herein, refers to the
dissociation equilibrium constant of a particular ABP-antigen
interaction. K.sub.D=k.sub.d/k.sub.a. In some embodiments, the
affinity of an ABP is described in terms of the K.sub.D for an
interaction between such ABP and its antigen. For clarity, as known
in the art, a smaller K.sub.D value indicates a higher affinity
interaction, while a larger K.sub.D value indicates a lower
affinity interaction.
[0184] The term "K.sub.A" (M.sup.-1), as used herein, refers to the
association equilibrium constant of a particular ABP-antigen
interaction. K.sub.A=k.sub.a/k.sub.d.
[0185] An "affinity matured" ABP is an ABP with one or more
alterations (e.g., in one or more CDRs or FRs) relative to a parent
ABP (i.e., an ABP from which the altered ABP is derived or
designed) that result in an improvement in the affinity of the ABP
for its antigen, compared to the parent ABP which does not possess
the alteration(s). In some embodiments, an affinity matured ABP has
nanomolar or picomolar affinity for the target antigen. Affinity
matured ABPs may be produced using a variety of methods known in
the art. For example, Marks et al. (Bio/Technology, 1992,
10:779-783, incorporated by reference in its entirety) describes
affinity maturation by V.sub.H and V.sub.L domain shuffling. Random
mutagenesis of CDR and/or framework residues is described by, for
example, Barbas et al. (Proc. Nat. Acad. Sci. U.S.A., 1994,
91:3809-3813); Schier et al., Gene, 1995, 169:147-155; Yelton et
al., J. Immunol., 1995, 155:1994-2004; Jackson et al., J. Immunol.,
1995, 154:3310-33199; and Hawkins et al, J. Mol. Biol., 1992,
226:889-896; each of which is incorporated by reference in its
entirety.
[0186] An "immunoconjugate" is an ABP conjugated to one or more
heterologous molecule(s), such as a therapeutic or diagnostic
agent.
[0187] "Effector functions" refer to those biological activities
mediated by the Fc region of an antibody, which activities may vary
depending on the antibody isotype. Examples of antibody effector
functions include C1q binding to activate complement dependent
cytotoxicity (CDC), Fc receptor binding to activate
antibody-dependent cellular cytotoxicity (ADCC), and antibody
dependent cellular phagocytosis (ADCP).
[0188] When used herein in the context of two or more ABPs, the
term "competes with" or "cross-competes with" indicates that the
two or more ABPs compete for binding to an antigen (e.g., NRP-1).
In one exemplary assay, NRP-1 is coated on a surface and contacted
with a first NRP-1 ABP, after which a second NRP-1 ABP is added. In
another exemplary assay, a first NRP-1 ABP is coated on a surface
and contacted with NRP-1, and then a second NRP-1 ABP is added. If
the presence of the first NRP-1 ABP reduces binding of the second
NRP-1 ABP, in either assay, then the ABPs compete with each other.
The term "competes with" also includes combinations of ABPs where
one ABP reduces binding of another ABP, but where no competition is
observed when the ABPs are added in the reverse order. However, in
some embodiments, the first and second ABPs inhibit binding of each
other, regardless of the order in which they are added. In some
embodiments, one ABP reduces binding of another ABP to its antigen
by at least 25%, at least 50%, at least 60%, at least 70%, at least
80%, at least 85%, at least 90%, or at least 95%. A skilled artisan
can select the concentrations of the antibodies used in the
competition assays based on the affinities of the ABPs for NRP-1
and the valency of the ABPs. The assays described in this
definition are illustrative, and a skilled artisan can utilize any
suitable assay to determine if antibodies compete with each other.
Suitable assays are described, for example, in Cox et al.,
"Immunoassay Methods," in Assay Guidance Manual [Internet], Updated
Dec. 24, 2014 (www.ncbi.nlm.nih.gov/books/NBK92434/; accessed Sep.
29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et
al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each of which is
incorporated by reference in its entirety.
[0189] The term "epitope" means a portion of an antigen that
specifically binds to an ABP. Epitopes frequently consist of
surface-accessible amino acid residues and/or sugar side chains and
may have specific three-dimensional structural characteristics, as
well as specific charge characteristics. Conformational and
non-conformational epitopes are distinguished in that the binding
to the former but not the latter may be lost in the presence of
denaturing solvents. An epitope may comprise amino acid residues
that are directly involved in the binding, and other amino acid
residues, which are not directly involved in the binding. The
epitope to which an ABP binds can be determined using known
techniques for epitope determination such as, for example, testing
for ABP binding to NRP-1 variants with different point-mutations,
or to chimeric NRP-1 variants.
[0190] Percent "identity" between a polypeptide sequence and a
reference sequence, is defined as the percentage of amino acid
residues in the polypeptide sequence that are identical to the
amino acid residues in the reference sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared.
[0191] A "conservative substitution" or a "conservative amino acid
substitution," refers to the substitution an amino acid with a
chemically or functionally similar amino acid. Conservative
substitution tables providing similar amino acids are well known in
the art. By way of example, the groups of amino acids provided in
Tables 2-4 are, in some embodiments, considered conservative
substitutions for one another.
TABLE-US-00002 TABLE 2 Selected groups of amino acids that are
considered conservative substitutions for one another, in certain
embodiments. Acidic Residues D and E Basic Residues K, R, and H
Hydrophilic Uncharged Residues S, T, N, and Q Aliphatic Uncharged
Residues G, A, V, L, and I Non-polar Uncharged Residues C, M, and P
Aromatic Residues F, Y, and W
TABLE-US-00003 TABLE 3 Additional selected groups of amino acids
that are considered conservative substitutions for one another, in
certain embodiments. Group 1 A, S, and T Group 2 D and E Group 3 N
and Q Group 4 R and K Group 5 I, L, and M Group 6 F, Y, and W
TABLE-US-00004 TABLE 4 Further selected groups of amino acids that
are considered conservative substitutions for one another, in
certain embodiments. Group A A and G Group B D and E Group C N and
Q Group D R, K, and H Group E I, L, M, V Group F F, Y, and W Group
G S and T Group H C and M
[0192] Additional conservative substitutions may be found, for
example, in Creighton, Proteins: Structures and Molecular
Properties 2nd ed. (1993) W. H. Freeman & Co., New York, N.Y.
An ABP generated by making one or more conservative substitutions
of amino acid residues in a parent ABP is referred to as a
"conservatively modified variant."
[0193] The term "amino acid" refers to the twenty common naturally
occurring amino acids. Naturally occurring amino acids include
alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic
acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine
(Gln; Q), Glycine (Gly; G); histidine (His; H), isoleucine (Ile;
I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M),
phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S),
threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and
valine (Val; V).
[0194] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0195] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which an
exogenous nucleic acid has been introduced, and the progeny of such
cells. Host cells include "transformants" (or "transformed cells")
and "transfectants" (or "transfected cells"), which each include
the primary transformed or transfected cell and progeny derived
therefrom. Such progeny may not be completely identical in nucleic
acid content to a parent cell, and may contain mutations.
[0196] The term "treating" (and variations thereof such as "treat"
or "treatment") refers to clinical intervention in an attempt to
alter the natural course of a disease or condition in a subject in
need thereof. Treatment can be performed both for prophylaxis and
during the course of clinical pathology. Desirable effects of
treatment include preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, preventing metastasis,
decreasing the rate of disease progression, amelioration or
palliation of the disease state, and remission or improved
prognosis.
[0197] As used herein, the term "therapeutically effective amount"
or "effective amount" refers to an amount of an ABP or
pharmaceutical composition provided herein that, when administered
to a subject, is effective to treat a disease or disorder.
[0198] As used herein, the term "subject" means a mammalian
subject. Exemplary subjects include humans, monkeys, dogs, cats,
mice, rats, cows, horses, camels, goats, rabbits, and sheep. In
certain embodiments, the subject is a human. In some embodiments,
the subject has a disease or condition that can be treated with an
ABP provided herein. In some aspects, the disease or condition is a
cancer. In some aspects, the disease or condition is a viral
infection.
[0199] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic or
diagnostic products (e.g., kits) that contain information about the
indications, usage, dosage, administration, combination therapy,
contraindications and/or warnings concerning the use of such
therapeutic or diagnostic products.
[0200] The term "cytotoxic agent," as used herein, refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction.
[0201] A "chemotherapeutic agent" refers to a chemical compound
useful in the treatment of cancer. Chemotherapeutic agents include
"anti-hormonal agents" or "endocrine therapeutics" which act to
regulate, reduce, block, or inhibit the effects of hormones that
can promote the growth of cancer.
[0202] The term "cytostatic agent" refers to a compound or
composition which arrests growth of a cell either in vitro or in
vivo. In some embodiments, a cytostatic agent is an agent that
reduces the percentage of cells in S phase. In some embodiments, a
cytostatic agent reduces the percentage of cells in S phase by at
least about 20%, at least about 40%, at least about 60%, or at
least about 80%.
[0203] The term "tumor" refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues. The terms "cancer," "cancerous,"
"cell proliferative disorder," "proliferative disorder" and "tumor"
are not mutually exclusive as referred to herein. The terms "cell
proliferative disorder" and "proliferative disorder" refer to
disorders that are associated with some degree of abnormal cell
proliferation. In some embodiments, the cell proliferative disorder
is a cancer. In some aspects, the tumor is a solid tumor. In some
aspects, the tumor is a hematologic malignancy.
[0204] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective
in treating a subject, and which contains no additional components
which are unacceptably toxic to the subject in the amounts provided
in the pharmaceutical composition.
[0205] The terms "modulate" and "modulation" refer to reducing or
inhibiting or, alternatively, activating or increasing, a recited
variable.
[0206] The terms "increase" and "activate" refer to an increase of
10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,
2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold,
100-fold, or greater in a recited variable.
[0207] The terms "reduce" and "inhibit" refer to a decrease of 10%,
20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold,
3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or
greater in a recited variable.
[0208] The term "agonize" refers to the activation of receptor
signaling to induce a biological response associated with
activation of the receptor. An "agonist" is an entity that binds to
and agonizes a receptor.
[0209] The term "antagonize" refers to the inhibition of receptor
signaling to inhibit a biological response associated with
activation of the receptor. An "antagonist" is an entity that binds
to and antagonizes a receptor. An antagonist in one embodiment
blocks 100% of binding of a ligand to its receptor; in other
embodiments, an antagonist may reduce binding by 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, or 100% of binding of a ligand to its receptor.
[0210] The term "a semaphorin molecule" as used herein in
connection with agonists of the NRP-1:semaphorin axis of Tregs
encompasses transmembrane semaphorin molecules involved in
interaction with NRP-1 on Tregs (e.g., Sema3a, Sema4a), various
surface- and bead-immobilized versions of such molecules, as well
as multimers, derivatives, mutants, analogs, and fragments of such
molecules which can be used to enhance a function or increase
stability of Tregs. Non-limiting examples of such agonist
semaphorin molecules include, for example, IgM-derived semaphorin
fusion proteins that assemble multimeric complexes incapable of
fixing complement, that crosslink NRP-1.
[0211] The term "neuropilin-1 (NRP-1):semaphorin axis of a
regulatory T cell (Treg)" as used herein refers to the signaling
pathway initiated by semaphorin (e.g., a semaphorin expressed by a
cell such as, e.g., a conventional T cell, or a recombinant
semaphorin), ligation of NRP-1, and the subsequent downstream
signaling.
[0212] The term "effector T cell" includes T helper (i.e., CD4+)
cells and cytotoxic (i.e., CD8+) T cells. CD4+ effector T cells
contribute to the development of several immunologic processes,
including maturation of B cells into plasma cells and memory B
cells, and activation of cytotoxic T cells and macrophages. CD8+
effector T cells destroy virus-infected cells and tumor cells. See
Seder and Ahmed, Nature Immunol., 2003, 4:835-842, incorporated by
reference in its entirety, for additional information on effector T
cells.
[0213] The term "regulatory T cell" includes cells that regulate
immunological tolerance, for example, by suppressing effector T
cells. In some aspects, the regulatory T cell has a CD4+CD25+Foxp3+
phenotype. In some aspects, the regulatory T cell has a CD8+CD25+
phenotype. See Nocentini et al., Br. J. Pharmacol., 2012,
165:2089-2099, incorporated by reference in its entirety, for
additional information on regulatory T cells expressing NRP-1.
[0214] The term "dendritic cell" refers to a professional
antigen-presenting cell capable of activating a naive T cell and
stimulating growth and differentiation of a B cell.
2. NRP-1 Antigen-Binding Proteins
[0215] 2.1 NRP-1 Binding and Target Cells
[0216] Provided herein are ABPs that specifically bind to NRP-1. In
some aspects, the NRP-1 is hNRP-1 (SEQ ID NO:130). In some aspects,
the NRP-1 is cNRP-1 (SEQ ID NO:132). In some aspects, the NRP-1 is
mNRP-1 with the sequence provided in SEQ ID NO:134. In some
aspects, the NRP-1 is rNRP-1 with the sequence provided in SEQ ID
NO:135.
[0217] In some embodiments, the ABPs provided herein specifically
bind to the extracellular domain of NRP-1.
[0218] In some embodiments, the ABPs provided herein specifically
bind to the extracellular domain of NRP-1 and the extracellular
domain of PD-1, PD-L1, or PD-L2, i.e., are bispecific
antibodies.
[0219] In some embodiments, an ABP provided herein is an antibody.
In some embodiments, an ABP provided herein is an antibody
fragment. In some embodiments, an ABP provided herein is an
alternative scaffold.
[0220] The NRP-1 may be expressed on the surface of any suitable
target cell. In some embodiments, the target cell is a T cell. In
some embodiments, the target cell is an effector T cell. In some
embodiments, the target cell is a regulatory T cell. In some
embodiments, the target cell is a natural killer (NK) cell. In some
embodiments, the target cell is a natural killer T (NKT) cell. In
some embodiments, the target cell is a macrophage. In other
embodiments, the target cell is a dendritic cell. In one
embodiment, the dendritic cell is a plasmacytoid dendritic
cell.
[0221] In some embodiments, the NRP-1 is associated with another
receptor on the surface of the cell. In some embodiments, the NRP-1
is part of a co-receptor complex. In one embodiment, the NRP-1 is
associated with a plexin. In some embodiments, the NRP-1 is
associated with a VEGF receptor.
[0222] In some embodiments, the ABPs provided herein comprise an
immunoglobulin molecule. In some embodiments, the ABPs provided
herein consist of an immunoglobulin molecule. In some embodiments,
the ABPs provided herein consist essentially of an immunoglobulin
molecule. In some aspects, the immunoglobulin molecule comprises an
antibody. In some aspects, the immunoglobulin molecule consists of
an antibody. In some aspects, the immunoglobulin molecule consists
essentially of an antibody.
[0223] In some embodiments, the ABPs provided herein comprise a
light chain. In some aspects, the light chain is a kappa light
chain. In some aspects, the light chain is a lambda light
chain.
[0224] In some embodiments, the ABPs provided herein comprise a
heavy chain. In some aspects, the heavy chain is an IgA. In some
aspects, the heavy chain is an IgD. In some aspects, the heavy
chain is an IgE. In some aspects, the heavy chain is an IgG. In
some aspects, the heavy chain is an IgM. In some aspects, the heavy
chain is an IgG1. In some aspects, the heavy chain is an IgG2. In
some aspects, the heavy chain is an IgG3. In some aspects, the
heavy chain is an IgG4. In some aspects, the heavy chain is an
IgA1. In some aspects, the heavy chain is an IgA2.
[0225] In some embodiments, the ABPs provided herein comprise an
antibody fragment. In some embodiments, the ABPs provided herein
consist of an antibody fragment. In some embodiments, the ABPs
provided herein consist essentially of an antibody fragment. In
some aspects, the antibody fragment is an Fv fragment. In some
aspects, the antibody fragment is a Fab fragment. In some aspects,
the antibody fragment is a F(ab').sub.2fragment. In some aspects,
the antibody fragment is a Fab' fragment. In some aspects, the
antibody fragment is an scFv (sFv) fragment. In some aspects, the
antibody fragment is an scFv-Fc fragment. In some aspects, the
antibody fragment is a fragment of a single domain antibody.
[0226] In some embodiments, an antibody fragment provided herein is
derived from an illustrative antibody provided herein. In some
embodiments, an antibody fragments provided herein is not derived
from an illustrative antibody provided herein and may, for example,
be isolated de novo according to the methods provided herein for
obtaining antibody fragments.
[0227] In some embodiments, an antibody fragment provided
specifically binds hNRP-1. In some embodiments, an antibody
fragment provided herein specifically binds cNRP-1. In some
embodiments, an antibody fragment provided herein specifically
binds mNRP-1. In some embodiments, an antibody fragment provided
herein specifically binds hNRP-1 and cNRP-1. In some embodiments,
an antibody fragment provided herein specifically binds hNRP-1 and
mNRP-1. In some embodiments, an antibody fragment provided herein
specifically binds cNRP-1 and mNRP-1. In some embodiments, an
antibody fragment provided herein specifically binds hNRP-1, cNRP-1
and mNRP-1.
[0228] In some embodiments, an antibody fragment provided herein
retains the ability to antagonize NRP-1, as measured by one or more
assays or biological effects described herein. In some embodiments,
an antibody fragment provided herein retains the ability to prevent
NRP-1 from interacting with one or more of its ligands, as
described herein.
[0229] In some embodiments, an antibody fragment provided herein
competes for binding to NRP-1 with an antibody selected from MAB1,
MAB2, MAB3, MAB4, MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11,
MAB12, MAB13, MAB14, or MAB15, each as provided in Appendix A of
this disclosure.
[0230] In some embodiments, the ABPs provided herein are specific
for cell surface NRP-1.
[0231] In some embodiments, the ABPs provided herein are
specifically block NRP-1 binding to a transmembrane semaphorin
polypeptide.
[0232] In some embodiments, the ABPs provided herein block the
interaction between a NRP-1 polypeptide and a vascular endothelial
cell growth factor (VEGF) polypeptide. In one embodiment, the VEGF
polypeptide is VEGFA.
[0233] In some embodiments, the anti-NRP-1 antibody blocks SEMA3
binding.
[0234] In some embodiments, the anti-NRP-1 antibody blocks SEMA4
binding.
[0235] In some embodiments, the antibody blocks interaction between
a NRP-1 polypeptide and SEMA3.
[0236] In some embodiments, the antibody blocks interaction between
a NRP-1 polypeptide and VEGF.
[0237] In some embodiments, the ABPs provided herein are capable of
inhibiting Treg suppression in a human subject.
[0238] In some embodiments, the ABPs provided herein co-stimulate
an effector T cell in combination with antigen presentation from an
antigen-presenting cell.
[0239] In some embodiments, the ABPs provided herein inhibit the
suppression of an effector T cell by a regulatory T cell.
[0240] In some embodiments, the ABPs provided herein reduce the
number of effector T cells in a tissue or in systemic
circulation.
[0241] In some embodiments, a fragment of an antibody provided
herein binds the same epitope of NRP-1 as such antibody.
[0242] In some embodiments, the ABPs provided herein are monoclonal
antibodies. In some embodiments, the ABPs provided herein are
polyclonal antibodies.
[0243] In some embodiments, the ABPs provided herein comprise a
chimeric antibody. In some embodiments, the ABPs provided herein
consist of a chimeric antibody. In some embodiments, the ABPs
provided herein consist essentially of a chimeric antibody. In some
embodiments, the ABPs provided herein comprise a humanized
antibody. In some embodiments, the ABPs provided herein consist of
a humanized antibody. In some embodiments, the ABPs provided herein
consist essentially of a humanized antibody. In some embodiments,
the ABPs provided herein comprise a human antibody. In some
embodiments, the ABPs provided herein consist of a human antibody.
In some embodiments, the ABPs provided herein consist essentially
of a human antibody.
[0244] In some embodiments, the ABPs provided herein are affinity
matured. In some aspects, the affinity matured ABPs are affinity
matured ABPs derived from an illustrative ABP provided herein.
[0245] In some embodiments, the ABPs provided herein comprise an
alternative scaffold. In some embodiments, the ABPs provided herein
consist of an alternative scaffold. In some embodiments, the ABPs
provided herein consist essentially of an alternative scaffold. Any
suitable alternative scaffold may be used. In some aspects, the
alternative scaffold is selected from an Adnectin.TM., an iMab, an
Anticalin.RTM., an EETI-II/AGRP, a Kunitz domain, a thioredoxin
peptide aptamer, an Affibody, DARPin, an Affilin, a Tetranectin, a
Fynomer, and an Avimer.
[0246] In some embodiments, an ABP provided herein specifically
blocks binding of NRP-1 to a transmembrane semaphorin polypeptide.
In some aspects, the ABP inhibits binding of NRP-1 to a
transmembrane semaphorin polypeptide by at least about 50%. In some
aspects, the ABP inhibits binding of NRP-1 to a transmembrane
semaphorin polypeptide by at least about 75%. In some aspects, the
ABP inhibits binding of NRP-1 to a transmembrane semaphorin
polypeptide by at least about 90%. In some aspects, the ABP
inhibits binding of NRP-1 to a transmembrane semaphorin polypeptide
by at least about 95%. In some embodiments, the semaphorin
polypeptide is a SEMA3 polypeptide. In other embodiments, the
semaphorin polypeptide is a SEMA4 polypeptide.
[0247] In some embodiments, an ABP of the invention is an ABP that
competes with an illustrative ABP provided herein. In some aspects,
the ABP that competes with the illustrative ABP provided herein
binds the same epitope as an illustrative ABP provided herein.
[0248] It is known that when an antibody is expressed in cells, the
antibody is modified after translation. Examples of the
posttranslational modification include cleavage of lysine at the C
terminal of the heavy chain by a carboxypeptidase; modification of
glutamine or glutamic acid at the N terminal of the heavy chain and
the light chain to pyroglutamic acid by pyroglutamylation;
glycosylation; oxidation; deamidation; and glycation, and it is
known that such posttranslational modifications occur in various
antibodies (See Journal of Pharmaceutical Sciences, 2008, Vol. 97,
p. 2426-2447, incorporated by reference in its entirety). In some
embodiments, an ABP of the invention is an antibody or
antigen-binding fragment thereof which has undergone
posttranslational modification. Examples of an antibody or
antigen-binding fragment thereof which have undergone
posttranslational modification include an antibody or
antigen-binding fragments thereof which have undergone
pyroglutamylation at the N terminal of the heavy chain variable
region, pyroglutamylation at the N terminal of the light chain
variable region, and/or deletion of lysine at the C terminal of the
heavy chain. It is known in the art that such posttranslational
modification due to pyroglutamylation at the N terminal and
deletion of lysine at the C terminal does not have any influence on
the activity of the antibody or fragment thereof (Analytical
Biochemistry, 2006, Vol. 348, p. 24-39, incorporated by reference
in its entirety).
[0249] In some embodiments, an ABP of the invention is an
anti-human NRP-1 antibody or an antigen-binding fragment thereof,
comprising a heavy chain variable region comprising a CDR-H3
consisting of SEQ ID NO:47, a CDR-H2 consisting of SEQ ID NO:30,
and a CDR-H1 consisting of SEQ ID NO:14; and a light chain variable
region comprising a CDR-L3 consisting of SEQ ID NO:81, a CDR-L2
consisting of SEQ ID NO:71, and a CDR-L1 consisting of SEQ ID
NO:63
[0250] In one embodiment, the anti-human NRP-1 antibody or an
antigen-binding fragment thereof, comprising a heavy chain variable
region consisting of SEQ ID NO:96, and a light chain variable
region consisting of SEQ ID NO:104.
[0251] In one embodiment, the anti-human NRP-1 antibody or the
antigen-binding fragment thereof, comprising a heavy chain variable
region consisting of SEQ ID NO:96 in which E of the amino acid
number 1 is modified to pyroglutamate, and a light chain variable
region consisting of SEQ ID NO:104.
[0252] In one embodiment, the anti-human NRP-1 antibody comprising
a heavy chain consisting of SEQ ID NO:118, and a light chain
consisting of SEQ ID NO:126.
[0253] In one embodiment, the anti-human NRP-1 antibody comprising
a heavy chain consisting of SEQ ID NO:118 in which E of the amino
acid number 1 is modified to pyroglutamate, and a light chain
consisting of SEQ ID NO:126.
[0254] In one embodiment, the anti-human NRP-1 antibody comprising
a heavy chain consisting of the amino acid sequence of amino acid
numbers 1 to 453 of SEQ ID NO:118, and a light chain consisting of
SEQ ID NO:126.
[0255] In one embodiment, the anti-human NRP-1 antibody comprising
a heavy chain consisting of the amino acid sequence of amino acid
numbers 1 to 453 of SEQ ID NO:118 in which E of the amino acid
number 1 is modified to pyroglutamate, and a light chain consisting
of SEQ ID NO:126.
[0256] 2.2 NRP-1 Antagonism
[0257] In some embodiments, the ABPs provided herein antagonize
NRP-1 upon binding.
[0258] In some embodiments, antagonism of NRP-1 by an ABP provided
herein results in activation of an effector T cell. In some
aspects, the effector T cell is a CD8+ T cell. In some aspects, the
effector T cell is a CD4+ T cell.
[0259] In some embodiments, antagonism of NRP-1 by an ABP provided
herein results in activation of an NK cell. In some embodiments,
antagonism of NRP-1 by an ABP provided herein results in activation
of an NKT cell. In some embodiments, the NKT cell is an
IL-17-secreting cell.
[0260] In some embodiments, antagonism of NRP-1 by an ABP provided
herein results in a reduction of the inhibitory activity of a
regulatory T cell toward an effector T cell.
[0261] In some embodiments, antagonism of NRP-1 by an ABP provided
herein results in increased secretion of IL-2, IL-6, GM-CSF, TNF,
LT-.alpha., and/or IFN-.gamma. by a target cell.
[0262] In some embodiments, antagonism of NRP-1 by an ABP provided
herein increases the proliferation, survival, and/or function of an
effector T cell. In some aspects, the effector T cell is a CD4+
effector T cell. In some aspects, the effector T cell is a CD8+
effector T cell.
[0263] In some embodiments, antagonism of NRP-1 by an ABP provided
herein abrogates suppression of an effector T cell by a regulatory
T cell. In some aspects, the regulatory T cell is a CD4+CD25+Foxp3+
regulatory T cell. In some aspects, the regulatory T cell is a
CD8+CD25+ regulatory T cell.
[0264] In some embodiments, antagonism of NRP-1 by an ABP provided
herein results in an enhancement of an immune response.
[0265] In some embodiments, antagonism of NRP-1 by an ABP provided
herein results in the prevention of a tumor. In some embodiments,
antagonism of NRP-1 by an ABP provided herein results in the delay
of onset of a tumor. In some embodiments, antagonism of NRP-1 by an
ABP provided herein results in a reduction of the size of a tumor.
In some embodiments, antagonism of NRP-1 by an ABP provided herein
results in elimination of a tumor. In some embodiments, antagonism
of NRP-1 by an ABP provided herein results in a reduction in the
number of metastases.
[0266] In some embodiments, antagonism of NRP-1 by an ABP provided
herein results in the prevention of a viral disease. In some
embodiments, antagonism of NRP-1 by an ABP provided herein results
in the delay of onset of a viral disease. In some embodiments,
antagonism of NRP-1 by an ABP provided herein results in a
reduction of the viral load in a subject. In some embodiments,
antagonism of NRP-1 by an ABP provided herein results in the
elimination of a viral infection.
[0267] 2.3 Affinity and Kinetics of Antigen-Binding Proteins for
NRP-1; Potency
[0268] In some embodiments, the affinity of an ABP provided herein
for NRP-1 as indicated by K.sub.D, is less than about 10.sup.-5 M,
less than about 10.sup.-6 M, less than about 10.sup.-7M, less than
about 10.sup.-8M, less than about 10.sup.-9 M, less than about
10.sup.-10 M, less than about 10.sup.-11 M, or less than about
10.sup.-12 M. In some embodiments, the affinity of the ABP is
between about 10.sup.-7 M and 10.sup.-12 M. In some embodiments,
the affinity of the ABP is between about 10.sup.-7 M and 10.sup.-11
M. In some embodiments, the affinity of the ABP is between about
10.sup.-7M and 10.sup.-10 M. In some embodiments, the affinity of
the ABP is between about 10.sup.-7 M and 10.sup.-9 M. In some
embodiments, the affinity of the ABP is between about 10.sup.-7 M
and 10.sup.-8 M. In some embodiments, the affinity of the ABP is
between about 10.sup.-8 M and 10.sup.-12 M. In some embodiments,
the affinity of the ABP is between about 10.sup.-8M and 10.sup.-11
M. In some embodiments, the affinity of the ABP is between about
10.sup.-9 M and 10.sup.-11 M. In some embodiments, the affinity of
the ABP is between about 10.sup.-10 M and 10.sup.-11 M.
[0269] 2.3.1 Glycosylation Variants
[0270] In certain embodiments, an ABP provided herein may be
altered to increase, decrease or eliminate the extent to which it
is glycosylated. Glycosylation of polypeptides is typically either
"N-linked" or "O-linked."
[0271] "N-linked" glycosylation refers to the attachment of a
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and
asparagine-X-threonine, where X is any amino acid except proline,
are the recognition sequences for enzymatic attachment of the
carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site.
[0272] "O-linked" glycosylation refers to the attachment of one of
the sugars N-acetylgalactosamine, galactose, or xylose to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
[0273] Addition or deletion of N-linked glycosylation sites to or
from an ABP provided herein may be accomplished by altering the
amino acid sequence such that one or more of the above-described
tripeptide sequences is created or removed. Addition or deletion of
O-linked glycosylation sites may be accomplished by addition,
deletion, or substitution of one or more serine or threonine
residues in or to (as the case may be) the sequence of an ABP.
[0274] In some embodiments, an ABP provided herein comprises a
glycosylation motif that is different from a naturally occurring
ABP. Any suitable naturally occurring glycosylation motif can be
modified in the ABPs provided herein. The structural and
glycosylation properties of immunoglobulins, for example, are known
in the art and summarized, for example, in Schroeder and Cavacini,
J. Allergy Clin. Immunol., 2010, 125:S41-52, incorporated by
reference in its entirety.
[0275] In some embodiments, an ABP provided herein comprises an
IgG1 Fc region with modification to the oligosaccharide attached to
asparagine 297 (Asn 297). Naturally occurring IgG1 antibodies
produced by mammalian cells typically comprise a branched,
biantennary oligosaccharide that is generally attached by an
N-linkage to Asn 297 of the C.sub.H2 domain of the Fc region. See
Wright et al., TIBTECH, 1997, 15:26-32, incorporated by reference
in its entirety. The oligosaccharide attached to Asn 297 may
include various carbohydrates such as mannose, N-acetyl glucosamine
(GlcNAc), galactose, and sialic acid, as well as a fucose attached
to a GlcNAc in the "stem" of the biantennary oligosaccharide
structure.
[0276] In some embodiments, the oligosaccharide attached to Asn 297
is modified to create ABPs having altered ADCC. In some
embodiments, the oligosaccharide is altered to improve ADCC. In
some embodiments, the oligosaccharide is altered to reduce
ADCC.
[0277] In some aspects, an ABP provided herein comprises an IgG1
domain with reduced fucose content at position Asn 297 compared to
a naturally occurring IgG1 domain. Such Fc domains are known to
have improved ADCC. See Shields et al., J. Biol. Chem., 2002,
277:26733-26740, incorporated by reference in its entirety. In some
aspects, such ABPs do not comprise any fucose at position Asn 297.
The amount of fucose may be determined using any suitable method,
for example as described in WO 2008/077546, incorporated by
reference in its entirety.
[0278] In some embodiments, an ABP provided herein comprises a
bisected oligosaccharide, such as a biantennary oligosaccharide
attached to the Fc region of the ABP that is bisected by GlcNAc.
Such ABP variants may have reduced fucosylation and/or improved
ADCC function. Examples of such ABP variants are described, for
example, in WO 2003/011878; U.S. Pat. No. 6,602,684; and U.S. Pat.
Pub. No. 2005/0123546; each of which is incorporated by reference
in its entirety.
[0279] Other illustrative glycosylation variants which may be
incorporated into the ABPs provided herein are described, for
example, in U.S. Pat. Pub. Nos. 2003/0157108, 2004/0093621,
2003/0157108, 2003/0115614, 2002/0164328, 2004/0093621,
2004/0132140, 2004/0110704, 2004/0110282, 2004/0109865;
International Pat. Pub. Nos. 2000/61739, 2001/29246, 2003/085119,
2003/084570, 2005/035586, 2005/035778; 2005/053742, 2002/031140;
Okazaki et al., J. Mol. Biol., 2004, 336:1239-1249; and
Yamane-Ohnuki et al., Biotech. Bioeng., 2004, 87: 614-622; each of
which is incorporated by reference in its entirety.
[0280] In some embodiments, an ABP provided herein comprises an Fc
region with at least one galactose residue in the oligosaccharide
attached to the Fc region. Such ABP variants may have improved CDC
function. Examples of such ABP variants are described, for example,
in WO 1997/30087; WO 1998/58964; and WO 1999/22764; each of which
his incorporated by reference in its entirety.
[0281] Examples of cell lines capable of producing defucosylated
ABPs include Lec13 CHO cells, which are deficient in protein
fucosylation (see Ripka et al., Arch. Biochem. Biophys., 1986,
249:533-545; U.S. Pat. Pub. No. 2003/0157108; WO 2004/056312; each
of which is incorporated by reference in its entirety), and
knockout cell lines, such as alpha-1,6-fucosyltransferase gene or
FUT8 knockout CHO cells (see Yamane-Ohnuki et al., Biotech.
Bioeng., 2004, 87: 614-622; Kanda et al., Biotechnol. Bioeng.,
2006, 94:680-688; and WO 2003/085107; each of which is incorporated
by reference in its entirety).
[0282] In some embodiments, an ABP provided herein is an
aglycosylated ABP. An aglycosylated ABP can be produced using any
method known in the art or described herein. In some aspects, an
aglycosylated ABP is produced by modifying the ABP to remove all
glycosylation sites. In some aspects, the glycosylation sites are
removed only from the Fc region of the ABP. In some aspects, an
aglycosylated ABP is produced by expressing the ABP in an organism
that is not capable of glycosylation, such as E. coli, or by
expressing the ABP in a cell-free reaction mixture.
[0283] In some embodiments, an ABP provided herein has a constant
region with reduced effector function compared to a native IgG1
antibody. In some embodiments, the affinity of a constant region of
an Fc region of an ABP provided herein for Fc receptor is less than
the affinity of a native IgG1 constant region for such Fc
receptor.
[0284] 2.4 NRP-1 Domains
[0285] NRP-1 has both a transmembrane and a truncated form. The
transmembrane form is as follows. Following a short stretch of
secretion signal, NRP-1 consists of four different domains: two
repeats of CUB domain (a1/a2), two repeats of FV/VIII domain
(b1/b2), a MAM (c) domain, and a fourth domain (d) that contains
transmembrane and relatively short 40 to 43 amino acid cytoplasmic
region. The first CUB domains have significant homology with
complement factor C1s/C1r, Bone Morphogenetic Protein 1(BMP1), and
Tolloid proteins. The second FV/VIII domain shares the homology
with coagulation factor FV/VIII, one of the receptor type tyrosine
kinase DDR, and discoidin-1. The third domain MAM is the
abbreviation of meprin, A5 (former name of NRP), and receptor
protein-tyrosine phosphatase mu and kappa. In one embodiment, an
ABP provided herein binds to the al domain. In another embodiment,
an ABP provided herein binds to the a2 domain. In another
embodiment, an ABP provided herein binds to the b1 domain. In
another embodiment, an ABP provided herein binds to the b2 domain.
In one embodiment, an ABP provided herein binds to more than one
domain
[0286] 1.1. Fc Region Amino Acid Sequence Variants
[0287] In certain embodiments, an ABP provided herein comprises an
Fc region with one or more amino acid substitutions, insertions, or
deletions in comparison to a naturally occurring Fc region. In some
aspects, such substitutions, insertions, or deletions yield ABPs
with altered stability, glycosylation, or other characteristics. In
some aspects, such substitutions, insertions, or deletions yield
aglycosylated ABPs.
[0288] In some aspects, the Fc region of an ABP provided herein is
modified to yield an ABP with altered affinity for an Fc receptor,
or an ABP that is more immunologically inert. In some embodiments,
the ABP variants provided herein possess some, but not all,
effector functions. Such ABPs may be useful, for example, when the
half-life of the ABP is important in vivo, but when certain
effector functions (e.g., complement activation and ADCC) are
unnecessary or deleterious.
[0289] In some embodiments, the Fc region of an ABP provided herein
is a human IgG4 Fc region comprising one or more of the hinge
stabilizing mutations S228P and L235E. See Aalberse et al.,
Immunology, 2002, 105:9-19, incorporated by reference in its
entirety. In some embodiments, the Fc region of an ABP provided
herein is a human IgG4 Fc region comprising the hinge stabilizing
mutations S228P. In some embodiments, the IgG4 Fc region comprises
one or more of the following mutations: E233P, F234V, and L235A.
See Armour et al., Mol. Immunol., 2003, 40:585-593, incorporated by
reference in its entirety. In some embodiments, the IgG4 Fc region
comprises a deletion at position G236.
[0290] In some embodiments, the Fc region of an ABP provided herein
is a human IgG1 Fc region comprising one or more mutations to
reduce Fc receptor binding. In some aspects, the one or more
mutations are in residues selected from 5228 (e.g., S228A), L234
(e.g., L234A), L235 (e.g., L235A), D265 (e.g., D265A), and N297
(e.g., N297A). In some aspects, the ABP comprises a PVA236
mutation. PVA236 means that the amino acid sequence ELLG (SEQ ID
NO: 147), from amino acid position 233 to 236 of IgG1 or EFLG (SEQ
ID NO: 148) of IgG4, is replaced by PVA. See U.S. Pat. No.
9,150,641, incorporated by reference in its entirety.
[0291] In some embodiments, the Fc region of an ABP provided herein
is modified as described in Armour et al., Eur. J. Immunol., 1999,
29:2613-2624; WO 1999/058572; and/or U.K. Pat. App. No. 98099518;
each of which is incorporated by reference in its entirety.
[0292] In some embodiments, the Fc region of an ABP provided herein
is a human IgG2 Fc region comprising one or more of mutations A330S
and P331S.
[0293] In some embodiments, the Fc region of an ABP provided herein
has an amino acid substitution at one or more positions selected
from 238, 265, 269, 270, 297, 327 and 329. See U.S. Pat. No.
6,737,056, incorporated by reference in its entirety. Such Fc
mutants include Fc mutants with substitutions at two or more of
amino acid positions 265, 269, 270, 297 and 327, including the
so-called "DANA" Fc mutant with substitution of residues 265 and
297 with alanine. See U.S. Pat. No. 7,332,581, incorporated by
reference in its entirety. In some embodiments, the ABP comprises
an alanine at amino acid position 265. In some embodiments, the ABP
comprises an alanine at amino acid position 297.
[0294] In certain embodiments, an ABP provided herein comprises an
Fc region with one or more amino acid substitutions which improve
ADCC, such as a substitution at one or more of positions 298, 333,
and 334 of the Fc region. In some embodiments, an ABP provided
herein comprises an Fc region with one or more amino acid
substitutions at positions 239, 332, and 330, as described in Lazar
et al., Proc. Natl. Acad. Sci. USA, 2006, 103:4005-4010,
incorporated by reference in its entirety.
[0295] In some embodiments, an ABP provided herein comprises one or
more alterations that improves or diminishes C1q binding and/or
CDC. See U.S. Pat. No. 6,194,551; WO 99/51642; and Idusogie et al.,
J. Immunol., 2000, 164:4178-4184; each of which is incorporated by
reference in its entirety.
[0296] In some embodiments, an ABP provided herein comprises one or
more alterations to increase half-life. ABPs with increased
half-lives and improved binding to the neonatal Fc receptor (FcRn)
are described, for example, in Hinton et al., J. Immunol., 2006,
176:346-356; and U.S. Pat. No. 7,361,740; each of which is
incorporated by reference in its entirety. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
250, 256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340,
356, 360, 362, 376, 378, 380, 382, 413, 424, 428, and 434 of an
IgG.
[0297] In some embodiments, an ABP provided herein comprises one or
more Fc region variants as described in U.S. Pat. Nos. 7,371,826
5,648,260, and 5,624,821; Duncan and Winter, Nature, 1988,
322:738-740; and WO 94/29351; each of which is incorporated by
reference in its entirety.
[0298] 1.2. Pyroglutamate
[0299] As is known in the art, both glutamate (E) and glutamine (Q)
at the N-termini of recombinant proteins can cyclize spontaneously
to form pyroglutamate (pE) in vitro and in vivo. See Liu et al., J.
Biol. Chem., 2011, 286:11211-11217, incorporated by reference in
its entirety.
[0300] In some embodiments, provided herein are ABPs comprising a
polypeptide sequence having a pE residue at the N-terminal
position. In some embodiments, provided herein are ABPs comprising
a polypeptide sequence in which the N-terminal residue has been
converted from Q to pE. In some embodiments, provided herein are
ABPs comprising a polypeptide sequence in which the N-terminal
residue has been converted from E to pE.
[0301] In some embodiments, provided herein are ABPs comprising
V.sub.H sequences having a pE residue at the N-terminal position.
In some embodiments, provided herein are ABPs comprising a V.sub.H
sequence in which the N-terminal residue has been converted from Q
to pE. In some embodiments, provided herein is an ABP comprising a
V.sub.H sequence selected from SEQ ID Nos:85-90, 97-99, wherein the
N-terminal Q residue has been converted to pE. In some embodiments,
provided herein is a composition comprising an ABP, wherein the ABP
comprises a V.sub.H selected from SEQ ID NOs:85-90, 97-99, in which
at least about 20%, at least about 40%, at least about 60%, at
least about 80%, at least about 90%, at least about 95%, or at
least about 99% of the N-terminal residues of such V.sub.H in such
composition have been converted from Q to pE.
[0302] In some embodiments, provided herein are ABPs comprising
V.sub.H sequences having a pE residue at the N-terminal position.
In some embodiments, provided herein are ABPs comprising a V.sub.H
sequence in which the N-terminal residue has been converted from E
to pE. In some embodiments, provided herein is an ABP comprising a
V.sub.H sequence selected from SEQ ID Nos:91-96, wherein the
N-terminal E residue has been converted to pE. In some embodiments,
provided herein is a composition comprising an ABP, wherein the ABP
comprises a V.sub.H selected from SEQ ID NOs:91-96, in which at
least about 20%, at least about 40%, at least about 60%, at least
about 80%, at least about 90%, at least about 95%, or at least
about 99% of the N-terminal residues of such V.sub.H in such
composition have been converted from E to pE.
[0303] In some embodiments, provided herein are ABPs comprising
V.sub.L sequences having a pE residue at the N-terminal position.
In some embodiments, provided herein are ABPs comprising a V.sub.L
sequence in which the N-terminal residue has been converted from E
to pE. In some embodiments, provided herein is an ABP comprising a
V.sub.L sequence set forth in SEQ ID No:120, wherein the N-terminal
E residue has been converted to pE. In some embodiments, provided
herein is a composition comprising an ABP, wherein the ABP
comprises a V.sub.L set forth in SEQ ID NO:120, in which at least
about 20%, at least about 40%, at least about 60%, at least about
80%, at least about 90%, at least about 95%, or at least about 99%
of the N-terminal residues of such V.sub.L in such composition have
been converted from E to pE.
[0304] In some embodiments, provided herein are ABPs comprising
heavy chain sequences having a pE residue at the N-terminal
position. In some embodiments, provided herein are ABPs comprising
a heavy chain sequence in which the N-terminal residue has been
converted from Q to pE. In some embodiments, provided herein is an
ABP comprising a heavy chain sequence selected from SEQ ID
Nos:107-112, 119-121, wherein the N-terminal Q residue has been
converted to pE. In some embodiments, provided herein is a
composition comprising an ABP, wherein the ABP comprises a heavy
chain selected from SEQ ID NOs:107-112, 119-121, in which at least
about 20%, at least about 40%, at least about 60%, at least about
80%, at least about 90%, at least about 95%, or at least about 99%
of the N-terminal residues of such heavy chain in such composition
have been converted from Q to pE.
[0305] In some embodiments, provided herein are ABPs comprising
heavy chain sequences having a pE residue at the N-terminal
position. In some embodiments, provided herein are ABPs comprising
a heavy chain sequence in which the N-terminal residue has been
converted from E to pE. In some embodiments, provided herein is an
ABP comprising a heavy chain sequence selected from SEQ ID
Nos:113-118, wherein the N-terminal E residue has been converted to
pE. In some embodiments, provided herein is a composition
comprising an ABP, wherein the ABP comprises a heavy chain selected
from SEQ ID NOs:113-118, in which at least about 20%, at least
about 40%, at least about 60%, at least about 80%, at least about
90%, at least about 95%, or at least about 99% of the N-terminal
residues of such heavy chain in such composition have been
converted from E to pE.
[0306] In some embodiments, provided herein are ABPs comprising
light chain sequences having a pE residue at the N-terminal
position. In some embodiments, provided herein are ABPs comprising
a light chain sequence in which the N-terminal residue has been
converted from E to pE. In some embodiments, provided herein is an
ABP comprising a kappa light chain sequence selected from SEQ ID
NOs:124-125, wherein the N-terminal E residue has been converted to
pE. In some embodiments, provided herein is a composition
comprising an ABP, wherein the ABP comprises a kappa light chain
selected from SEQ ID NOs:124-125, in which at least about 20%, at
least about 40%, at least about 60%, at least about 80%, at least
about 90%, at least about 95%, or at least about 99% of the
N-terminal residues of such light chain in such composition have
been converted from E to pE.
[0307] 1.3. Cysteine Engineered Antigen-Binding Protein
Variants
[0308] In certain embodiments, provided herein are cysteine
engineered ABPs, also known as "thioMAbs," in which one or more
residues of the ABP are substituted with cysteine residues. In
particular embodiments, the substituted residues occur at solvent
accessible sites of the ABP. By substituting such residues with
cysteine, reactive thiol groups are introduced at solvent
accessible sites of the ABP and may be used to conjugate the ABP to
other moieties, such as drug moieties or linker-drug moieties, for
example, to create an immunoconjugate.
[0309] In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 of the light chain;
A118 of the heavy chain Fc region; and 5400 of the heavy chain Fc
region. Cysteine engineered ABPs may be generated as described, for
example, in U.S. Pat. No. 7,521,541, which is incorporated by
reference in its entirety.
[0310] 2. Methods of Making NRP-1 Antigen-Binding Proteins
[0311] 2.1. NRP-1 Antigen Preparation
[0312] The NRP-1 antigen used for isolation of the ABPs provided
herein may be intact NRP-1 or a fragment of NRP-1. The NRP-1
antigen may be, for example, in the form of an isolated protein or
a protein expressed on the surface of a cell.
[0313] In some embodiments, the NRP-1 antigen is a non-naturally
occurring variant of NRP-1, such as a NRP-1 protein having an amino
acid sequence or post-translational modification that does not
occur in nature.
[0314] In some embodiments, the NRP-1 antigen is truncated by
removal of, for example, intracellular or membrane-spanning
sequences, or signal sequences. In some embodiments, the NRP-1
antigen is fused at its C-terminus to a human IgG1 Fc domain or a
polyhistidine tag.
[0315] 2.2. Methods of Making Monoclonal Antibodies
[0316] Monoclonal antibodies may be obtained, for example, using
the hybridoma method first described by Kohler et al., Nature,
1975, 256:495-497 (incorporated by reference in its entirety),
and/or by recombinant DNA methods (see e.g., U.S. Pat. No.
4,816,567, incorporated by reference in its entirety). Monoclonal
antibodies may also be obtained, for example, using phage or
yeast-based libraries. See e.g., U.S. Pat. Nos. 8,258,082 and
8,691,730, each of which is incorporated by reference in its
entirety.
[0317] In the hybridoma method, a mouse or other appropriate host
animal is immunized to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
protein used for immunization. Alternatively, lymphocytes may be
immunized in vitro. Lymphocytes are then fused with myeloma cells
using a suitable fusing agent, such as polyethylene glycol, to form
a hybridoma cell. See Goding J. W., Monoclonal Antibodies:
Principles and Practice 3.sup.rd ed. (1986) Academic Press, San
Diego, Calif., incorporated by reference in its entirety.
[0318] The hybridoma cells are seeded and grown in a suitable
culture medium that contains one or more substances that inhibit
the growth or survival of the unfused, parental myeloma cells. For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine (HAT medium), which substances prevent
the growth of HGPRT-deficient cells.
[0319] Useful myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive media conditions, such
as the presence or absence of HAT medium. Among these, preferred
myeloma cell lines are murine myeloma lines, such as those derived
from MOP-21 and MC-11 mouse tumors (available from the Salk
Institute Cell Distribution Center, San Diego, Calif.), and SP-2 or
X63-Ag8-653 cells (available from the American Type Culture
Collection, Rockville, Md.). Human myeloma and mouse-human
heteromyeloma cell lines also have been described for the
production of human monoclonal antibodies. See e.g., Kozbor, J.
Immunol., 1984, 133:3001, incorporated by reference in its
entirety.
[0320] After the identification of hybridoma cells that produce
antibodies of the desired specificity, affinity, and/or biological
activity, selected clones may be subcloned by limiting dilution
procedures and grown by standard methods. See Goding, supra.
Suitable culture media for this purpose include, for example, D-MEM
or RPMI-1640 medium. In addition, the hybridoma cells may be grown
in vivo as ascites tumors in an animal
[0321] DNA encoding the monoclonal antibodies may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
monoclonal antibodies). Thus, the hybridoma cells can serve as a
useful source of DNA encoding antibodies with the desired
properties. Once isolated, the DNA may be placed into expression
vectors, which are then transfected into host cells such as
bacteria (e.g., E. coli), yeast (e.g., Saccharomyces or Pichia
sp.), COS cells, Chinese hamster ovary (CHO) cells, or myeloma
cells that do not otherwise produce antibody, to produce the
monoclonal antibodies.
[0322] In another aspect is provided a method for producing an
anti-human NRP-1 antibody or an antigen-binding fragment thereof,
comprising culturing host cell(s) selected from the group
consisting of (a) to (c) below to express an anti-human NRP-1
antibody or an antigen-binding fragment thereof: (a) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the heavy chain variable region
of the anti-human NRP-1 antibody or the antigen-binding fragment
thereof provided herein and a polynucleotide comprising a base
sequence encoding the light chain variable region of the antibody
or the antigen-binding fragment thereof; (b) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the heavy chain variable region
of the anti-human NRP-1 antibody or the antigen-binding fragment
thereof provided herein and an expression vector comprising a
polynucleotide comprising a base sequence encoding the light chain
variable region of the antibody or the antigen-binding fragment
thereof; and (c) a host cell transformed with an expression vector
comprising a polynucleotide comprising a base sequence encoding the
heavy chain variable region of the anti-human NRP-1 antibody or the
antigen-binding fragment thereof provided herein and a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the light chain variable region
of the antibody or the antigen-binding fragment thereof.
[0323] In another aspect is provided a method for producing an
anti-human NRP-1 antibody, comprising culturing host cell(s)
selected from the group consisting of (a) to (c) below to express
an anti-human NRP-1 antibody: (a) a host cell transformed with an
expression vector comprising a polynucleotide comprising a base
sequence encoding the heavy chain of the anti-human NRP-1 antibody
provided herein and a polynucleotide comprising a base sequence
encoding the light chain of the antibody; (b) a host cell
transformed with an expression vector comprising a polynucleotide
comprising a base sequence encoding the heavy chain of the
anti-human NRP-1 antibody provided herein and an expression vector
comprising a polynucleotide comprising a base sequence encoding the
light chain of the antibody; and (c) a host cell transformed with
an expression vector comprising a polynucleotide comprising a base
sequence encoding the heavy chain of the anti-human NRP-1 antibody
provided herein and a host cell transformed with an expression
vector comprising a polynucleotide comprising a base sequence
encoding the light chain of the antibody.
[0324] 2.3. Methods of Making Chimeric Antibodies
[0325] Illustrative methods of making chimeric antibodies are
described, for example, in U.S. Pat. No. 4,816,567; and Morrison et
al., Proc. Natl. Acad. Sci. USA, 1984, 81:6851-6855; each of which
is incorporated by reference in its entirety. In some embodiments,
a chimeric antibody is made by using recombinant techniques to
combine a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) with a human constant region.
[0326] 2.4. Methods of Making Humanized Antibodies
[0327] Humanized antibodies may be generated by replacing most, or
all, of the structural portions of a non-human monoclonal antibody
with corresponding human antibody sequences. Consequently, a hybrid
molecule is generated in which only the antigen-specific variable,
or CDR, is composed of non-human sequence. Methods to obtain
humanized antibodies include those described in, for example,
Winter and Milstein, Nature, 1991, 349:293-299; Rader et al., Proc.
Nat. Acad. Sci. U.S.A., 1998, 95:8910-8915; Steinberger et al., J.
Biol. Chem., 2000, 275:36073-36078; Queen et al., Proc. Natl. Acad.
Sci. U.S.A., 1989, 86:10029-10033; and U.S. Pat. Nos. 5,585,089,
5,693,761, 5,693,762, and 6,180,370; each of which is incorporated
by reference in its entirety.
[0328] 2.5. Methods of Making Human Antibodies
[0329] Human antibodies can be generated by a variety of techniques
known in the art, for example by using transgenic animals (e.g.,
humanized mice). See, e.g., Jakobovits et al., Proc. Natl. Acad.
Sci. U.S.A., 1993, 90:2551; Jakobovits et al., Nature, 1993,
362:255-258; Bruggermann et al., Year in Immuno., 1993, 7:33; and
U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807; each of which is
incorporated by reference in its entirety. Human antibodies can
also be derived from phage-display libraries (see e.g., Hoogenboom
et al., J. Mol. Biol., 1991, 227:381-388; Marks et al., J. Mol.
Biol., 1991, 222:581-597; and U.S. Pat. Nos. 5,565,332 and
5,573,905; each of which is incorporated by reference in its
entirety). Human antibodies may also be generated by in vitro
activated B cells (see e.g., U.S. Pat. Nos. 5,567,610 and
5,229,275, each of which is incorporated by reference in its
entirety). Human antibodies may also be derived from yeast-based
libraries (see e.g., U.S. Pat. No. 8,691,730, incorporated by
reference in its entirety).
[0330] 2.6. Methods of Making Antibody Fragments
[0331] The antibody fragments provided herein may be made by any
suitable method, including the illustrative methods described
herein or those known in the art. Suitable methods include
recombinant techniques and proteolytic digestion of whole
antibodies. Illustrative methods of making antibody fragments are
described, for example, in Hudson et al., Nat. Med., 2003,
9:129-134, incorporated by reference in its entirety. Methods of
making scFv antibodies are described, for example, in Pluckthun, in
The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458; each of which
is incorporated by reference in its entirety.
[0332] 2.7. Methods of Making Alternative Scaffolds
[0333] The alternative scaffolds provided herein may be made by any
suitable method, including the illustrative methods described
herein or those known in the art. For example, methods of preparing
Adnectins.TM. are described in Emanuel et al., mAbs, 2011, 3:38-48,
incorporated by reference in its entirety. Methods of preparing
iMabs are described in U.S. Pat. Pub. No. 2003/0215914,
incorporated by reference in its entirety. Methods of preparing
Anticalins.RTM. are described in Vogt and Skerra, Chem. Biochem.,
2004, 5:191-199, incorporated by reference in its entirety. Methods
of preparing Kunitz domains are described in Wagner et al.,
Biochem. & Biophys. Res. Comm., 1992, 186:118-1145,
incorporated by reference in its entirety. Methods of preparing
thioredoxin peptide aptamers are provided in Geyer and Brent, Meth.
Enzymol., 2000, 328:171-208, incorporated by reference in its
entirety. Methods of preparing Affibodies are provided in
Fernandez, Curr. Opinion in Biotech., 2004, 15:364-373,
incorporated by reference in its entirety. Methods of preparing
DARPins are provided in Zahnd et al., J. Mol. Biol., 2007,
369:1015-1028, incorporated by reference in its entirety. Methods
of preparing Affilins are provided in Ebersbach et al., J. Mol.
Biol., 2007, 372:172-185, incorporated by reference in its
entirety. Methods of preparing Tetranectins are provided in
Graversen et al., J. Biol. Chem., 2000, 275:37390-37396,
incorporated by reference in its entirety. Methods of preparing
Avimers are provided in Silverman et al., Nature Biotech., 2005,
23:1556-1561, incorporated by reference in its entirety. Methods of
preparing Fynomers are provided in Silacci et al., J. Biol. Chem.,
2014, 289:14392-14398, incorporated by reference in its
entirety.
[0334] Further information on alternative scaffolds is provided in
Binz et al., Nat. Biotechnol., 2005 23:1257-1268; and Skerra,
Current Opin. in Biotech., 2007 18:295-304, each of which is
incorporated by reference in its entirety.
[0335] 2.8. Methods of Making Variants
[0336] In some embodiments, an ABP provided herein is an affinity
matured variant of a parent ABP, which may be generated, for
example, using phage display-based affinity maturation techniques.
Briefly, one or more CDR residues may be mutated and the variant
ABPs, or portions thereof, displayed on phage and screened for
affinity. Such alterations may be made in CDR "hotspots," or
residues encoded by codons that undergo mutation at high frequency
during the somatic maturation process (see Chowdhury, Methods Mol.
Biol., 2008, 207:179-196, incorporated by reference in its
entirety), and/or residues that contact the antigen.
[0337] Any suitable method can be used to introduce variability
into a polynucleotide sequence(s) encoding an ABP, including
error-prone PCR, chain shuffling, and oligonucleotide-directed
mutagenesis such as trinucleotide-directed mutagenesis (TRIM). In
some aspects, several CDR residues (e.g., 4-6 residues at a time)
are randomized CDR residues involved in antigen binding may be
specifically identified, for example, using alanine scanning
mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often
targeted for mutation.
[0338] The introduction of diversity into the variable regions
and/or CDRs can be used to produce a secondary library. The
secondary library is then screened to identify ABP variants with
improved affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, for
example, in Hoogenboom et al., Methods in Molecular Biology, 2001,
178:1-37, incorporated by reference in its entirety.
[0339] 2.9. Vectors, Host Cells, and Recombinant Methods
[0340] Also provided are isolated nucleic acids encoding NRP-1
ABPs, vectors comprising the nucleic acids, and host cells
comprising the vectors and nucleic acids, as well as recombinant
techniques for the production of the ABPs.
[0341] In another aspect is provided a polynucleotide comprising a
base sequence encoding the heavy chain variable region of the
anti-human NRP-1 antibody or the antigen-binding fragment thereof
provided herein. In another aspect is provided a polynucleotide
comprising a base sequence encoding the light chain variable region
of the anti-human NRP-1 antibody or the antigen-binding fragment
thereof provided herein.
[0342] In another aspect is provided a polynucleotide comprising a
base sequence encoding the heavy chain of the anti-human NRP-1
antibody provided herein. In another aspect is provided a
polynucleotide comprising a base sequence encoding the light chain
of the anti-human NRP-1 antibody provided herein.
[0343] For recombinant production of an ABP, the nucleic acid(s)
encoding it may be isolated and inserted into a replicable vector
for further cloning (i.e., amplification of the DNA) or expression.
In some aspects, the nucleic acid may be produced by homologous
recombination, for example as described in U.S. Pat. No. 5,204,244,
incorporated by reference herein in its entirety.
[0344] In another aspect is provided an expression vector
comprising (a) a polynucleotide comprising a base sequence encoding
the heavy chain variable region of the anti-human NRP-1 antibody or
the antigen-binding fragment thereof provided herein and/or (b) a
polynucleotide comprising a base sequence encoding the light chain
variable region of the anti-human NRP-1 antibody or the
antigen-binding fragment thereof.
[0345] In another aspect is provided an expression vector
comprising (a) a polynucleotide comprising a base sequence encoding
the heavy chain of the anti-human NRP-1 antibody provided herein
and/or (b) a polynucleotide comprising a base sequence encoding the
light chain of the anti-human NRP-1 antibody.
[0346] Many different vectors are known in the art. The vector
components generally include one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence, for example as described in U.S. Pat. No. 5,534,615,
incorporated by reference in its entirety.
[0347] Illustrative examples of suitable host cells are provided
below. These host cells are not meant to be limiting, and any
suitable host cell may be used to produce the ABPs provided
herein.
[0348] In another aspect is provided a host cell transformed with
an expression vector selected from the group consisting of (a) to
(d): (a) a host cell transformed with an expression vector
comprising a polynucleotide comprising a base sequence encoding the
heavy chain variable region of the anti-human NRP-1 antibody or the
antigen-binding fragment thereof provided herein, and a
polynucleotide comprising a base sequence encoding the light chain
variable region of the antibody or the antigen-binding fragment
thereof; (b) a host cell transformed with an expression vector
comprising a polynucleotide comprising a base sequence encoding the
heavy chain variable region of the anti-human NRP-1 antibody or the
antigen-biding fragment thereof provided herein and an expression
vector comprising a polynucleotide comprising a base sequence
encoding the light chain variable region of the antibody or the
antigen-binding fragment thereof; (c) a host cell transformed with
an expression vector comprising a polynucleotide comprising a base
sequence encoding the heavy chain variable region of the anti-human
NRP-1 antibody or the antigen-binding fragment thereof provided
herein; and (d) a host cell transformed with an expression vector
comprising a polynucleotide comprising a base sequence encoding the
light chain variable region of the anti-human NRP-1 antibody or the
antigen-binding fragment thereof provided herein.
[0349] In another aspect is provided host cell transformed with an
expression vector selected from the group consisting of (a) to (d):
(a) a host cell transformed with an expression vector comprising a
polynucleotide comprising a base sequence encoding the heavy chain
of the anti-human NRP-1 antibody provided herein and a
polynucleotide comprising a base sequence encoding the light chain
of the antibody; (b) a host cell transformed with an expression
vector comprising a polynucleotide comprising a base sequence
encoding the heavy chain of the anti-human NRP-1 antibody provided
herein and an expression vector comprising a polynucleotide
comprising a base sequence encoding the light chain of the
antibody; (c) a host cell transformed with an expression vector
comprising a polynucleotide comprising a base sequence encoding the
heavy chain of the anti-human NRP-1 antibody provided herein; and
(d) a host cell transformed with an expression vector comprising a
polynucleotide comprising a base sequence encoding the light chain
of the anti-human NRP-1 antibody provided herein.
[0350] Suitable host cells include any prokaryotic (e.g.,
bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic
(e.g., mammalian) cells. Suitable prokaryotes include eubacteria,
such as Gram-negative or Gram-positive organisms, for example,
Enterobacteriaceae such as Escherichia (E. coli), Enterobacter,
Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia
(S. marcescans), Shigella, Bacilli (B. subtilis and B.
licheniformis), Pseudomonas (P. aeruginosa), and Streptomyces. One
useful E. coli cloning host is E. coli 294, although other strains
such as E. coli B, E. coli X1776, and E. coli W3110 are also
suitable.
[0351] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are also suitable cloning or expression
hosts for NRP-1 ABP-encoding vectors. Saccharomyces cerevisiae, or
common baker's yeast, is a commonly used lower eukaryotic host
microorganism. However, a number of other genera, species, and
strains are available and useful, such as Schizosaccharomyces
pombe, Kluyveromyces (K. lactis, K. fragilis, K. bulgaricus K.
wickeramii, K. waltii, K. drosophilarum, K. thermotolerans, and K.
marxianus), Yarrowia, Pichia pastoris, Candida (C. albicans),
Trichoderma reesia, Neurospora crassa, Schwanniomyces (S.
occidentalis), and filamentous fungi such as, for example
Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A.
niger).
[0352] The host cells used to produce the NRP-1 ABPs disclosed
herein may be cultured in a variety of media. Commercially
available media such as, for example, Ham's F10, Minimal Essential
Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium
(DMEM) are suitable for culturing the host cells. In addition, any
of the media described in Ham et al., Meth. Enz., 1979, 58:44;
Barnes et al., Anal. Biochem., 1980, 102:255; and U.S. Pat. Nos.
4,767,704, 4,657,866, 4,927,762, 4,560,655, and 5,122,469; or WO
90/03430 and WO 87/00195 may be used. Each of the foregoing
references is incorporated herein by reference in its entirety.
[0353] Any of these media may be supplemented as necessary with
hormones and/or other growth factors (such as insulin, transferrin,
or epidermal growth factor), salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides (such as adenosine and thymidine), antibiotics, trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art.
[0354] The culture conditions, such as temperature, pH, and the
like, are those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled
artisan.
[0355] When using recombinant techniques, the ABP can be produced
intracellularly, in the periplasmic space, or directly secreted
into the medium. If the ABP is produced intracellularly, as a first
step, the particulate debris, either host cells or lysed fragments,
is removed, for example, by centrifugation or ultrafiltration. For
example, Carter et al. (Bio/Technology, 1992, 10:163-167,
incorporated by reference in its entirety) describes a procedure
for isolating ABPs which are secreted to the periplasmic space of
E. coli. Briefly, cell paste is thawed in the presence of sodium
acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF)
over about 30 min. Cell debris can be removed by
centrifugation.
[0356] In some embodiments, the ABP is produced in a cell-free
system. In some aspects, the cell-free system is an in vitro
transcription and translation system as described in Yin et al.,
mAbs, 2012, 4:217-225, incorporated by reference in its entirety.
In some aspects, the cell-free system utilizes a cell-free extract
from a eukaryotic cell or from a prokaryotic cell. In some aspects,
the prokaryotic cell is E. coli. Cell-free expression of the ABP
may be useful, for example, where the ABP accumulates in a cell as
an insoluble aggregate, or where yields from periplasmic expression
are low.
[0357] Where the ABP is secreted into the medium, supernatants from
such expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon.RTM. or Millipore.RTM. Pellcon.RTM. ultrafiltration unit.
A protease inhibitor such as PMSF may be included in any of the
foregoing steps to inhibit proteolysis and antibiotics may be
included to prevent the growth of adventitious contaminants.
[0358] The ABP composition prepared from the cells can be purified
using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being a particularly useful purification
technique. The suitability of protein A as an affinity ligand
depends on the species and isotype of any immunoglobulin Fc domain
that is present in the ABP. Protein A can be used to purify ABPs
that comprise human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth., 1983, 62:1-13, incorporated by
reference in its entirety). Protein G is useful for all mouse
isotypes and for human .gamma.3 (Guss et al., EMBO J., 1986,
5:1567-1575, incorporated by reference in its entirety).
[0359] The matrix to which the affinity ligand is attached is most
often agarose, but other matrices are available. Mechanically
stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the ABP
comprises a C.sub.H3 domain, the BakerBond ABX.RTM. resin is useful
for purification.
[0360] Other techniques for protein purification, such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin Sepharose.RTM., chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available, and can be applied by one
of skill in the art.
[0361] Following any preliminary purification step(s), the mixture
comprising the ABP of interest and contaminants may be subjected to
low pH hydrophobic interaction chromatography using an elution
buffer at a pH between about 2.5 to about 4.5, generally performed
at low salt concentrations (e.g., from about 0 to about 0.25 M
salt).
3. Assays
[0362] A variety of assays known in the art may be used to identify
and characterize the NRP-1 ABPs provided herein.
[0363] 3.1. Binding, Competition, and Epitope Mapping Assays
[0364] Specific antigen-binding activity of the ABPs provided
herein may be evaluated by any suitable method, including using
SPR, BLI, RIA, KinExA, flow cytometry, and MSD-SET. Additionally,
antigen-binding activity may be evaluated by ELISA assays and
western blot assays.
[0365] Assays for measuring competition between two ABPs, or an ABP
and another molecule (e.g., one or more ligands of NRP-1) are
described elsewhere in this disclosure and, for example, in Harlow
and Lane, Antibodies: A Laboratory Manual ch. 14, 1988, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y, incorporated by
reference in its entirety.
[0366] Assays for mapping the epitopes to which the ABPs provided
herein bind are described, for example, in Morris "Epitope Mapping
Protocols," in Methods in Molecular Biology vol. 66, 1996, Humana
Press, Totowa, N.J., incorporated by reference in its entirety. In
some embodiments, the epitope is determined by peptide competition.
In some embodiments, the epitope is determined by mass
spectrometry. In some embodiments, the epitope is determined by
crystallography.
[0367] 3.2. NRP-1 Antagonism Assays
[0368] In some embodiments, the ABPs provided herein are screened
to identify or characterize ABPs with antagonistic activity against
NRP-1. Any suitable assay may be used to identify or characterize
such ABPs. In some aspects, the assay measures the amount of a
cytokine secreted by an effector T cell after contacting the
effector T cell with an ABP provided herein. In some aspects, the
cytokine is selected from IL-2, IL-6, LT-.alpha., TNF, GM-CSF,
IFN.gamma., and combinations thereof. In some aspects, the cytokine
is selected from sCD40L, VEGF, TGF-.alpha., RANTES, PDGF-AB/BB,
PDGF-AA, MIP-1.beta., MIP-1.alpha., MDC (CCL22), MCP-3, MCP-1,
IP-10, IL-17A, IL-2R.alpha., IL-15, IL-13, IL-12 (p70), IL-12
(p40), IL-10, IL-9, IL-8, IL-7, IL-5, IL-4, IL-3, IL-2,
IL-2R.alpha., IL-1RA, IL-1.beta., IL-1.alpha., IFN.gamma.,
IFN.alpha.2, GRO, GM-CSF, G-CSF, fractalkine, Flt-3 ligand, FGF-2,
eotaxin, EGF, and combinations thereof.
[0369] In some embodiments, the effector cells are co-stimulated
with an agonist of CD3, to promote the secretion of cytokines by
the effector cell. In some aspects, the CD3 agonist is provided at
a submaximal level.
[0370] In some aspects, such assays may measure the proliferation
of an effector T cell after contacting the effector T cell with an
ABP provided herein. In some aspects, proliferation of the effector
T cell is measured by dilution of a dye (e.g., carboxyfluorescein
diacetate succinimidyl ester; CFSE), by tritiated thymidine uptake,
by luminescent cell viability assays, or by other assays known in
the art.
[0371] In some aspects, such assays may measure the
differentiation, cytokine production, viability (e.g., survival),
proliferation, or suppressive activity of a regulatory T cell after
contacting the regulatory T cell with an ABP provided herein.
[0372] In some aspects, such assays may measure the cytotoxic
activity of an NK cell after contacting the NK cell with an ABP
provided herein. In some aspects, the cytotoxic activity of the NK
cell is measured using a cytotoxicity assay that quantifies
NK-mediated killing of target cells (e.g., a K562 cell line). See
Jang et al., Ann. Clin. Lab. Sci., 2012, 42:42-49, incorporated by
reference in its entirety.
[0373] In some aspects, such assays may measure the amount of
granzyme B. In some aspects, such assays may measure the amount of
perforin.
[0374] 3.3. Assays for Effector Functions
[0375] Effector function following treatment with the ABPs provided
herein may be evaluated using a variety of in vitro and in vivo
assays known in the art, including those described in Ravetch and
Kinet, Annu. Rev. Immunol., 1991, 9:457-492; U.S. Pat. Nos.
5,500,362, 5,821,337; Hellstrom et al., Proc. Nat'l Acad. Sci. USA,
1986, 83:7059-7063; Hellstrom et al., Proc. Nat'l Acad. Sci. USA,
1985, 82:1499-1502; Bruggemann et al., J. Exp. Med., 1987,
166:1351-1361; Clynes et al., Proc. Nat'l Acad. Sci. USA, 1998,
95:652-656; WO 2006/029879; WO 2005/100402; Gazzano-Santoro et al.,
J. Immunol. Methods, 1996, 202:163-171; Cragg et al., Blood, 2003,
101:1045-1052; Cragg et al. Blood, 2004, 103:2738-2743; and Petkova
et al., Int'l. Immunol., 2006, 18:1759-1769; each of which is
incorporated by reference in its entirety.
4. Pharmaceutical Compositions
[0376] The ABPs provided herein can be formulated in any
appropriate pharmaceutical composition and administered by any
suitable route of administration. Suitable routes of administration
include, but are not limited to, the intraarterial, intradermal,
intramuscular, intraperitoneal, intravenous, nasal, parenteral,
pulmonary, and subcutaneous routes.
[0377] In another aspect is provided a pharmaceutical composition
comprising an anti-human NRP-1 antibody or an antigen-binding
fragment thereof provided herein and pharmaceutically acceptable
excipients.
[0378] In another aspect is provided a pharmaceutical composition
comprising plural kinds of anti-human NRP-1 antibodies or
antigen-binding fragments thereof provided herein. For example, the
pharmaceutical composition comprises an antibody or an
antigen-binding fragment thereof, which does not undergo
posttranslational modification and an antibody or an
antigen-binding fragment thereof derived from posttranslational
modification of the antibody or the antigen-binding fragment
thereof.
[0379] In one embodiment, the pharmaceutical composition comprises
at least two kinds of anti-human NRP-1 antibodies selected from (1)
to (4): (1) an anti-human NRP-1 antibody comprising a heavy chain
consisting of SEQ ID NO:118, and a light chain consisting of SEQ ID
NO:126, (2) an anti-human NRP-1 antibody comprising a heavy chain
consisting of SEQ ID NO:118 in which E of the amino acid number 1
is modified to pyroglutamate, and a light chain consisting of SEQ
ID NO:126, (3) an anti-human NRP-1 antibody comprising a heavy
chain consisting of the amino acid sequence of amino acid numbers 1
to 453 of SEQ ID NO:118, and a light chain consisting of SEQ ID
NO:126; and (4) an anti-human NRP-1 antibody comprising a heavy
chain consisting of the amino acid sequence of amino acid numbers 1
to 453 of SEQ ID NO:118 in which E of the amino acid number 1 is
modified to pyroglutamate, and a light chain consisting of SEQ ID
NO:126.
[0380] In one embodiment, the pharmaceutical composition comprises
an anti-human NRP-1 antibody comprising a heavy chain consisting of
SEQ ID NO:118, and a light chain consisting of SEQ ID NO:126, an
anti-human NRP-1 antibody comprising a heavy chain consisting of
the amino acid sequence of amino acid numbers 1 to 453 of SEQ ID
NO:118, and a light chain consisting of SEQ ID NO:126, and a
pharmaceutically acceptable excipient.
[0381] The pharmaceutical composition may comprise one or more
pharmaceutical excipients. Any suitable pharmaceutical excipient
may be used, and one of ordinary skill in the art is capable of
selecting suitable pharmaceutical excipients. Accordingly, the
pharmaceutical excipients provided below are intended to be
illustrative, and not limiting. Additional pharmaceutical
excipients include, for example, those described in the Handbook of
Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009),
incorporated by reference in its entirety.
[0382] In some embodiments, the pharmaceutical composition
comprises an anti-foaming agent. Any suitable anti-foaming agent
may be used. In some aspects, the anti-foaming agent is selected
from an alcohol, an ether, an oil, a wax, a silicone, a surfactant,
and combinations thereof. In some aspects, the anti-foaming agent
is selected from a mineral oil, a vegetable oil, ethylene bis
stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a
long chain fatty alcohol, a fatty acid soap, a fatty acid ester, a
silicon glycol, a fluorosilicone, a polyethylene
glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon
dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate,
ethyl alcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol,
simethicone, and combinations thereof.
[0383] In some embodiments, the pharmaceutical composition
comprises a cosolvent. Illustrative examples of cosolvents include
ethanol, poly(ethylene) glycol, butylene glycol, dimethylacetamide,
glycerin, propylene glycol, and combinations thereof.
[0384] In some embodiments, the pharmaceutical composition
comprises a buffer. Illustrative examples of buffers include
acetate, borate, carbonate, lactate, malate, phosphate, citrate,
hydroxide, diethanolamine, monoethanolamine, glycine, methionine,
guar gum, monosodium glutamate, and combinations thereof.
[0385] In some embodiments, the pharmaceutical composition
comprises a carrier or filler. Illustrative examples of carriers or
fillers include lactose, maltodextrin, mannitol, sorbitol,
chitosan, stearic acid, xanthan gum, guar gum, and combinations
thereof.
[0386] In some embodiments, the pharmaceutical composition
comprises a surfactant. Illustrative examples of surfactants
include d-alpha tocopherol, benzalkonium chloride, benzethonium
chloride, cetrimide, cetylpyridinium chloride, docusate sodium,
glyceryl behenate, glyceryl monooleate, lauric acid, macrogol 15
hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene
alkyl ethers, polyoxyethylene sorbitan fatty acid esters,
polyoxyethylene stearates, polyoxylglycerides, sodium lauryl
sulfate, sorbitan esters, vitamin E polyethylene(glycol) succinate,
and combinations thereof.
[0387] In some embodiments, the pharmaceutical composition
comprises an anti-caking agent. Illustrative examples of
anti-caking agents include calcium phosphate (tribasic),
hydroxymethyl cellulose, hydroxypropyl cellulose, magnesium oxide,
and combinations thereof.
[0388] Other excipients that may be used with the pharmaceutical
compositions include, for example, albumin, antioxidants,
antibacterial agents, antifungal agents, bioabsorbable polymers,
chelating agents, controlled release agents, diluents, dispersing
agents, dissolution enhancers, emulsifying agents, gelling agents,
ointment bases, penetration enhancers, preservatives, solubilizing
agents, solvents, stabilizing agents, sugars, and combinations
thereof. Specific examples of each of these agents are described,
for example, in the Handbook of Pharmaceutical Excipients, Rowe et
al. (Eds.) 6th Ed. (2009), The Pharmaceutical Press, incorporated
by reference in its entirety.
[0389] In some embodiments, the pharmaceutical composition
comprises a solvent. In some aspects, the solvent is saline
solution, such as a sterile isotonic saline solution or dextrose
solution. In some aspects, the solvent is water for injection.
[0390] In some embodiments, the pharmaceutical compositions are in
a particulate form, such as a microparticle or a nanoparticle.
Microparticles and nanoparticles may be formed from any suitable
material, such as a polymer or a lipid. In some aspects, the
microparticles or nanoparticles are micelles, liposomes, or
polymersomes.
[0391] Further provided herein are anhydrous pharmaceutical
compositions and dosage forms comprising an ABP, since water can
facilitate the degradation of some ABPs.
[0392] Anhydrous pharmaceutical compositions and dosage forms
provided herein can be prepared using anhydrous or low moisture
containing ingredients and low moisture or low humidity conditions.
Pharmaceutical compositions and dosage forms that comprise lactose
and at least one active ingredient that comprises a primary or
secondary amine can be anhydrous if substantial contact with
moisture and/or humidity during manufacturing, packaging, and/or
storage is expected.
[0393] An anhydrous pharmaceutical composition should be prepared
and stored such that its anhydrous nature is maintained.
Accordingly, anhydrous compositions can be packaged using materials
known to prevent exposure to water such that they can be included
in suitable formulary kits. Examples of suitable packaging include,
but are not limited to, hermetically sealed foils, plastics, unit
dose containers (e.g., vials), blister packs, and strip packs.
[0394] 4.1. Parenteral Dosage Forms
[0395] In certain embodiments, the ABPs provided herein are
formulated as parenteral dosage forms. Parenteral dosage forms can
be administered to subjects by various routes including, but not
limited to, subcutaneous, intravenous (including infusions and
bolus injections), intramuscular, and intraarterial. Because their
administration typically bypasses subjects' natural defenses
against contaminants, parenteral dosage forms are typically,
sterile or capable of being sterilized prior to administration to a
subject. Examples of parenteral dosage forms include, but are not
limited to, solutions ready for injection, dry (e.g., lyophilized)
products ready to be dissolved or suspended in a pharmaceutically
acceptable vehicle for injection, suspensions ready for injection,
and emulsions.
[0396] Suitable vehicles that can be used to provide parenteral
dosage forms are well known to those skilled in the art. Examples
include, but are not limited to: Water for Injection USP; aqueous
vehicles such as, but not limited to, Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium
Chloride Injection, and Lactated Ringer's Injection; water miscible
vehicles such as, but not limited to, ethyl alcohol, polyethylene
glycol, and polypropylene glycol; and non-aqueous vehicles such as,
but not limited to, corn oil, cottonseed oil, peanut oil, sesame
oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
[0397] Excipients that increase the solubility of one or more of
the ABPs disclosed herein can also be incorporated into the
parenteral dosage forms.
[0398] In some embodiments, the parenteral dosage form is
lyophilized. Exemplary lyophilized formulations are described, for
example, in U.S. Pat. Nos. 6,267,958 and 6,171,586; and WO
2006/044908; each of which is incorporated by reference in its
entirety.
5. Dosage and Unit Dosage Forms
[0399] In human therapeutics, the doctor will determine the
posology which he considers most appropriate according to a
preventive or curative treatment and according to the age, weight,
condition and other factors specific to the subject to be
treated.
[0400] In certain embodiments, a composition provided herein is a
pharmaceutical composition or a single unit dosage form.
Pharmaceutical compositions and single unit dosage forms provided
herein comprise a prophylactically or therapeutically effective
amount of one or more prophylactic or therapeutic ABPs.
[0401] The amount of the ABP or composition which will be effective
in the prevention or treatment of a disorder or one or more
symptoms thereof will vary with the nature and severity of the
disease or condition, and the route by which the ABP is
administered. The frequency and dosage will also vary according to
factors specific for each subject depending on the specific therapy
(e.g., therapeutic or prophylactic agents) administered, the
severity of the disorder, disease, or condition, the route of
administration, as well as age, body, weight, response, and the
past medical history of the subject. Effective doses may be
extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0402] In certain embodiments, exemplary doses of a composition
include milligram or microgram amounts of the ABP per kilogram of
subject or sample weight (e.g., about 10 micrograms per kilogram to
about 50 milligrams per kilogram, about 100 micrograms per kilogram
to about 25 milligrams per kilogram, or about 100 micrograms per
kilogram to about 10 milligrams per kilogram). In certain
embodiment, the dosage of the ABP provided herein, based on weight
of the ABP, administered to prevent, treat, manage, or ameliorate a
disorder, or one or more symptoms thereof in a subject is 0.1
mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10
mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, or more of
a subject's body weight. It may be necessary to use dosages of the
ABP outside the ranges disclosed herein in some cases, as will be
apparent to those of ordinary skill in the art. Furthermore, it is
noted that the clinician or treating physician will know how and
when to interrupt, adjust, or terminate therapy in conjunction with
subject response.
[0403] Different therapeutically effective amounts may be
applicable for different diseases and conditions, as will be
readily known by those of ordinary skill in the art. Similarly,
amounts sufficient to prevent, manage, treat or ameliorate such
disorders, but insufficient to cause, or sufficient to reduce,
adverse effects associated with the ABPs provided herein are also
encompassed by the dosage amounts and dose frequency schedules
provided herein. Further, when a subject is administered multiple
dosages of a composition provided herein, not all of the dosages
need be the same. For example, the dosage administered to the
subject may be increased to improve the prophylactic or therapeutic
effect of the composition or it may be decreased to reduce one or
more side effects that a particular subject is experiencing.
[0404] In certain embodiments, treatment or prevention can be
initiated with one or more loading doses of an ABP or composition
provided herein followed by one or more maintenance doses.
[0405] In certain embodiments, a dose of an ABP or composition
provided herein can be administered to achieve a steady-state
concentration of the ABP in blood or serum of the subject. The
steady-state concentration can be determined by measurement
according to techniques available to those of skill or can be based
on the physical characteristics of the subject such as height,
weight and age.
[0406] In certain embodiments, administration of the same
composition may be repeated and the administrations may be
separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15
days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months.
In other embodiments, administration of the same composition may be
repeated and the composition may be given once weekly, once every
two weeks, once every three weeks, or once every four weeks. In
certain embodiments, the first dose administered to the patient may
be a "loading dose." A loading dose may be a higher dose than
subsequent doses.
[0407] As discussed in more detail elsewhere in this disclosure, an
ABP provided herein may optionally be administered with one or more
additional agents useful to prevent or treat a disease or disorder.
The effective amount of such additional agents may depend on the
amount of ABP present in the formulation, the type of disorder or
treatment, and the other factors known in the art or described
herein.
6. Therapeutic Applications
[0408] For therapeutic applications, the ABPs of the invention are
administered to a mammal, generally a human, in a pharmaceutically
acceptable dosage form such as those known in the art and those
discussed above. For example, the ABPs of the invention may be
administered to a human intravenously as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, or intratumoral routes. The ABPs also are suitably
administered by peritumoral, intralesional, or perilesional routes,
to exert local as well as systemic therapeutic effects. The
intraperitoneal route may be particularly useful, for example, in
the treatment of ovarian tumors.
[0409] The ABPs provided herein may be useful for the treatment of
any disease or condition involving NRP-1. In some embodiments, the
disease or condition is a disease or condition that can benefit
from treatment with an anti-NRP-1 ABP. In some embodiments, the
disease or condition is a tumor. In some embodiments, the disease
or condition is a cell proliferative disorder. In some embodiments,
the disease or condition is a cancer.
[0410] In some embodiments, the ABPs provided herein are provided
for use as a medicament. In some embodiments, the ABPs provided
herein are provided for use in the manufacture or preparation of a
medicament. In some embodiments, the medicament is for the
treatment of a disease or condition that can benefit from an
anti-NRP-1 ABP. In some embodiments, the disease or condition is a
tumor. In some embodiments, the disease or condition is a cell
proliferative disorder. In some embodiments, the disease or
condition is a cancer. In some embodiments, the disease or
condition is a viral infection.
[0411] In some embodiments, provided herein is a method of treating
a disease or condition in a subject in need thereof by
administering an effective amount of an ABP provided herein to the
subject. In some aspects, the disease or condition is a cancer. In
some aspects, the disease or condition is a viral infection.
[0412] Any suitable cancer may be treated with the ABPs provided
herein. Illustrative suitable cancers include, for example, acute
lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),
adrenocortical carcinoma, anal cancer, appendix cancer,
astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer,
bladder cancer, bone cancer, breast cancer, bronchial tumor,
carcinoma of unknown primary origin, cardiac tumor, cervical
cancer, chordoma, colon cancer, colorectal cancer,
craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial
cancer, ependymoma, esophageal cancer, esthesioneuroblastoma,
fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor,
gallbladder cancer, gastric cancer, gastrointestinal carcinoid
tumor, gastrointestinal stromal tumor, gestational trophoblastic
disease, glioma, head and neck cancer, hepatocellular cancer,
histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular
melanoma, islet cell tumor,
[0413] Kaposi sarcoma, kidney cancer, Langerhans cell
histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver
cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia,
malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma,
mesothelioma, metastatic squamous neck cancer with occult primary,
midline tract carcinoma involving NUT gene, mouth cancer, multiple
endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides,
myelodysplastic syndrome, myelodysplastic/myeloproliferative
neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal
cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal
cancer, osteosarcoma, ovarian cancer, pancreatic cancer,
papillomatosis, paraganglioma, parathyroid cancer, penile cancer,
pharyngeal cancer, pheochromocytomas, pituitary tumor,
pleuropulmonary blastoma, primary central nervous system lymphoma,
prostate cancer, rectal cancer, renal cell cancer, renal pelvis and
ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland
cancer, Sezary syndrome, skin cancer, small cell lung cancer, small
intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach
cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat
cancer, thymoma and thymic carcinoma, thyroid cancer, urethral
cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms
tumor.
[0414] In some embodiments, provided herein is a method of
antagonizing NRP-1 in a target cell of a subject in need thereof by
administering an effective amount of an ABP provided herein to the
subject. In some aspects, antagonism of NRP-1 by an ABP provided
herein results in increased secretion of IL-2, LT-.alpha., IL-6,
TNF, GM-CSF, IFN.gamma. or combinations thereof by a target
cell.
[0415] In some embodiments, provided herein is a method of
increasing the proliferation, survival, and/or function of an
effector T cell in a subject in need thereof by administering an
effective amount of an ABP provided herein to the subject. In some
aspects, the effector T cell is a CD4+ effector T cell. In some
aspects, the effector T cell is a CD8+ effector T cell.
[0416] In some embodiments, provided herein is a method of
abrogating suppression of an effector T cell by a regulatory T cell
in a subject in need thereof by administering an effective amount
of an ABP provided herein to the subject. In some aspects, the
regulatory T cell is a CD4+CD25+Foxp3+ regulator T cell. In some
aspects, the regulatory T cell is a CD8+CD25+ regulatory T
cell.
[0417] In some embodiments, provided herein is a method of
increasing the activity of a natural killer (NK) cell, a natural
killer T (NKT) cell, a macrophage, or a dendritic cell (e.g., a
plasmacytoid dendritic cell) in a subject in need thereof by
administering an effective amount of an ABP provided herein to the
subject.
[0418] In some embodiments, provided herein is a method of treating
a subject having a cancer without concomitant platelet reduction.
In some aspects, the method does not result in a substantive amount
of thrombocytopenia in the subject.
[0419] In some embodiments, provided herein is a method of
enhancing an immune response in a subject in need thereof by
administering an effective amount of an ABP provided herein to the
subject.
[0420] In some embodiments, provided herein is a method delaying
the onset of a tumor in a subject in need thereof by administering
an effective amount of an ABP provided herein to the subject.
[0421] In some embodiments, provided herein is a method preventing
the onset of a tumor in a subject in need thereof by administering
an effective amount of an ABP provided herein to the subject.
[0422] In some embodiments, provided herein is a method of delaying
the onset of a cancer in a subject in need thereof by administering
an effective amount of an ABP provided herein to the subject.
[0423] In some embodiments, provided herein is a method of
preventing the onset of a cancer in a subject in need thereof by
administering an effective amount of an ABP provided herein to the
subject.
[0424] In some embodiments, provided herein is a method of reducing
the size of a tumor in a subject in need thereof by administering
an effective amount of an ABP provided herein to the subject.
[0425] In some embodiments, provided herein is a method of reducing
the number of metastases in a subject in need thereof by
administering an effective amount of an ABP provided herein to the
subject.
[0426] In some embodiments, provided herein is a method of reducing
viral titer a subject in need thereof by administering an effective
amount of an ABP provided herein to the subject.
[0427] In some embodiments, provided herein is a method for
extending the period of overall survival, median survival time, or
progression-free survival in a subject in need thereof by
administering an effective amount of an ABP provided herein to the
subject.
[0428] In some embodiments, provided herein is a method for
treating a subject who has become resistant to a standard of care
therapeutic by administering an effective amount of an ABP provided
herein to the subject. In some embodiments, the standard-of-care
therapeutic to which the subject has become resistant is a PD-1
inhibitor. In other embodiments, the standard-of-care therapeutic
to which the subject has become resistant is a PD-L1 inhibitor. In
other embodiments, the standard-of-care therapeutic to which the
subject has become resistant is a CTLA-4 inhibitor.
7. Combination Therapies
[0429] In some embodiments, an ABP provided herein is administered
with at least one additional therapeutic agent. Any suitable
additional therapeutic agent may be administered with an ABP
provided herein. In some aspects, the additional therapeutic agent
is selected from radiation, a cytotoxic agent, a chemotherapeutic
agent, a cytostatic agent, an anti-hormonal agent, an EGFR
inhibitor, an immunostimulatory agent, an anti-angiogenic agent,
and combinations thereof.
[0430] In some embodiments, the additional therapeutic agent
comprises an immunostimulatory agent.
[0431] In some embodiments, the immunostimulatory agent is an agent
that blocks signaling of an inhibitory receptor of an immune cell,
or a ligand thereof. In some aspects, the inhibitory receptor or
ligand is selected from PVRIG, VISTA, CCR4, CD27, CTLA-4, PD-1,
PD-L1, LAG-3, Tim3, TIGIT, neuritin, BTLA, KIR, and combinations
thereof. In some aspects, the agent is selected from an anti-PD-1
antibody (e.g., pembrolizumab or nivolumab), and anti-PD-L1
antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g.,
ipilimumab), and combinations thereof. In some aspects, the agent
is pembrolizumab. In some aspects, the agent is nivolumab. In some
aspects, the agent is atezolizumab.
[0432] In some embodiments, the additional therapeutic agent is an
agent that inhibits the interaction between PD-1 and PD-L1. In some
aspects, the additional therapeutic agent that inhibits the
interaction between PD-1 and PD-L1 is selected from an antibody, a
peptidomimetic and a small molecule. In some aspects, the
additional therapeutic agent that inhibits the interaction between
PD-1 and PD-L1 is selected from pembrolizumab, nivolumab,
atezolizumab, avelumab, durvalumab, BMS-936559, sulfamonomethoxine
1, and sulfamethizole 2. In some embodiments, the additional
therapeutic agent that inhibits the interaction between PD-1 and
PD-L1 is any therapeutic known in the art to have such activity,
for example as described in Weinmann et al., Chem Med Chem, 2016,
14:1576 (DOI: 10.1002/cmdc.201500566), incorporated by reference in
its entirety. In some embodiments, the agent that inhibits the
interaction between PD-1 and PD-L1 is formulated in the same
pharmaceutical composition an ABP provided herein. In some
embodiments, the agent that inhibits the interaction between PD-1
and PD-L1 is formulated in a different pharmaceutical composition
from an ABP provided herein. In some embodiments, the agent that
inhibits the interaction between PD-1 and PD-L1 is administered
prior to administration of an ABP provided herein. In some
embodiments, the agent that inhibits the interaction between PD-1
and PD-L1 is administered after administration of an ABP provided
herein. In some embodiments, the agent that inhibits the
interaction between PD-1 and PD-L1 is administered
contemporaneously with an ABP provided herein, but the agent and
ABP are administered in separate pharmaceutical compositions.
[0433] In some embodiments, the immunostimulatory agent is an agent
that, when administered alone and at its recommended dosage,
results in a certain amount of thrombocytopenia in the subject. In
some aspects, such an agent may be administered in combination with
an ABP provided herein at a reduced dosage. Such combination
therapy may be safely administered without resulting in substantive
platelet deterioration or thrombocytopenia.
[0434] In some embodiments, the immunostimulatory agent is an
agonist of a co-stimulatory receptor of an immune cell. In some
aspects, the co-stimulatory receptor is selected from OX40, ICOS,
CD28, CD37, GITR, CD40, and 4-1BB, and combinations thereof. In
some embodiments, the agonist is an antibody.
[0435] In some embodiments, the immunostimulatory agent is a
cytokine. In some aspects, the cytokine is selected from IL-2,
IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.
[0436] In some embodiments, the immunostimulatory agent is an
oncolytic virus. In some aspects, the oncolytic virus is selected
from a herpes simplex virus, a vesicular stomatitis virus, an
adenovirus, a Newcastle disease virus, a vaccinia virus, and a
maraba virus.
[0437] In some embodiments, the immunostimulatory agent is a T cell
with a chimeric antigen receptor (CAR-T cell). In some embodiments,
the immunostimulatory agent is a bi- or multi-specific T
cell-directed antibody. In some embodiments, the immunostimulatory
agent is an anti-TGF-.beta. antibody. In some embodiments, the
immunostimulatory agent is a TGF-.beta. trap.
[0438] In some embodiments, the additional therapeutic agent is a
vaccine to a tumor antigen. Any suitable antigen may be targeted by
the vaccine, provided that it is present in a tumor treated by the
methods provided herein. In some aspects, the tumor antigen is a
tumor antigen that is overexpressed in comparison its expression
levels in normal tissue. In some aspects, the tumor antigen is
selected from cancer testis antigen, differentiation antigen,
NY-ESO-1, MAGE-A1, MART, and combinations thereof.
[0439] Further examples of additional therapeutic agents include a
taxane (e.g., paclitaxel or docetaxel); a platinum agent (e.g.,
carboplatin, oxaliplatin, and/or cisplatin); a topoisomerase
inhibitor (e.g., irinotecan, topotecan, etoposide, and/or
mitoxantrone); folinic acid (e.g., leucovorin); or a nucleoside
metabolic inhibitor (e.g., fluorouracil, capecitabine, and/or
gemcitabine). In some embodiments, the additional therapeutic agent
is folinic acid, 5-fluorouracil, and/or oxaliplatin. In some
embodiments, the additional therapeutic agent is 5-fluorouracil and
irinotecan. In some embodiments, the additional therapeutic agent
is a taxane and a platinum agent. In some embodiments, the
additional therapeutic agent is paclitaxel and carboplatin. In some
embodiments, the additional therapeutic agent is pemetrexate. In
some embodiments, the additional therapeutic agent is a targeted
therapeutic such as an EGFR, RAF or MEK-targeted agent.
[0440] The additional therapeutic agent may be administered by any
suitable means. In some embodiments, an ABP provided herein and the
additional therapeutic agent are included in the same
pharmaceutical composition. In some embodiments, an ABP provided
herein and the additional therapeutic agent are included in
different pharmaceutical compositions.
[0441] In embodiments where an ABP provided herein and the
additional therapeutic agent are included in different
pharmaceutical compositions, administration of the ABP can occur
prior to, simultaneously, and/or following, administration of the
additional therapeutic agent. In some aspects, administration of an
ABP provided herein and the additional therapeutic agent occur
within about one month of each other. In some aspects,
administration of an ABP provided herein and the additional
therapeutic agent occur within about one week of each other. In
some aspects, administration of an ABP provided herein and the
additional therapeutic agent occur within about one day of each
other. In some aspects, administration of an ABP provided herein
and the additional therapeutic agent occur within about twelve
hours of each other. In some aspects, administration of an ABP
provided herein and the additional therapeutic agent occur within
about one hour of each other.
8. Kits
[0442] Also provided are kits comprising the ABPs provided herein.
The kits may be used for the treatment, prevention, and/or
diagnosis of a disease or disorder, as described herein.
[0443] In some embodiments, the kit comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
and IV solution bags. The containers may be formed from a variety
of materials, such as glass or plastic. The container holds a
composition that is by itself, or when combined with another
composition, effective for treating, preventing and/or diagnosing a
disease or disorder. The container may have a sterile access port.
For example, if the container is an intravenous solution bag or a
vial, it may have a port that can be pierced by a needle. At least
one active agent in the composition is an ABP provided herein. The
label or package insert indicates that the composition is used for
treating the selected condition.
[0444] In some embodiments, the kit comprises (a) a first container
with a first composition contained therein, wherein the first
composition comprises an ABP provided herein; and (b) a second
container with a second composition contained therein, wherein the
second composition comprises a further therapeutic agent. The kit
in this embodiment of the invention may further comprise a package
insert indicating that the compositions can be used to treat a
particular condition.
[0445] Alternatively, or additionally, the kit may further comprise
a second (or third) container comprising a
pharmaceutically-acceptable excipient. In some aspects, the
excipient is a buffer. The kit may further include other materials
desirable from a commercial and user standpoint, including filters,
needles, and syringes.
EXAMPLES
[0446] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided herein.
Example 1. Antibody Selection
Materials and Methods
[0447] Antigens were biotinylated using the EZ-Link
Sulfo-NHS-Biotinylation Kit from Pierce. Goat F(ab').sub.2
anti-human kappa-FITC (LC-FITC), ExtrAvidin-PE (EA-PE) and
Streptavidin-AF633 (SA-633) were obtained from Southern Biotech,
Sigma, and Molecular Probes, respectively. Streptavidin MicroBeads
and MACS LC separation columns were purchased from Miltenyi Biotec.
Goat anti-human IgG-PE (Human-PE) was obtained from Southern
Biotech.
Naive Discovery
[0448] Eight naive human synthetic yeast libraries each of
.about.10.sup.9 diversity were propagated as previously described
(see, e.g., Y. Xu et al, Addressing polyspecificity of antibodies
selected from an in vitro yeast presentation system: a FACS-based,
high-throughput selection and analytical tool. PEDS 26.10, 663-70
(2013); WO2009036379; WO2010105256; and WO2012009568.) For the
first two rounds of selection, a magnetic bead sorting technique
utilizing the Miltenyi MACS system was performed, as previously
described (see, e.g., Siegel et al, High efficiency recovery and
epitope-specific sorting of an scFv yeast display library." J
Immunol Methods 286(1-2), 141-153 (2004).) Briefly, yeast cells
(.about.10.sup.10 cells/library) were incubated with 5 ml of 100 nM
biotinylated antigen for 30 min at 30.degree. C. in wash buffer
(phosphate-buffered saline (PBS)/0.1% bovine serum albumin (BSA)).
After washing once with 40 ml ice-cold wash buffer, the cell pellet
was resuspended in 20 mL wash buffer, and Streptavidin MicroBeads
(500 .mu.l) were added to the yeast and incubated for 15 min at
4.degree. C. Next, the yeast were pelleted, resuspended in 20 mL
wash buffer, and loaded onto a Miltenyi LS column. After the 20 mL
were loaded, the column was washed 3 times with 3 ml wash buffer.
The column was then removed from the magnetic field, and the yeast
were eluted with 5 mL of growth media and then grown overnight. The
following rounds of selection were performed using flow cytometry.
Approximately 2.times.10.sup.7 yeast were pelleted, washed three
times with wash buffer, and incubated at 30.degree. C. with either
decreasing concentrations of biotinylated antigen (100 to 1 nM)
under equilibrium conditions, 100 nM biotinylated antigens of
different species in order to obtain species cross-reactivity, or
with a poly-specificity depletion reagent (PSR) to remove
non-specific antibodies from the selection. For the PSR depletion,
the libraries were incubated with a 1:10 dilution of biotinylated
PSR reagent as previously described (see, e.g., Y. Xu et al,
Addressing polyspecificity of antibodies selected from an in vitro
yeast presentation system: a FACS-based, high-throughput selection
and analytical tool. PEDS 26.10, 663-70 (2013).) Yeast were then
washed twice with wash buffer and stained with LC-FITC (diluted
1:100) and either SA-633 (diluted 1:500) or EAPE (diluted 1:50)
secondary reagents for 15 min at 4.degree. C. After washing twice
with wash buffer, the cell pellets were resuspended in 0.3 mL wash
buffer and transferred to strainer-capped sort tubes. Sorting was
performed using a FACS ARIA sorter (BD Biosciences) and sort gates
were determined to select for antibodies with desired
characteristics. Selection rounds were repeated until a population
with all of the desired characteristics was obtained. After the
final round of sorting, yeast were plated and individual colonies
were picked for characterization.
[0449] Antibody Optimization
[0450] Optimization of antibodies was performed via a light chain
diversification protocol, and then by introducing diversities into
the heavy chain and light chain variable regions as described
below. A combination of some of these approaches was used for each
antibody.
[0451] Light chain batch diversification protocol: Heavy chain
plasmids from a naive selection output were extracted from the
yeast via smash and grab, propagated in and subsequently purified
from E. coli, and transformed into a light chain library with a
diversity of 5.times.10.sup.6. Selections were performed with one
round of MACS and four rounds of FACS employing the same conditions
as the naive discovery.
[0452] CDRH1 and CDRH2 selection: The CDRH3 of a single antibody
was recombined into a premade library with CDRH1 and CDRH2 variants
of a diversity of 1.times.10.sup.8 and selections were performed
with one round of MACS and four rounds of FACS as described in the
naive discovery. For each FACS round the libraries were looked at
for PSR binding, species cross-reactivity, and affinity pressure,
and sorting was performed in order to obtain a population with the
desired characteristics.
[0453] V.sub.H Mutant selection: The heavy chain variable region
(V.sub.H) was mutagenized via error prone PCR. The library was then
created by transforming this mutagenized V.sub.H and the heavy
chain expression vector into yeast already containing the light
chain plasmid of the parent. Selections were performed similar to
previous cycles using FACS sorting for two rounds. For each FACS
round the libraries were looked at for PSR binding, species
cross-reactivity, and affinity pressure, and sorting was performed
in order to obtain a population with the desired
characteristics.
Antibody Production and Purification
[0454] Yeast clones were grown to saturation and then induced for
48 h at 30.degree. C. with shaking. After induction, yeast cells
were pelleted and the supernatants were harvested for purification.
IgGs were purified using a Protein A column and eluted with acetic
acid, pH 2.0. Fab fragments were generated by papain digestion and
purified over KappaSelect.RTM. (GE Healthcare LifeSciences).
ForteBio K.sub.D Measurements
[0455] ForteBio affinity measurements were performed on an Octet
RED384 generally as previously described (see, e.g., Estep et al,
High throughput solution-based measurement of antibody-antigen
affinity and epitope binning. Mabs 5(2), 270-278 (2013)). Briefly,
ForteBio affinity measurements were performed by loading IgGs
on-line onto AHQ sensors. Sensors were equilibrated off-line in
assay buffer for 30 min and then monitored on-line for 60 seconds
for baseline establishment. Sensors with loaded IgGs were exposed
to 100 nM antigen for 3 minutes, and afterwards were transferred to
assay buffer for 3 min for off-rate measurement. For monovalent
affinity assessment Fabs were used instead of IgGs. For this
assessment, the unbiotinylated Fc fusion antigen was loaded on-line
onto the AHQ sensors. Sensors were equilibrated off-line in assay
buffer for 30 min and then monitored on-line for 60 seconds for
baseline establishment. Sensors with loaded antigen were exposed to
200 nM Fab for 3 minutes, and afterwards they were transferred to
assay buffer for 3 min for off-rate measurement. All kinetics were
analyzed using the 1:1 binding model.
ForteBio Epitope Binning/Ligand Blocking
[0456] Epitope binning/ligand blocking was performed using a
standard sandwich format cross-blocking assay. Control anti-target
IgG was loaded onto AHQ sensors and unoccupied Fc-binding sites on
the sensor were blocked with an irrelevant human IgG1 antibody. The
sensors were then exposed to 100 nM target antigen followed by a
second anti-target antibody or ligand. Additional binding by the
second antibody or ligand after antigen association indicates an
unoccupied epitope (non-competitor), while no binding indicates
epitope blocking (competitor or ligand blocking).
Size Exclusion Chromatography
[0457] A TSKgel.RTM. SuperSW mAb HTP column (22855) was used for
fast SEC analysis of mammalian produced mAbs at 0.4 mL/min with a
cycle time of 6 min/run. 200 mM Sodium Phosphate and 250 mM Sodium
Chloride was used as the mobile phase.
Dynamic Scanning Fluorimetry
[0458] 10 .mu.L of 20.times.Sypro Orange is added to 20 .mu.L of
0.2-1 mg/mL mAb or Fab solution. A RT-PCR instrument (BioRad CFX96
RT PCR) is used to ramp the sample plate temperature from 40 to 95
C at 0.5 C increment, with 2 min equilibrate at each temperature.
The negative of first derivative for the raw data is used to
extract Tm.
Example 2. Antibody Characterization
[0459] ForteBio K.sub.D Measurements: Quantitative binding of
antibodies to recombinant monomeric human, mouse, or cynomolgus
monkey NRP-1 was measured using biolayer interferometry (BLI) using
FORTEBIO.RTM.. Affinity measurements of selected antibodies were
performed generally as described in Estep et al., Mabs, 2013,
5:270-278, incorporated by reference in its entirety. FORTEBIO
affinity measurements were performed by loading IgGs (human IgG1
N297A) on-line onto AHQ sensors. Sensors were equilibrated off-line
in assay buffer for 30 min and then monitored on-line for 60
seconds for baseline establishment. Sensors with loaded IgGs were
exposed to a single concentration of antigen (100 nM) for 3
minutes. Afterwards they were transferred to assay buffer for 3
minutes for off-rate measurement. Kinetics were analyzed using the
1:1 binding model. A summary of K.sub.D measurements for antibodies
binding a single concentration of human, cynomolgus monkey, and
mouse NRP-1 is shown in Table 5 below.
[0460] Additional K.sub.D measurements were performed with eight
antibodies (human IgG4 S228P) using multi-concentration kinetics.
The binding affinities for human NRP-1-His, cynomolgus monkey
NRP-1-His, and mouse NRP-1-His were measured using an Octet QKe
instrument (ForteBio). A strategy of capturing antibodies on
sensors followed by association/dissociation of monomeric NRP-1
proteins was used to avoid avidity effects in the assay. The BLI
analysis was performed at 30.degree. C. using 1.times. kinetics
buffer (ForteBio) as assay buffer. Anti-Human IgG Fc Capture (AHC)
biosensors (ForteBio) were first presoaked in assay buffer for
greater than 5 minutes. Test antibody (5 .mu.g/mL) was captured on
the sensor for 250 seconds. Sensors were then dipped in assay
buffer for 60 seconds to establish a baseline before measuring
binding to each NRP-1 protein. Sensors were then dipped into
varying concentrations of human NRP-1-His (93.3 to 0.7 nM, 2-fold
dilutions in assay buffer), cynomolgus monkey NRP-1-His (93.3 to
1.5 nM, 2-fold dilutions in assay buffer), or mouse NRP-1-His (93.3
to 1.5 nM, 2-fold dilutions in assay buffer) for 250 seconds to
measure association. Dissociation of NRP-1 was then measured by
dipping sensors into assay buffer for 600 seconds. Agitation at all
steps was 1000 rpm. Kinetic parameters were generated with Octet
Data Analysis Software Version 8.2.0.7 using reference subtraction
(antibody "binding" to buffer), dissociation based inter-step
correction, 1 to 1 binding model, and global fit (Rmax unlinked by
sensor). K.sub.D values are shown in Table 6.
[0461] MSD-SET K.sub.D Measurements:
[0462] Solution equilibrium affinity measurements of selected
antibodies binding human NRP-1 were performed generally as
previously described. See Estep et al., supra, incorporated by
reference in its entirety. Briefly, solution equilibrium titrations
(SET) were performed in PBS+0.1% IgG-Free BSA (PBSF) with antigen
held constant at 10-100 pM and incubated with 3- to 5-fold serial
dilutions of Fab or mAbs starting at 10 pM-10 nM. Antibodies (20 nM
in PBS) were coated onto standard bind MSD-ECL plates overnight at
4.degree. C. or at room temperature for 30 min. Plates were then
blocked by BSA for 30 min with shaking at 700 rpm, followed by
three washes with wash buffer (PBSF+0.05% Tween.RTM. 20). SET
samples were applied and incubated on the plates for 150s with
shaking at 700 rpm followed by one wash. Antigen captured on a
plate was detected with 250 ng/mL sulfotag-labeled streptavidin in
PBSF by incubation on the plate for 3 min. The plates were washed
three times with wash buffer and then read on the MSD Sector Imager
2400 instrument using 1.times. Read Buffer T with surfactant. The
percent free antigen was plotted as a function of titrated antibody
in Prism and fit to a quadratic equation to extract the K.sub.D. To
improve throughput, liquid handling robots were used throughout
MSD-SET experiments, including SET sample preparation.
TABLE-US-00005 TABLE 5 Antibody Binding Affinities - Single
Concentration Kinetics ForteBio ForteBio ForteBio MSD Fab IgG
K.sub.D IgG K.sub.D IgG K.sub.D K.sub.D Human NRP-1 Cyno NRP-1
Mouse NRP-1 Human NRP-1 His (M) His (M) His (M) His (M) MAB
Monovalent Monovalent Monovalent Monovalent 1 1.87E-09 2.16E-09
2.12E-09 3.20E-10 2 1.86E-09 2.43E-09 1.94E-09 2.30E-10 3 1.08E-09
1.19E-09 9.90E-10 6.00E-11 4 8.51E-10 9.25E-10 7.46E-10 4.60E-11 5
3.23E-09 4.09E-09 5.06E-09 2.80E-10 6 4.72E-09 5.54E-09 6.98E-09
4.50E-10 7 1.12E-08 1.09E-08 1.47E-08 N.D. 8 6.13E-10 6.42E-10
5.52E-10 9.60E-11 9 6.45E-10 6.43E-10 5.66E-10 1.90E-11 10 8.68E-10
8.66E-10 7.46E-10 6.40E-11 11 4.85E-10 4.80E-10 4.46E-10 2.10E-11
12 4.81E-10 4.69E-10 4.40E-10 2.60E-11 13 1.41E-09 1.58E-09
7.42E-09 5.40E-10 14 1.12E-09 1.10E-09 5.00E-09 2.80E-10 15
8.51E-10 9.20E-09 5.41E-08 1.80E-10
TABLE-US-00006 TABLE 6 Antibody Binding Affinities - Multiple
Concentration Kinetics ForteBio ForteBio ForteBio IgG K.sub.D IgG
K.sub.D IgG K.sub.D Human NRP-1 Cyno NRP-1 Mouse NRP-1 His (M) His
(M) His (M) MAB Monovalent Monovalent Monovalent MAB2 I111T*
2.8E-09 5.5E-09 4.6E-09 IgG4 S228P MAB2 IgG4 S228P 2.4E-09 4.5E-09
5.1E-09 MAB3 IgG4 S228P 3.7E-09 7.3E-09 4.4E-09 MAB4 IgG4 S228P
3.1E-09 4.5E-09 2.3E-09 MAB5 IgG4 S228P 8.4E-09 1.2E-08 6.6E-09
MAB12 IgG4 S228P 1.2E-10 1.9E-10 1.6E-10 MAB13 IgG4 S228P 9.6E-10
9.4E-10 3.7E-09 MAB14 IgG4 S228P 8.7E-10 7.4E-10 2.7E-09
Example 3. Anti-Tumor Efficacy of Nine Anti-NRP-1 MABs Alone and in
Combination with a PD-1 or PD-L1 Antibody
[0463] Nine optimized antibodies were evaluated for anti-tumor
efficacy using immunocompetent mice. The assay was conducted with a
panel of murine versions of MABs 2, 3, 4, 5, 7, 12, 13, 14, and 15,
as well as an IgG control and SEC10 (SEQ ID NOS 141-142) as a
comparator. The antibodies were tested as chimeric mouse IgG2a
antibodies containing the N297A mutation which abolishes ADCC and
CDC effector functions. Anti-tumor efficacy was measured using the
mouse colon CT26 syngeneic tumor model grown in female BALB/c mice.
3.times.10.sup.5 mouse CT26 cells were implanted subcutaneously on
Day 1. The mice were randomized based on body weight and antibodies
were administered intraperitoneally at the indicated dose on the
same day as tumor cell implantation. The anti-NRP-1 antibodies were
administered as a monotherapy at 500 .mu.g/dose or in combination
with an anti-PD-1 immune checkpoint inhibitor which was used at 200
.mu.g/dose. FIG. 1A shows the monotherapy effect of antibodies in
the CT26 model, and FIG. 1B shows the effect of combination of
anti-NRP-1 antibodies with anti-PD-1. The black arrows along the
horizontal axis indicate the treatment days of the antibodies. The
average tumor volume from 10 mice per group is shown for each
treatment group.
[0464] FIG. 1C shows a subset of data from FIGS. 1A and 1B
comparing mMAB12 alone and in combination with an anti-PD-1
checkpoint antibody in the mouse colon CT26 syngeneic tumor model.
mMAB12 at 500 .mu.g/animal inhibited tumor growth by 61.6% TGI
(tumor growth inhibition) compared to control antibody-treated
mice. This effect was statistically significant by Student's t test
(p<0.05). The anti-PD-1 checkpoint antibody administered at 200
.mu.g/animal was less efficacious than mMAB12 (37.8% TGI,
p<0.05). However, the combination of mMAB12 with the PD-1
antibody resulted in additive anti-tumor efficacy (79.0% TGI,
p<0.001) compared to the monotherapy treatments. The effect of
the combination was statistically significant when compared to PD-1
and mMAB12 (p<0.05 in both cases). There was no untoward
toxicity exhibited by the treated mice which all gained weight over
the course of the treatment, except for one non-treatment-related
expired mouse in the mMAB12 group.
[0465] The same nine antibodies were evaluated in a second tumor
model, the mouse colon MC38 syngeneic model. 5.times.10.sup.5 mouse
MC38 cells were implanted subcutaneously into female C57Bl/6 mice.
The mice were randomized into treatment groups when the tumors
reached an average tumor volume of 60 mm.sup.3 to 90 mm.sup.3
followed by initiation of treatment on Day 1. The anti-NRP-1
antibodies were administered as a monotherapy at 500 .mu.g/dose or
in combination with an anti-PD-L1 immune checkpoint inhibitor which
was used at 250 .mu.g/dose. The anti-PD-L1 antibody works in the
same immune checkpoint pathway as the PD-1 antibody. FIG. 2A shows
the monotherapy effect of antibodies in the MC38 model, and FIG. 2B
shows the effect of combination of anti-NRP-1 antibodies with
anti-PD-L1. The black arrows along the horizontal axis indicate the
treatment days. The average tumor volume from 10 mice per group is
shown for each treatment group.
[0466] The anti-tumor efficacy of mMAB12 in the MC38 syngeneic
colon mouse tumor model is shown in FIG. 2C. The mMAB12 at 500
.mu.g/animal inhibited tumor growth by 77.3% TGI (p<0.05)
compared to control antibody-treated mice. The MC38 model is very
sensitive to PD-1 antibody blockade. Therefore, an antibody against
PD-L1 at 250 .mu.g/animal which works in the same immune checkpoint
pathway as the PD-1 antibody was used to demonstrate potential
combination benefits. As expected, PD-L1 monotherapy blocked tumor
growth at 77.5% TGI (p<0.05). However, the combination of mMAB12
with the PD-L1 antibody did not demonstrate additional anti-tumor
benefits (76.2% TGI). As with the CT26 model there was no untoward
toxicity exhibited by the treated mice which all gained weight over
the course of the treatment. Four antibodies (MABs 2, 5, 12, and
13) were selected based on their efficacies in the CT26 and MC38
studies and retested in the MC38 model under the same conditions
(alone and in combination with anti-PD-L1). The findings in the
repeat MC38 study confirmed the above findings for efficacy and
tolerability.
Example 4. Evaluation of Blockade of NRP-1 Ligands
[0467] Quantitative ligand blocking studies, measuring the ability
of antibodies to block the binding of recombinant human SEMA3A and
human VEGFA to recombinant human NRP-1, was measured by a blocking
ELISA. To measure the ability of antibody to block the SEMA3A/NRP-1
interaction, the assay plate was coated with human SEMA3A at 2.5
.mu.g/mL in PBS, overnight at 4.degree. C. Biotinylated human NRP-1
(500 ng/mL in 1% BSA/PBS) was incubated with test antibody
(30-0.002 .mu.g/mL, 4-fold dilution in 1% BSA/PBS) prior to
addition to the assay plate, and then HRP conjugated streptavidin
(1:200 in 1% BSA/PBS) was used for detection of NRP-1 bound to
SEMA3A. Briefly, to measure the ability of antibody to block the
VEGFA/NRP-1 interaction, the assay plate was coated with human
NRP-1 at 2.5 .mu.g/mL in PBS, overnight at 4.degree. C. Test
antibody (30-0.002 .mu.g/mL, 4-fold dilution in 1% BSA/PBS) was
incubated with VEGFA (125 ng/mL) prior to addition to the assay
plate, biotinylated anti-VEGFA antibody (0.2 .mu.g/mL in 1%
BSA/PBS) was added, and then HRP conjugated streptavidin (1:200 in
1% BSA/PBS) was used for detection of VEGFA bound to NRP-1. The
IC.sub.50 values for 15 IgG1 format test antibodies blocking
SEMA3A/VEGFA binding are shown in Table 7.
TABLE-US-00007 TABLE 7 IC.sub.50 values for Blocking Assays with
IgG1 Format Antibodies SEMA3A/NRP-1 VEGFA/NRP-1 Blocking Blocking
MAB IC.sub.50 (nM) IC.sub.50 (nM) 1 2.9 No Blocking 2 3.1 No
Blocking 3 0.6 No Blocking 4 3.9 No Blocking 5 5.9 No Blocking 6
1.8 7.4 7 1.7 6.9 8 2.0 7.3 9 1.8 6.5 10 1.5 6.7 11 0.8 5.9 12 0.8
6.0 13 3.4 No Blocking 14 3.1 No Blocking 15 No Blocking No
Blocking
[0468] Eight MABs were converted to IgG4 S228P format and the assay
was repeated. A summary of the averages is shown in Table 8.
TABLE-US-00008 TABLE 8 Averages for Blocking Assays with IgG4
Format Antibodies SEMA3A/NRP-1 VEGFA/NRP-1 Blocking Blocking MAB
IC50 (nM) n IC50 (nM) n MAB2 I111T* IgG4 2.8 2 No Blocking 1 S228P
MAB2 IgG4 S228P 2.6 2 No Blocking 2 MAB3 IgG4 S228P 2.0 2 No
Blocking 2 MAB4 IgG4 S228P 2.3 2 No Blocking 2 MAB5 IgG4 S228P 2.9
2 No Blocking 2 MAB12 IgG4 S228P 1.2 2 3.2 2 MAB13 IgG4 S228P 0.9 2
2.9 2 MAB14 IgG4 S228P 0.6 2 2.5 2 *humanizing site-directed
mutation
Example 5. Epitope Binning of MAB12 vs SEC10
[0469] Epitope binning for MAB12 and SEC10 was measured using
BioLayer Interferometry (BLI) using an Octet.RTM. QKe instrument
(ForteBio.RTM.). MAB12 or SEC10 at 5 .mu.g/mL was immobilized on
anti-human Fc AHC sensors for 300 seconds. Sensors were then dipped
in kinetics buffer for baseline determination. Next, sensors were
dipped in human IgG at 200 .mu.g/ml for 400 seconds to saturate all
the IgG Fc binding sites on the sensors. After baseline
determination, the sensors were exposed to 100 nM human NRP-1-HIS
for 300 seconds to allow for antigen binding. Finally, sensors were
transferred to wells containing 20 .mu.g/mL of either MAB12 or
SEC10 for 300 seconds to analyze antibody binding. If the test
antibody showed clear binding in the last step, it was considered a
non-competitor (different epitope bin), and if the test antibody
did not show clear binding, it was considered a competitor (same
epitope bin).
[0470] Results are shown in FIG. 3. Capturing MAB12 and then
binding NRP-1 does not prevent SEC10 from also binding NRP-1 (top
panel). Similarly, capturing SEC10 and then binding NRP-1 does not
prevent MAB12 from also binding NRP-1 (bottom panel). Self-binning
(e.g., capture MAB12, bind NRP-1, test binding of MAB12) served as
a positive control for binning. These data show that MAB12 and
SEC10 can simultaneously bind NRP-1, and must therefore bind to
different epitopes.
Example 6. Binding of Anti-NRP-1 Antibodies to NRP-1 Domains
[0471] In order to understand the approximate binding domain for
antibodies binding to human NRP-1, the ability of antibodies to
bind fragments of NRP-1 that contained different domains of NRP-1
extracellular region was measured by BLI using an Octet.RTM. QKe
instrument (ForteBio.RTM.). Recombinant human NRP-1-Fc fusion
proteins consisted of a1, a1a2, a1a2b1, a2b1b2, or a1a2b1b2
domains, and the differences in antibody binding to each protein
led to determination of which primary domain the antibody binds.
The BLI analysis was performed at 29.degree. C. or 30.degree. C.
using 1.times. kinetics buffer (ForteBio) as the assay buffer.
Briefly, antibodies (5 .mu.g/mL) were captured onto anti-human IgG
Fc (AHC) biosensors for 250 seconds. Then sensors were dipped into
assay buffer (100 seconds) to achieve a baseline prior to measuring
binding to each NRP-1 protein. A quenching step using human IgG Fc
(150 nM, 250 nM or 500 nM, depending on the experiment) for 250
seconds was performed next. Sensors were then dipped into each
NRP-1 protein at 500 nM for 300 seconds, followed by dissociation
of each NRP-1 protein in assay buffer for 900 or 1000 seconds.
Agitation was performed at 900 rpm or 1000 rpm for all steps,
depending on the experiment.
[0472] Table 9 shows the results of the assays described above. The
binding domain for each antibody is shown in the far-right
column.
TABLE-US-00009 TABLE 9 NRP1 Domain Binding Specificity Binding
Antibody a1 a1a2 a1a2b1 a2b1b2 a1a2b1b2 Domain MAB1 - + + + + a2
MAB2 - + + + + a2 MAB3 + + + + + a1 MAB4 + + + + + a1 MAB5 - + + +
+ a2 MAB6 - + + + + a2 MAB7 - - - + + b2 MAB8 - - + + + b1 MAB9 - -
+ + + b1 MAB10 - - + + + b1 MAB11 - - + + + b1 MAB12 - - + + + b1
MAB13 - - + + + b1 MAB14 - - + + + b1 MAB15 + + + - + a1 SEC10* -
-/+ + + + b1 with weak a2 SEC3** + + + - + a1 MAB59941*** - - - + +
b2 *SEQ ID Nos 141-142 **Described in Appleton, et. al., The EMBO
Journal (2007) 26, 4902-4912. ***Described in Delgoffe G M, Woo
S-R, Turnis M E, Gravano D M, Guy C, Overacre A E, et al. Stability
and function of regulatory T cells is maintained by a
neuropilin-1-semaphorin-4a axis. Nature 501(7466): 252-6. Available
from R&D Systems.
Example 7: Mutational Analysis for Epitope Determination
[0473] To identify the epitope for MAB12 binding to the b1 domain
of human NRP1, single point mutations were made within the human
NRP1 b1 domain Either alanine substitutions or NRP2 specific
residues were used (MAB12 does not bind NRP2). Proteins were
expressed in HEK293 cells, secreted as soluble protein, purified on
Ni-NTA resin, and characterized by SDS-PAGE. Binding was assessed
by Bio-Layer Interferometry (BLI) using the Octet platform. MAB12
was captured on anti-human Fc sensors, washed, and exposed to
either monomeric wild type human NRP1 b1 domain or to monomeric
mutant NRP1 bl. Residues considered part of the binding epitope
demonstrated reduced binding (e.g., a K.sub.D more than 5-fold
poorer than that of binding to wild type human NRP1 b1) or no
binding. Single point mutants P317A, D320A, T349A, K352G, Y353A,
Y354A, and T413A resulted in reduced binding, whereas K351N and
E412H resulted in no binding.
Example 8: Structure Determination of MAB12 Complexed with NRP1
[0474] The binding epitope was also identified through
crystallographic studies. MAB12 Fab was complexed with human NRP1
b1, purified by size exclusion chromatography and concentrated to
10 mg/ml. Crystals were grown out of 42% PEG200, HEPES pH 7. X-ray
data was collected at Argonne National Laboratories (GM/CA CAT
23ID-D) and processed using CCP4 and Phenix. NRP1 b1 residues
within a contact distance of 3.8 .ANG. from the heavy and light
chain were considered part of the binding epitope and include Y297,
T316, D320, E348, T349, K350, K351, K352, Y353, Y354, E412, T413,
G414 and 1415.
Example 9: Analysis of Amino Acid Modifications of MAB12
[0475] Analysis of the amino acid modifications of purified MAB12
suggested that the deletion of lysine at the C terminal of the
heavy chain occurred in most of the purified antibodies and that
the pyroglutamylation of glutamic acid at the N terminal of the
light chain occurred in some of the purified antibodies.
INCORPORATION BY REFERENCE
[0476] The entire disclosures of all patent and non-patent
publications cited herein are each incorporated by reference in
their entireties for all purposes.
OTHER EMBODIMENTS
[0477] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in this application, in applications claiming
priority from this application, or in related applications. Such
claims, whether directed to a different invention or to the same
invention, and whether broader, narrower, equal, or different in
scope in comparison to the original claims, also are regarded as
included within the subject matter of the inventions of the present
disclosure.
TABLE-US-00010 APPENDIX A SEQUENCE REFERENCE TABLE SEQ ID NO
Molecule Region Sequence 1 MAB1 VH FR1 QVQLVQSGAGVKKPGASVKVSCKASG 2
MAB2 VH FR1 QVQLVQSGAEVKKPGASVKVSCKASG 3 MAB3 VH FR1
QAQLVQSGAEVKKPGASVKVSCKASG 2 MAB4 VH FR1 QVQLVQSGAEVKKPGASVKVSCKASG
2 MAB5 VH FR1 QVQLVQSGAEVKKPGASVKVSCKASG 4 MAB6 VH FR1
QVQLVQSGAKVKKPGASVKVSCKASG 5 MAB7 VH FR1 EVQLVESGGGLVQPGGSLRLSCAASG
6 MAB8 VH FR1 EVQLLESGGGLVQPGGSLRLSCAASG 6 MAB9 VH FR1
EVQLLESGGGLVQPGGSLRLSCAASG 6 MAB10 VH FR1
EVQLLESGGGLVQPGGSLRLSCAASG 6 MAB11 VH FR1
EVQLLESGGGLVQPGGSLRLSCAASG 6 MAB12 VH FR1
EVQLLESGGGLVQPGGSLRLSCAASG 7 MAB13 VH FR1
QVQLQQWGAGLLKPSETLSLTCAVYG 7 MAB14 VH FR1
QVQLQQWGAGLLKPSETLSLTCAVYG 7 MAB15 VH FR1
QVQLQQWGAGLLKPSETLSLTCAVYG 8 MAB1 VH CDR1 YTFRSYYML 8 MAB2 VH CDR1
YTFRSYYML 9 MAB3 VH CDR1 YTFSRYYMH 9 MAB4 VH CDR1 YTFSRYYMH 10 MAB5
VH CDR1 YTFTSYYMH 10 MAB6 VH CDR1 YTFTSYYMH 11 MAB7 VH CDR1
FTFSSYWME 12 MAB8 VH CDR1 FTFASYAMV 13 MAB9 VH CDR1 FTFKSYAMV 14
MAB10 VH CDR1 FTFSSVAMV 14 MAB11 VH CDR1 FTFSSVAMV 14 MAB12 VH CDR1
FTFSSVAMV 15 MAB13 VH CDR1 GSFRGYYWE 15 MAB14 VH CDR1 GSFRGYYWE 16
MAB15 VH CDR1 GSFVKYYWS 17 MAB1 VH FR2 WVRQAPGQGLEWMG 17 MAB2 VH
FR2 WVRQAPGQGLEWMG 17 MAB3 VH FR2 WVRQAPGQGLEWMG 17 MAB4 VH FR2
WVRQAPGQGLEWMG 17 MAB5 VH FR2 WVRQAPGQGLEWMG 18 MAB6 VH FR2
WVRQVPGQGLEWMG 19 MAB7 VH FR2 WVRQAPGKGLEWVA 20 MAB8 VH FR2
WVRQAPGKGLEWVS 20 MAB9 VH FR2 WVRQAPGKGLEWVS 20 MAB10 VH FR2
WVRQAPGKGLEWVS 20 MAB11 VH FR2 WVRQAPGKGLEWVS 20 MAB12 VH FR2
WVRQAPGKGLEWVS 21 MAB13 VH FR2 WIRQPPGKGLEWIG 22 MAB14 VH FR2
WSRQPPGKGLEWIG 21 MAB15 VH FR2 WIRQPPGKGLEWIG 23 MAB1 VH CDR2
IIDPSDGSTSYAQKFQG 23 MAB2 VH CDR2 IIDPSDGSTSYAQKFQG 24 MAB3 VH CDR2
IINPLGGSTLYAQKFQG 24 MAB4 VH CDR2 IINPLGGSTLYAQKFQG 25 MAB5 VH CDR2
IINPQGGDTSYAQKFQG 25 MAB6 VH CDR2 IINPQGGDTSYAQKFQG 26 MAB7 VH CDR2
RIKRDGSEKYYVDSVKG 27 MAB8 VH CDR2 IISGSGGSTYYADSVKG 28 MAB9 VH CDR2
IISGSGGATYYADSVKG 29 MAB10 VH CDR2 AISGSGGATYYADSVKG 30 MAB11 VH
CDR2 AISGSGGATYYADSVEG 30 MAB12 VH CDR2 AISGSGGATYYADSVEG 31 MAB13
VH CDR2 EISHSGSTNYNPSLKS 31 MAB14 VH CDR2 EISHSGSTNYNPSLKS 32 MAB15
VH CDR2 DIWHSGMTNYNPSLKS 33 MAB1 VH FR3
RVTMTRDTPTSTVYMELSSLRSEDTAVYYC 34 MAB2 VH FR3
RVTMTRDASTSTVYMELSSLRSEDTAVYYC 35 MAB3 VH FR3
RVTMTRDTSTSTVYMELSSLRSEDTAVYYC 35 MAB4 VH FR3
RVTMTRDTSTSTVYMELSSLRSEDTAVYYC 35 MAB5 VH FR3
RVTMTRDTSTSTVYMELSSLRSEDTAVYYC 35 MAB6 VH FR3
RVTMTRDTSTSTVYMELSSLRSEDTAVYYC 36 MAB7 VH FR3
RFTISRDNAKNSLYLQMNSLRAEDTAVYYC 37 MAB8 VH FR3
RFTISRDNSKNTLYLQMNSLRAEDTAVYYC 37 MAB9 VH FR3
RFTISRDNSKNTLYLQMNSLRAEDTAVYYC 37 MAB10 VH FR3
RFTISRDNSKNTLYLQMNSLRAEDTAVYYC 38 MAB11 VH FR3
RFTISRDNSKNTLYLQMSSLRAEDTAVYYC 37 MAB12 VH FR3
RFTISRDNSKNTLYLQMNSLRAEDTAVYYC 39 MAB13 VH FR3
RVTISVDTSKNQFSLKLSSVTAADTAVYYC 40 MAB14 VH FR3
RVTISVDTSKNQFSLKLSPVTAADTAVYYC 39 MAB15 VH FR3
RVTISVDTSKNQFSLKLSSVTAADTAVYYC 41 MAB1 VH CDR3 ARGARRITGYGMDV 41
MAB2 VH CDR3 ARGARRITGYGMDV 42 MAB3 VH CDR3 ARDLGYYGSGMHA 43 MAB4
VH CDR3 ARDLGYYGSGMHV 44 MAB5 VH CDR3 ARDRGMYYASGFGP 45 MAB6 VH
CDR3 ARDRGMYYASGFNP 46 MAB7 VH CDR3 ARDQGYKTPTDFDL 47 MAB8 VH CDR3
AKDPGYDSSRYYYSNYGMDV 47 MAB9 VH CDR3 AKDPGYDSSRYYYSNYGMDV 47 MAB10
VH CDR3 AKDPGYDSSRYYYSNYGMDV 47 MAB11 VH CDR3 AKDPGYDSSRYYYSNYGMDV
47 MAB12 VH CDR3 AKDPGYDSSRYYYSNYGMDV 48 MAB13 VH CDR3
ARARPYREPYGMDV 48 MAB14 VH CDR3 ARARPYREPYGMDV 49 MAB15 VH CDR3
ARGPGYDSSGYSRRFDP 50 MAB1 VH FR4 WGQGTTVTVSS 51 MAB2 VH FR4
WGQGTTVIVSS 52 MAB3 VH FR4 WGQGTLVTVSS 52 MAB4 VH FR4 WGQGTLVTVSS
52 MAB5 VH FR4 WGQGTLVTVSS 52 MAB6 VH FR4 WGQGTLVTVSS 53 MAB7 VH
FR4 WGRGTLVTVSS 50 MAB8 VH FR4 WGQGTTVTVSS 50 MAB9 VH FR4
WGQGTTVTVSS 50 MAB10 VH FR4 WGQGTTVTVSS 50 MAB11 VH FR4 WGQGTTVTVSS
50 MAB12 VH FR4 WGQGTTVTVSS 50 MAB13 VH FR4 WGQGTTVTVSS 50 MAB14 VH
FR4 WGQGTTVTVSS 52 MAB15 VH FR4 WGQGTLVTVSS 54 MAB1 VL FR1
DIQMTQSPSSVSASVGDRVTITC 54 MAB2 VL FR1 DIQMTQSPSSVSASVGDRVTITC 54
MAB3 VL FR1 DIQMTQSPSSVSASVGDRVTITC 54 MAB4 VL FR1
DIQMTQSPSSVSASVGDRVTITC 55 MAB5 VL FR1 EIVMTQSPGTLSLSPGERATLSC 55
MAB6 VL FR1 EIVMTQSPGTLSLSPGERATLSC 56 MAB7 VL FR1
DIQMTQSPSSLSASVGDRVTITC 56 MAB8 VL FR1 DIQMTQSPSSLSASVGDRVTITC 56
MAB9 VL FR1 DIQMTQSPSSLSASVGDRVTITC 56 MAB10 VL FR1
DIQMTQSPSSLSASVGDRVTITC 56 MAB11 VL FR1 DIQMTQSPSSLSASVGDRVTITC 56
MAB12 VL FR1 DIQMTQSPSSLSASVGDRVTITC 57 MAB13 VL FR1
DIQLTQSPSSVSASVGDRVTITC 57 MAB14 VL FR1 DIQLTQSPSSVSASVGDRVTITC 58
MAB15 VL FR1 DIQMTQSPSTLSASVGDRVTITC 59 MAB1 VL CDR1 RASQGISSWLA 59
MAB2 VL CDR1 RASQGISSWLA
60 MAB3 VL CDR1 RASQGISRWLA 60 MAB4 VL CDR1 RASQGISRWLA 61 MAB5 VL
CDR1 RASQSVSSSYLA 61 MAB6 VL CDR1 RASQSVSSSYLA 62 MAB7 VL CDR1
QASQDITNYLN 63 MAB8 VL CDR1 RASQSISSYLN 63 MAB9 VL CDR1 RASQSISSYLN
63 MAB10 VL CDR1 RASQSISSYLN 63 MAB11 VL CDR1 RASQSISSYLN 63 MAB12
VL CDR1 RASQSISSYLN 64 MAB13 VL CDR1 RASQDISSWLA 64 MAB14 VL CDR1
RASQDISSWLA 65 MAB15 VL CDR1 RASQSISSWLA 66 MAB1 VL FR2
WYQQKPGKAPKLLIY 66 MAB2 VL FR2 WYQQKPGKAPKLLIY 66 MAB3 VL FR2
WYQQKPGKAPKLLIY 66 MAB4 VL FR2 WYQQKPGKAPKLLIY 146 MAB5 VL FR2
WYQQKPGQAPRLLIY 146 MAB6 VL FR2 WYQQKPGQAPRLLIY 66 MAB7 VL FR2
WYQQKPGKAPKLLIY 66 MAB8 VL FR2 WYQQKPGKAPKLLIY 66 MAB9 VL FR2
WYQQKPGKAPKLLIY 66 MAB10 VL FR2 WYQQKPGKAPKLLIY 66 MAB11 VL FR2
WYQQKPGKAPKLLIY 66 MAB12 VL FR2 WYQQKPGKAPKLLIY 66 MAB13 VL FR2
WYQQKPGKAPKLLIY 66 MAB14 VL FR2 WYQQKPGKAPKLLIY 66 MAB15 VL FR2
WYQQKPGKAPKLLIY 67 MAB1 VL CDR2 AASNLQS 67 MAB2 VL CDR2 AASNLQS 68
MAB3 VL CDR2 AASSLQS 68 MAB4 VL CDR2 AASSLQS 69 MAB5 VL CDR2
GASNRAT 69 MAB6 VL CDR2 GASNRAT 70 MAB7 VL CDR2 DASNLET 71 MAB8 VL
CDR2 GASSLQS 71 MAB9 VL CDR2 GASSLQS 71 MAB10 VL CDR2 GASSLQS 71
MAB11 VL CDR2 GASSLQS 71 MAB12 VL CDR2 GASSLQS 68 MAB13 VL CDR2
AASSLQS 68 MAB14 VL CDR2 AASSLQS 72 MAB15 VL CDR2 KASSLES 73 MAB1
VL FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB2 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB3 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB4 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 74 MAB5 VL FR3
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 74 MAB6 VL FR3
GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 75 MAB7 VL FR3
GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC 73 MAB8 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB9 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB10 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB11 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB12 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB13 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 73 MAB14 VL FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 76 MAB15 VL FR3
GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC 77 MAB1 VL CDR3 QQASVFPFT 77 MAB2
VL CDR3 QQASVFPFT 78 MAB3 VL CDR3 QQANLLPFT 78 MAB4 VL CDR3
QQANLLPFT 79 MAB5 VL CDR3 QQLSSFPIT 79 MAB6 VL CDR3 QQLSSFPIT 80
MAB7 VL CDR3 QQSDVLPIT 81 MAB8 VL CDR3 QQTYSLYT 81 MAB9 VL CDR3
QQTYSLYT 81 MAB10 VL CDR3 QQTYSLYT 81 MAB11 VL CDR3 QQTYSLYT 81
MAB12 VL CDR3 QQTYSLYT 82 MAB13 VL CDR3 QQELAFPRT 82 MAB14 VL CDR3
QQELAFPRT 83 MAB15 VL CDR3 QQLNSYPPT 84 MAB1 VL FR4 FGGGTKVEIK 84
MAB2 VL FR4 FGGGTKVEIK 84 MAB3 VL FR4 FGGGTKVEIK 84 MAB4 VL FR4
FGGGTKVEIK 84 MAB5 VL FR4 FGGGTKVEIK 84 MAB6 VL FR4 FGGGTKVEIK 84
MAB7 VL FR4 FGGGTKVEIK 84 MAB8 VL FR4 FGGGTKVEIK 84 MAB9 VL FR4
FGGGTKVEIK 84 MAB10 VL FR4 FGGGTKVEIK 84 MAB11 VL FR4 FGGGTKVEIK 84
MAB12 VL FR4 FGGGTKVEIK 84 MAB13 VL FR4 FGGGTKVEIK 84 MAB14 VL FR4
FGGGTKVEIK 84 MAB15 VL FR4 FGGGTKVEIK 85 MAB1 VH Full
QVQLVQSGAGVKKPGASVKVSCKASGYTFRSYYMLWV
RQAPGQGLEWMGIIDPSDGSTSYAQKFQGRVTMTRDTPT
STVYMELSSLRSEDTAVYYCARGARRITGYGMDVWGQG TTVTVSS 86 MAB2 VH Full
QVQLVQSGAEVKKPGASVKVSCKASGYTFRSYYMLWVR
QAPGQGLEWMGIIDPSDGSTSYAQKFQGRVTMTRDASTS
TVYMELSSLRSEDTAVYYCARGARRITGYGMDVWGQGT TVIVSS 87 MAB3 VH Full
QAQLVQSGAEVKKPGASVKVSCKASGYTFSRYYMHWV
RQAPGQGLEWMGIINPLGGSTLYAQKFQGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARDLGYYGSGMHAWGQG TLVTVSS 88 MAB4 VH Full
QVQLVQSGAEVKKPGASVKVSCKASGYTFSRYYMHWV
RQAPGQGLEWMGIINPLGGSTLYAQKFQGRVTMTRDTST
STVYMELSSLRSEDTAVYYCARDLGYYGSGMHVWGQG TLVTVSS 89 MAB5 VH Full
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWV
RQAPGQGLEWMGIINPQGGDTSYAQKFQGRVTMTRDTS
TSTVYMELSSLRSEDTAVYYCARDRGMYYASGFGPWGQ GTLVTVSS 90 MAB6 VH Full
QVQLVQSGAKVKKPGASVKVSCKASGYTFTSYYMHWV
RQVPGQGLEWMGIINPQGGDTSYAQKFQGRVTMTRDTS
TSTVYMELSSLRSEDTAVYYCARDRGMYYASGFNPWGQ GTLVTVSS 91 MAB7 VH Full
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMEWVR
QAPGKGLEWVARIKRDGSEKYYVDSVKGRFTISRDNAK
NSLYLQMNSLRAEDTAVYYCARDQGYKTPTDFDLWGRG TLVTVSS 92 MAB8 VH Full
EVQLLESGGGLVQPGGSLRLSCAASGFTFASYAMVWVR
QAPGKGLEWVSIISGSGGSTYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMD VWGQGTTVTVSS 93 MAB9 VH Full
EVQLLESGGGLVQPGGSLRLSCAASGFTFKSYAMVWVR
QAPGKGLEWVSIISGSGGATYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMD VWGQGTTVTVSS 94 MAB10 VH Full
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSVAMVWVRQ
APGKGLEWVSAISGSGGATYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMD VWGQGTTVTVSS 95 MAB11 VH Full
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSVAMVWVRQ
APGKGLEWVSAISGSGGATYYADSVEGRFTISRDNSKNT
LYLQMSSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMDV WGQGTTVTVSS 96 MAB12 VH Full
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSVAMVWVRQ
APGKGLEWVSAISGSGGATYYADSVEGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMD VWGQGTTVTVSS 97 MAB13 VH Full
QVQLQQWGAGLLKPSETLSLTCAVYGGSFRGYYWEWIR QPPGKGLEWIGEISHSGS
TNYNPSLKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARARPYREPYGMDVWGQGTTVT VSS
98 MAB14 VH Full QVQLQQWGAGLLKPSETLSLTCAVYGGSFRGYYWEWSR
QPPGKGLEWIGEISHSGSTNYNPSLKSRVTISVDTSKNQFS
LKLSPVTAADTAVYYCARARPYREPYGMDVWGQGTTVT VSS 99 MAB15 VH Full
QVQLQQWGAGLLKPSETLSLTCAVYGGSFVKYYWSWIR
QPPGKGLEWIGDIWHSGMTNYNPSLKSRVTISVDTSKNQ
FSLKLSSVTAADTAVYYCARGPGYDSSGYSRRFDPWGQG TLVTVSS
100 MAB1 VL Full DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP
GKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYYCQQASVFPFTFGGGTKVEIK 100 MAB2 VL Full
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP
GKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYYCQQASVFPFTFGGGTKVEIK 101 MAB3 VL Full
DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQK
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYYCQQANLLPFTFGGGTKVEIK 101 MAB4 VL Full
DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQK
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYYCQQANLLPFTFGGGTKVEIK 102 MAB5 VL Full
EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK
PGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPE
DFAVYYCQQLSSFPITFGGGTKVEIK 102 MAB6 VL Full
EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK
PGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPE
DFAVYYCQQLSSFPITFGGGTKVEIK 103 MAB7 VL Full
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKP
GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPE
DIATYYCQQSDVLPITFGGGTKVEIK 104 MAB8 VL Full
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQTYSLYTFGGGTKVEIK
104 MAB9 VL Full DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQTYSLYTFGGGTKVEIK
104 MAB10 VL Full DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQTYSLYTFGGGTKVEIK
104 MAB11 VL Full DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQTYSLYTFGGGTKVEIK
104 MAB12 VL Full DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQTYSLYTFGGGTKVEIK
105 MAB13 VL Full DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKP
GKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYYCQQELAFPRTFGGGTKVEIK 105 MAB14 VL Full
DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKP
GKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYYCQQELAFPRTFGGGTKVEIK 106 MAB15 VL Full
DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP
GKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDD
FATYYCQQLNSYPPTFGGGTKVEIK 107 MAB1 HC Full-
QVQLVQSGAGVKKPGASVKVSCKASGYTFRSYYMLWV length
RQAPGQGLEWMGIIDPSDGSTSYAQKFQGRVTMTRDTPT IgG4
STVYMELSSLRSEDTAVYYCARGARRITGYGMDVWGQG S228P
TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 108 MAB2
HC Full- QVQLVQSGAEVKKPGASVKVSCKASGYTFRSYYMLWVR length
QAPGQGLEWMGIIDPSDGSTSYAQKFQGRVTMTRDASTS IgG4
TVYMELSSLRSEDTAVYYCARGARRITGYGMDVWGQGT S228P
TVIVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE
PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 109 MAB3
HC Full- QAQLVQSGAEVKKPGASVKVSCKASGYTFSRYYMHWV length
RQAPGQGLEWMGIINPLGGSTLYAQKFQGRVTMTRDTST IgG4
STVYMELSSLRSEDTAVYYCARDLGYYGSGMHAWGQG S228P
TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 110 MAB4
HC Full- QVQLVQSGAEVKKPGASVKVSCKASGYTFSRYYMHWV length
RQAPGQGLEWMGIINPLGGSTLYAQKFQGRVTMTRDTST IgG4
STVYMELSSLRSEDTAVYYCARDLGYYGSGMHVWGQG S228P
TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 111 MAB5
HC Full- QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWV length
RQAPGQGLEWMGIINPQGGDTSYAQKFQGRVTMTRDTS IgG4
TSTVYMELSSLRSEDTAVYYCARDRGMYYASGFGPWGQ S228P
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE
VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK 112
MAB6 HC Full- QVQLVQSGAKVKKPGASVKVSCKASGYTFTSYYMHWV length
RQVPGQGLEWMGIINPQGGDTSYAQKFQGRVTMTRDTS IgG4
TSTVYMELSSLRSEDTAVYYCARDRGMYYASGFNPWGQ S228P
GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP
EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE
VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK 113
MAB7 HC Full- EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMEWVR length
QAPGKGLEWVARIKRDGSEKYYVDSVKGRFTISRDNAK IgG4
NSLYLQMNSLRAEDTAVYYCARDQGYKTPTDFDLWGRG S228P
TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 114 MAB8
HC Full- EVQLLESGGGLVQPGGSLRLSCAASGFTFASYAMVWVR length
QAPGKGLEWVSIISGSGGSTYYADSVKGRFTISRDNSKNT IgG4
LYLQMNSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMD S228P
VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP
PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK
115 MAB9 HC Full- EVQLLESGGGLVQPGGSLRLSCAASGFTFKSYAMVWVR length
QAPGKGLEWVSIISGSGGATYYADSVKGRFTISRDNSKNT IgG4
LYLQMNSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMD S228P
VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP
PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK
116 MAB10 HC Full- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSVAMVWVRQ length
APGKGLEWVSAISGSGGATYYADSVKGRFTISRDNSKNT IgG4
LYLQMNSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMD S228P
VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP
PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK
117 MAB11 HC Full- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSVAMVWVRQ length
APGKGLEWVSAISGSGGATYYADSVEGRFTISRDNSKNT IgG4
LYLQMSSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMDV S228P
WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV
TVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPC
PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP
QVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK 118
MAB12 HC Full- EVQLLESGGGLVQPGGSLRLSCAASGFTFSSVAMVWVRQ length
APGKGLEWVSAISGSGGATYYADSVEGRFTISRDNSKNT IgG4
LYLQMNSLRAEDTAVYYCAKDPGYDSSRYYYSNYGMD S228P
VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP
PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ
EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK
119 MAB13 HC Full- QVQLQQWGAGLLKPSETLSLTCAVYGGSFRGYYWEWIR length
QPPGKGLEWIGEISHSGSTNYNPSLKSRVTISVDTSKNQFS IgG4
LKLSSVTAADTAVYYCARARPYREPYGMDVWGQGTTVT S228P
VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW
YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 120 MAB14 HC
Full- QVQLQQWGAGLLKPSETLSLTCAVYGGSFRGYYWEWSR length
QPPGKGLEWIGEISHSGSTNYNPSLKSRVTISVDTSKNQFS IgG4
LKLSPVTAADTAVYYCARARPYREPYGMDVWGQGTTVT S228P
VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVT
VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW
YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN
GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT
PPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 121 MAB15 HC
Full- QVQLQQWGAGLLKPSETLSLTCAVYGGSFVKYYWSWIR length
QPPGKGLEWIGDIWHSGMTNYNPSLKSRVTISVDTSKNQ IgG4
FSLKLSSVTAADTAVYYCARGPGYDSSGYSRRFDPWGQG S228P
TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS
SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ
FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLP
PSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE ALHNHYTQKSLSLSLGK 122 MAB1
LC Full- DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP length,
GKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQASVFPFTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 122 MAB2 LC
Full- DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP length,
GKAPKLLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQASVFPFTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 123 MAB3 LC
Full- DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQK length,
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQANLLPFTFGGGTKVEIKRTVAAPSVFIFPPS kappa
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS constant
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 123 MAB4
LC Full- DIQMTQSPSSVSASVGDRVTITCRASQGISRWLAWYQQK length,
PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQANLLPFTFGGGTKVEIKRTVAAPSVFIFPPS kappa
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS constant
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 124 MAB5
LC Full- EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK length,
PGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPE human
DFAVYYCQQLSSFPITFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 124 MAB6 LC
Full- EIVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQK length,
PGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPE human
DFAVYYCQQLSSFPITFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 125 MAB7 LC
Full- DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKP length,
GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPE human
DIATYYCQQSDVLPITFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 126 MAB8 LC
Full- DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP length,
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQTYSLYTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 126 MAB9 LC
Full- DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP length,
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQTYSLYTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 126 MAB10
LC Full- DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP length,
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQTYSLYTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 126 MAB11
LC Full- DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP length,
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQTYSLYTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 126 MAB12
LC Full- DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKP length,
GKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQTYSLYTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 127 MAB13
LC Full- DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKP length,
GKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQELAFPRTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 127 MAB14
LC Full- DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKP length,
GKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPE human
DFATYYCQQELAFPRTFGGGTKVEIKRTVAAPSVFIFPPSD kappa
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ constant
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 128 MAB15
LC Full- DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKP length
GKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDD
FATYYCQQLNSYPPTFGGGTKVEIKRTVAAPSVFIFPPSDE
QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 129 hNRP-1
GenBank ATGGAGAGGGGGCTGCCGCTCCTCTGCGCCGTGCTCGC Accession
CCTCGTCCTCGCCCCGGCCGGCGCTTTTCGCAACGATA No. NM_
AATGTGGCGATACTATAAAAATTGAAAGCCCCGGGTA 003873.5
CCTTACATCTCCTGGTTATCCTCATTCTTATCACCCAAG (corresponds
TGAAAAATGCGAATGGCTGATTCAGGCTCCGGACCCAT to NP_
ACCAGAGAATTATGATCAACTTCAACCCTCACTTCGAT 003864.4).
TTGGAGGACAGAGACTGCAAGTATGACTACGTGGAAG
TCTTCGATGGAGAAAATGAAAATGGACATTTTAGGGG
AAAGTTCTGTGGAAAGATAGCCCCTCCTCCTGTTGTGT
CTTCAGGGCCATTTCTTTTTATCAAATTTGTCTCTGACT
ACGAAACACATGGTGCAGGATTTTCCATACGTTATGAA
ATTTTCAAGAGAGGTCCTGAATGTTCCCAGAACTACAC
AACACCTAGTGGAGTGATAAAGTCCCCCGGATTCCCTG
AAAAATATCCCAACAGCCTTGAATGCACTTATATTGTC
TTTGCGCCAAAGATGTCAGAGATTATCCTGGAATTTGA
AAGCTTTGACCTGGAGCCTGACTCAAATCCTCCAGGGG
GGATGTTCTGTCGCTACGACCGGCTAGAAATCTGGGAT
GGATTCCCTGATGTTGGCCCTCACATTGGGCGTTACTG
TGGACAGAAAACACCAGGTCGAATCCGATCCTCATCG
GGCATTCTCTCCATGGTTTTTTACACCGACAGCGCGAT
AGCAAAAGAAGGTTTCTCAGCAAACTACAGTGTCTTGC
AGAGCAGTGTCTCAGAAGATTTCAAATGTATGGAAGCT
CTGGGCATGGAATCAGGAGAAATTCATTCTGACCAGAT
CACAGCTTCTTCCCAGTATAGCACCAACTGGTCTGCAG
AGCGCTCCCGCCTGAACTACCCTGAGAATGGGTGGACT
CCCGGAGAGGATTCCTACCGAGAGTGGATACAGGTAG
ACTTGGGCCTTCTGCGCTTTGTCACGGCTGTCGGGACA
CAGGGCGCCATTTCAAAAGAAACCAAGAAGAAATATT
ATGTCAAGACTTACAAGATCGACGTTAGCTCCAACGGG
GAAGACTGGATCACCATAAAAGAAGGAAACAAACCTG
TTCTCTTTCAGGGAAACACCAACCCCACAGATGTTGTG
GTTGCAGTATTCCCCAAACCACTGATAACTCGATTTGT
CCGAATCAAGCCTGCAACTTGGGAAACTGGCATATCTA
TGAGATTTGAAGTATACGGTTGCAAGATAACAGATTAT
CCTTGCTCTGGAATGTTGGGTATGGTGTCTGGACTTATT
TCTGACTCCCAGATCACATCATCCAACCAAGGGGACAG
AAACTGGATGCCTGAAAACATCCGCCTGGTAACCAGTC
GCTCTGGCTGGGCACTTCCACCCGCACCTCATTCCTAC
ATCAATGAGTGGCTCCAAATAGACCTGGGGGAGGAGA
AGATCGTGAGGGGCATCATCATTCAGGGTGGGAAGCA
CCGAGAGAACAAGGTGTTCATGAGGAAGTTCAAGATC
GGGTACAGCAACAACGGCTCGGACTGGAAGATGATCA
TGGATGACAGCAAACGCAAGGCGAAGTCTTTTGAGGG
CAACAACAACTATGATACACCTGAGCTGCGGACTTTTC
CAGCTCTCTCCACGCGATTCATCAGGATCTACCCCGAG
AGAGCCACTCATGGCGGACTGGGGCTCAGAATGGAGC
TGCTGGGCTGTGAAGTGGAAGCCCCTACAGCTGGACC
GACCACTCCCAACGGGAACTTGGTGGATGAATGTGAT
GACGACCAGGCCAACTGCCACAGTGGAACAGGTGATG
ACTTCCAGCTCACAGGTGGCACCACTGTGCTGGCCACA
GAAAAGCCCACGGTCATAGACAGCACCATACAATCAG
AGTTTCCAACATATGGTTTTAACTGTGAATTTGGCTGG
GGCTCTCACAAGACCTTCTGCCACTGGGAACATGACAA
TCACGTGCAGCTCAAGTGGAGTGTGTTGACCAGCAAG
ACGGGACCCATTCAGGATCACACAGGAGATGGCAACT
TCATCTATTCCCAAGCTGACGAAAATCAGAAGGGCAA
AGTGGCTCGCCTGGTGAGCCCTGTGGTTTATTCCCAGA
ACTCTGCCCACTGCATGACCTTCTGGTATCACATGTCT
GGGTCCCACGTCGGCACACTCAGGGTCAAACTGCGCTA
CCAGAAGCCAGAGGAGTACGATCAGCTGGTCTGGATG
GCCATTGGACACCAAGGTGACCACTGGAAGGAAGGGC
GTGTCTTGCTCCACAAGTCTCTGAAACTTTATCAGGTG
ATTTTCGAGGGCGAAATCGGAAAAGGAAACCTTGGTG
GGATTGCTGTGGATGACATTAGTATTAATAACCACATT
TCACAAGAAGATTGTGCAAAACCAGCAGACCTGGATA
AAAAGAACCCAGAAATTAAAATTGATGAAACAGGGAG
CACGCCAGGATACGAAGGTGAAGGAGAAGGTGACAAG
AACATCTCCAGGAAGCCAGGCAATGTGTTGAAGACCTT
AGACCCCATCCTCATCACCATCATAGCCATGAGTGCCC
TGGGGGTCCTCCTGGGGGCTGTCTGTGGGGTCGTGCTG
TACTGTGCCTGTTGGCATAATGGGATGTCAGAAAGAAA
CTTGTCTGCCCTGGAGAACTATAACTTTGAACTTGTGG
ATGGTGTGAAGTTGAAAAAAGACAAACTGAATACACA GAGTACTTATTCGGAGGCATGA 130
hNRP-1 Genbank MERGLPLLCAVLALVLAPAGAFRNDKCGDTIKIESPGYLT Protein NP_
SPGYPHSYHPSEKCEWLIQAPDPYQRIMINFNPHFDLEDR 003864.4.
DCKYDYVEVFDGENENGHFRGKFCGKIAPPPVVSSGPFLF
IKFVSDYETHGAGFSIRYEIFKRGPECSQNYTTPSGVIKSP
GFPEKYPNSLECTYIVFAPKMSEIILEFESFDLEPDSNPPGG
MFCRYDRLEIWDGFPDVGPHIGRYCGQKTPGRIRSSSGIL
SMVFYTDSAIAKEGFSANYSVLQSSVSEDFKCMEALGME
SGEIHSDQITASSQYSTNWSAERSRLNYPENGWTPGEDSY
REWIQVDLGLLRFVTAVGTQGAISKETKKKYYVKTYKID
VSSNGEDWITIKEGNKPVLFQGNTNPTDVVVAVFPKPLIT
RFVRIKPATWETGISMRFEVYGCKITDYPCSGMLGMVSG
LISDSQITSSNQGDRNWMPENIRLVTSRSGWALPPAPHSYI
NEWLQIDLGEEKIVRGIIIQGGKHRENKVFMRKFKIGYSN
NGSDWKMIMDDSKRKAKSFEGNNNYDTPELRTFPALSTR
FIRIYPERATHGGLGLRMELLGCEVEAPTAGPTTPNGNLV
DECDDDQANCHSGTGDDFQLTGGTTVLATEKPTVIDSTI
QSEFPTYGFNCEFGWGSHKTFCHWEHDNHVQLKWSVLT
SKTGPIQDHTGDGNFIYSQADENQKGKVARLVSPVVYSQ
NSAHCMTFWYHMSGSHVGTLRVKLRYQKPEEYDQLVW
MAIGHQGDHWKEGRVLLHKSLKLYQVIFEGEIGKGNLG
GIAVDDISINNHISQEDCAKPADLDKKNPEIKIDETGSTPG
YEGEGEGDKNISRKPGNVLKTLDPILITIIAMSALGVLLGA
VCGVVLYCACWHNGMSERNLSALENYNFELVDGVKLK KDKLNTQSTYSEA 131 cNRP-1 DNA:
ATGGAGAAGGGGTTGCCGCTCCTCTGCGCCGCGCTCGC Genbank
CCTCGCCCTCGCCCCGGCCGGCGCTTTTCGCAACGATA Acc No.
AATGTGGCGATACTATAAAAATTGAAAGCCCCGGGTA XM_
CCTTACATCTCCTGGTTATCCTCATTCTTATCACCCAAG 005564935.2
TGAAAAATGTGAATGGCTGATTCAGGCTCCGGACCCAT
ACCAGAGAATTATGATCAACTTCAACCCTCACTTCGAT
TTGGAGGACAGAGATTGCAAGTATGACTACGTGGAAG
TCTTCGATGGAGAAAATGAAAATGGACGTTTATGGGG
AAAGTTCTGTGGAAAGATAGCCCCTCCTCCTGTTGTGT
CTTCAGGGCAATTTCTTTTTATCAAATTTGTCTCTGACT
ACGAAACACACGGTGCAGGATTTTCCATACGTTATGAA
ATTTTCAAGAGAGGTCCTGAATGTTCCCAGAACTACAC
AACACCTAGTGGAGTGATAAAGTCCCCCGGATTCCCTG
AAAAATATCCCAACAGCCTTGAATGCACTTATATTGTC
TTTGCACCAAAGATGTCAGAGATTATCCTGGAATTTGA
AAGCTTTGACCTGGAGCCTGACTCAAATCCTCCAGGGG
GGATGTTCTGTCGCTACGACCGGCTGGAAATCTGGGAT
GGATTCCCTGACGTTGGCCCTCACATTGGGCGTTACTG
TGGACAGAAAACACCAGGTCGAATCCGATCCTCATCG
GGCATTCTCTCCATGGTTTTTTACACCGACAGCGCAAT
AGCAAAAGAAGGTTTCTCAGCAAACTACAGTGTCTTGC
AGAGCAGTGTCTCAGAAGATTTCAAATGTATGGAAGCT
GTGGGCATGGAATCAGGAGAAATTCATTCTGACCAGA
TCACAGCTTCTTCCCAGTACAGCACCAACTGGTCTGCA
GAGCGCTCCCGCCTGAACTATCCTGAGAATGGGTGGAC
TCCCGGAGAAGATTCCTACCGAGAGTGGATACAGGTG
GACTTGGGCCTTCTACGCTTCGTTACGGCTGTCGGGAC
ACAGGGCGCCATTTCAAAAGAAACCAAGAAGAAATAT
TATGTCAAGACTTACAAAATTGACATTAGCTCCAACGG
GGAAGACTGGATCACCATAAAAGAAGGAAACAAACCT
GTTCTCTTTCAGGGAAACACCAACCCCACAGACGTTGT
GGTTGCAGTATTCCCCAAGCCACTGATAACTCGATTTG
TCCGAATCAAGCCTGCAACTTGGGAAACTGGCATATCT
CTGAGATTTGAAGTATATGGTTGCAAGATAACAGATTA
TCCTTGCTCCGGAATGTTGGGTATGGTGTCTGGACTTA
TTTCTGACTCCCAGATCACATCATCCAACCAAGGGGAC
AGAAACTGGATGCCTGAAAACATCCGCCTGGTAACCA
GTCGCTCCGGCTGGGCACTGCCACCCGCACCTCATTCC
TACGTCAATGAGTGGCTCCAAATAGACCTGGGGGAGG
AGAAGATCGTGAGGGGCATCATCATTCAGGGTGGGAA
GCACCGAGAGAACAAGGTATTCATGAGGAAGTTCAAG
ATCGGGTACAGCAACAACGGCTCCGACTGGAAGATGA
TCATGGACGACAGCAAACGCAAGGCAAAGTCTTTTGA
GGGCAACAACAACTATGACACACCTGAGCTGCGGACT
TTTCCAGCTCTCTCCACGCGATTCATCAGGATCTACCCC
GAGAGAGCCACTCATGGCGGACTGGGGCTCCGAATGG
AGCTGCTGGGCTGTGAAGTGGAAGCCCCTACAGCTGG
ACCGACCACTCCCAACGGGAACCCGGTGGATGAATGT
GATGACGACCAGGCCAACTGCCACAGTGGAACAGGTG
ATGACTTCCAGCTCACAGGTGGCACCACTGTGCTGGCC
ACAGAAAAGCCCACGGTCATAGACAGCACCATACAAT
CAGAGTTTCCTACATATGGTTTTAACTGTGAATTTGGCT
GGGGCTCTCACAAGACCTTCTGCCACTGGGAACATGAC
AATCACGTGCAGCTCAAGTGGAGTGTGTTGACCAGCA
AGACGGGACCCATTCAGGATCACACAGGAGATGGCAA
CTTCATCTATTCCCAAGCTGATGAAAATCAGAAGGGCA
AAGTGGCTCGCCTGGTGAGCCCTGTGGTTTATTCCCAG
AACTCTGCCCACTGCATGACCTTCTGGTATCACATGTC
TGGGTCCCACGTCGGCACACTCAGGGTCAAACTGCGCT
ACCAGAAGCCAGAGGAGTACGATCAGCTGGTCTGGAT
GGCCATTGGACACCAAGGTGACCACTGGAAGGAAGGG
CGTGTCTTGCTTCACAAGTCTCTGAAACTTTATCAGGT
GATTTTCGAGGGCGAAATCGGAAAAGGAAACCTTGGT
GGGATTGCTGTGGATGACATTAGTATCAATAACCACAT
TTCACAAGAAGATTGTGCAAAACCAGCAGACCTGGAT
AAAAAGAACCCAGAAATTAAAATTGATGAAACAGGGA
GCACACCAGGATATGAAGGTGAAGGAGAAGGTGACAA
GAACATCTCCAGGAAACCAGGCAATGTGTTGAAGACC
TTAGACCCCATCCTCATCACCATCATAGCCATGAGCGC
CCTGGGGGTCCTCCTGGGGGCTGTGTGCGGGGTCGTGC
TGTACTGTGCCTGTTGGCATAATGGGATGTCAGAAAGA
AACTTGTCTGCCCTGGAGAACTATAACTTTGAACTTGT
GGACGGTGTGAAGTTGAAAAAAGACAAACTGAATACA CAGAGTACTTATTCGGAGGCATGA 132
cNRP-1 Protein: MEKGLPLLCAALALALAPAGAFRNDKCGDTIKIESPGYLT UniProtKB-
SPGYPHSYHPSEKCEWLIQAPDPYQRIMINFNPHFDLEDR G7PEQ1
DCKYDYVEVFDGENENGRLWGKFCGKIAPPPVVSSGQFL
FIKFVSDYETHGAGFSIRYEIFKRGPECSQNYTTPSGVIKSP
GFPEKYPNSLECTYIVFAPKMSEIILEFESFDLEPDSNPPGG
MFCRYDRLEIWDGFPDVGPHIGRYCGQKTPGRIRSSSGIL
SMVFYTDSAIAKEGFSANYSVLQSSVSEDFKCMEAVGME
SGEIHSDQITASSQYSTNWSAERSRLNYPENGWTPGEDSY
REWIQVDLGLLRFVTAVGTQGAISKETKKKYYVKTYKID
ISSNGEDWITIKEGNKPVLFQGNTNPTDVVVAVFPKPLITR
FVRIKPATWETGISLRFEVYGCKITDYPCSGMLGMVSGLI
SDSQITSSNQGDRNWMPENIRLVTSRSGWALPPAPHSYV
NEWLQIDLGEEKIVRGIIIQGGKHRENKVFMRKFKIGYSN
NGSDWKMIMDDSKRKAKSFEGNNNYDTPELRTFPALSTR
FIRIYPERATHGGLGLRMELLGCEVEAPTAGPTTPNGNPV
DECDDDQANCHSGTGDDFQLTGGTTVLATEKPTVIDSTI
QSEFPTYGFNCEFGWGSHKTFCHWEHDNHVQLKWSVLT
SKTGPIQDHTGDGNFIYSQADENQKGKVARLVSPVVYSQ
NSAHCMTFWYHMSGSHVGTLRVKLRYQKPEEYDQLVW
MAIGHQGDHWKEGRVLLHKSLKLYQVIFEGEIGKGNLG
GIAVDDISINNHISQEDCAKPADLDKKNPEIKIDETGSTPG
YEGEGEGDKNISRKPGNVLKTLDPILITIIAMSALGVLLGA
VCGVVLYCACWHNGMSERNLSALENYNFELVDGVKLK KDKLNTQSTYSEA 133 mNRP-1
GenBank ATGGAGAGGGGGCTGCCGTTGCTGTGCGCCACGCTCGC Acc. No.
CCTTGCCCTCGCCCTGGCGGGCGCTTTCCGCAGCGACA NM008737
AATGTGGCGGGACCATAAAAATCGAAAACCCAGGGTA
CCTCACATCTCCCGGTTACCCTCATTCTTACCATCCAAG
TGAGAAGTGTGAATGGCTAATCCAAGCTCCGGAACCCT
ACCAGAGAATCATGATCAACTTCAACCCACATTTCGAT
TTGGAGGACAGAGACTGCAAGTATGACTACGTGGAAG
TAATCGATGGGGAGAATGAAGGCGGCCGCCTGTGGGG
GAAGTTCTGTGGGAAGATTGCACCTTCTCCTGTGGTGT
CTTCAGGGCCCTTTCTCTTCATCAAATTTGTCTCTGACT
ATGAGACACATGGGGCAGGGTTTTCCATCCGCTATGAA
ATCTTCAAGAGAGGGCCCGAATGTTCTCAGAACTATAC
AGCACCTACTGGAGTGATAAAGTCCCCTGGGTTCCCTG
AAAAATACCCCAACAGCTTGGAGTGCACCTACATCATC
TTTGCACCAAAGATGTCTGAGATAATCCTGGAGTTTGA
AAGTTTTGACCTGGAGCAAGACTCGAATCCTCCCGGAG
GAATGTTCTGTCGCTATGACCGGCTGGAGATCTGGGAT
GGATTCCCTGAAGTTGGCCCTCACATTGGGCGTTATTG
TGGGCAGAAAACTCCTGGCCGGATCCGCTCCTCTTCAG
GCGTTCTATCCATGGTCTTTTACACTGACAGCGCAATA
GCAAAAGAAGGTTTCTCAGCCAACTACAGTGTGCTACA
GAGCAGCATCTCTGAAGATTTTAAGTGTATGGAGGCTC
TGGGCATGGAATCTGGAGAGATCCATTCTGATCAGATC
ACTGCATCTTCACAGTATGGTACCAACTGGTCTGTAGA
GCGCTCCCGCCTGAACTACCCTGAAAATGGGTGGACTC
CAGGAGAAGACTCCTACAAGGAGTGGATCCAGGTGGA
CTTGGGCCTCCTGCGATTCGTTACTGCTGTAGGGACAC
AGGGTGCCATTTCCAAGGAAACCAAGAAGAAATATTA
TGTCAAGACTTACAGAGTAGACATCAGCTCCAACGGA
GAGGACTGGATCTCCCTGAAAGAGGGAAATAAAGCCA
TTATCTTTCAGGGAAACACCAACCCCACAGATGTTGTC
TTAGGAGTTTTCTCCAAACCACTGATAACTCGATTTGT
CCGAATCAAACCTGTATCCTGGGAAACTGGTATATCTA
TGAGATTTGAAGTTTATGGCTGCAAGATAACAGATTAT
CCTTGCTCTGGAATGTTGGGCATGGTGTCTGGACTTAT
TTCAGACTCCCAGATTACAGCATCCAATCAAGCCGACA
GGAATTGGATGCCAGAAAACATCCGTCTGGTGACCAG
TCGTACCGGCTGGGCACTGCCACCCTCACCCCACCCAT
ACACCAATGAATGGCTCCAAGTGGACCTGGGAGATGA
GAAGATAGTAAGAGGTGTCATCATTCAGGGTGGGAAG
CACCGAGAAAACAAGGTGTTCATGAGGAAGTTCAAGA
TCGCCTATAGTAACAATGGCTCTGACTGGAAAACTATC
ATGGATGACAGCAAGCGCAAGGCTAAGTCGTTCGAAG
GCAACAACAACTATGACACACCTGAGCTTCGGACGTTT
TCACCTCTCTCCACAAGGTTCATCAGGATCTACCCTGA
GAGAGCCACACACAGTGGGCTTGGGCTGAGGATGGAG
CTACTGGGCTGTGAAGTGGAAGCACCTACAGCTGGAC
CAACCACACCCAATGGGAACCCAGTGGATGAGTGTGA
CGACGACCAGGCCAACTGCCACAGTGGCACAGGTGAT
GACTTCCAGCTCACAGGAGGCACCACTGTCCTGGCCAC
AGAGAAGCCAACCATTATAGACAGCACCATCCAATCA
GAGTTCCCGACATACGGTTTTAACTGCGAGTTTGGCTG
GGGCTCTCACAAGACATTCTGCCACTGGGAGCATGACA
GCCATGCACAGCTCAGGTGGAGTGTGCTGACCAGCAA
GACAGGGCCGATTCAGGACCATACAGGAGATGGCAAC
TTCATCTATTCCCAAGCTGATGAAAATCAGAAAGGCAA
AGTAGCCCGCCTGGTGAGCCCTGTGGTCTATTCCCAGA
GCTCTGCCCACTGTATGACCTTCTGGTATCACATGTCC
GGCTCTCATGTGGGTACACTGAGGGTCAAACTACGCTA
CCAGAAGCCAGAGGAATATGATCAACTGGTCTGGATG
GTGGTTGGGCACCAAGGAGACCACTGGAAAGAAGGAC
GTGTCTTGCTGCACAAATCTCTGAAACTATATCAGGTT
ATTTTTGAAGGTGAAATCGGAAAAGGAAACCTTGGTG
GAATTGCTGTGGATGATATCAGTATTAACAACCATATT
TCTCAGGAAGACTGTGCAAAACCAACAGACCTAGATA
AAAAGAACACAGAAATTAAAATTGATGAAACAGGGAG
CACTCCAGGATATGAAGGAGAAGGGGAAGGTGACAAG
AACATCTCCAGGAAGCCAGGCAATGTGCTTAAGACCCT
GGATCCCATCCTGATCACCATCATAGCCATGAGTGCCC
TGGGAGTACTCCTGGGTGCAGTCTGTGGAGTTGTGCTG
TACTGTGCCTGTTGGCACAATGGGATGTCAGAAAGGA
ACCTATCTGCCCTGGAGAACTATAACTTTGAACTTGTG
GATGGTGTAAAGTTGAAAAAAGATAAACTGAACCCAC AGAGTAATTACTCAGAGGCGTGA 134
mNRP-1 UniProtKB- MERGLPLLCATLALALALAGAFRSDKCGGTIKIENPGYLT P97333
SPGYPHSYHPSEKCEWLIQAPEPYQRIMINFNPHFDLEDR
DCKYDYVEVIDGENEGGRLWGKFCGKIAPSPVVSSGPFL
FIKFVSDYETHGAGFSIRYEIFKRGPECSQNYTAPTGVIKS
PGFPEKYPNSLECTYIIFAPKMSEIILEFESFDLEQDSNPPG
GMFCRYDRLEIWDGFPEVGPHIGRYCGQKTPGRIRSSSGV
LSMVFYTDSAIAKEGFSANYSVLQSSISEDFKCMEALGME
SGEIHSDQITASSQYGTNWSVERSRLNYPENGWTPGEDSY
KEWIQVDLGLLRFVTAVGTQGAISKETKKKYYVKTYRV
DISSNGEDWISLKEGNKAIIFQGNTNPTDVVLGVFSKPLIT
RFVRIKPVSWETGISMRFEVYGCKITDYPCSGMLGMVSG
LISDSQITASNQADRNWMPENIRLVTSRTGWALPPSPHPY
TNEWLQVDLGDEKIVRGVIIQGGKHRENKVFMRKFKIAY
SNNGSDWKTIMDDSKRKAKSFEGNNNYDTPELRTFSPLS
TRFIRIYPERATHSGLGLRMELLGCEVEAPTAGPTTPNGN
PVDECDDDQANCHSGTGDDFQLTGGTTVLATEKPTIIDST
IQSEFPTYGFNCEFGWGSHKTFCHWEHDSHAQLRWSVLT
SKTGPIQDHTGDGNFIYSQADENQKGKVARLVSPVVYSQ
SSAHCMTFWYHMSGSHVGTLRVKLRYQKPEEYDQLVW
MVVGHQGDHWKEGRVLLHKSLKLYQVIFEGEIGKGNLG
GIAVDDISINNHISQEDCAKPTDLDKKNTEIKIDETGSTPG
YEGEGEGDKNISRKPGNVLKTLDPILITIIAMSALGVLLGA
VCGVVLYCACWHNGMSERNLSALENYNFELVDGVKLK KDKLNPQSNYSEA 135 rNRP-1
UniProtKB- MERGLPLLCATLALALALAGAFRSDKCGGTIKIENPGYLT Q9QWJ9
SPGYPHSYHPSEKCEWLIQAPEPYQRIMINFNPHFDLEDR
DCKYDYVEVIDGENEGGRLWGKFCGKIAPSPVVSSGPFL
FIKFVSDYETHGAGFSIRYEIFKRGPECSQNYTAPTGVIKS
PGFPEKYPNSLECTYIIFAPKMSEIILEFESFDLEQDSNPPG
GVFCRYDRLEIWDGFPEVGPHIGRYCGQKTPGRIRSSSGIL
SMVFYTDSAIAKEGFSANYSVLQSSISEDFKCMEALGMES
GEIHSDQITASSQYGTNWSVERSRLNYPENGWTPGEDSY
REWIQVDLGLLRFVTAVGTQGAISKETKKKYYVKTYRV
DISSNGEDWITLKEGNKAIIFQGNTNPTDVVFGVFPKPLIT
RFVRIKPASWETGISMRFEVYGCKITDYPCSGMLGMVSG
LISDSQITASNQGDRNWMPENIRLVTSRTGWALPPSPHPYI
NEWLQVDLGDEKIVRGVIIQGGKHRENKVFMRKFKIAYS
NNGSDWKMIMDDSKRKAKSFEGNNNYDTPELRAFTPLS
TRFIRIYPERATHSGLGLRMELLGCEVEVPTAGPTTPNGN
PVDECDDDQANCHSGTGDDFQLTGGTTVLATEKPTIIDST
IQSEFPTYGFNCEFGWGSHKTFCHWEHDSHAQLRWRVLT
SKTGPIQDHTGDGNFIYSQADENQKGKVARLVSPVVYSQ
SSAHCMTFWYHMSGSHVGTLRVKLHYQKPEEYDQLVW
MVVGHQGDHWKEGRVLLHKSLKLYQVIFEGEIGKGNLG
GIAVDDISINNHIPQEDCAKPTDLDKKNTEIKIDETGSTPG
YEEGKGDKNISRKPGNVLKTLDPILITIIAMSALGVLLGAV
CGVVLYCACWHNGMSERNLSALENYNFELVDGVKLKK DKLNPQSNYSEA 136 MABs VHCDR2
X.sub.1ISGSGGX.sub.2TYYADSVX.sub.3G, wherein X.sub.1 is I or A,
X.sub.2 is 8-12 Consensus S or A, and X.sub.3 is K or E 137 MABs
VHCDR1 FTFX.sub.1SX.sub.2AMV, wherein X.sub.1 is A, K, or S,
X.sub.2 is Y or V 8-12 Consensus 138 MABs VHCDR3 ARDLGYYGSGMHX,
wherein X is A or V 3-4 Consensus 139 MABs VHCDR3 ARDRGMYYASGFXP,
wherein X is G or N 5-6 Consensus 140 Linker (GGGGS)N, wherein n is
an integer consensus 141 anti-NRP IgG1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ Antibody
APGKGLEWVSQISPAGGYTNYADSVKGRFTISADTSKNT SEC10
AYLQMNSLRAEDTAVYYCARGELPYYRMSKVMDVWGQ
GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC
PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGK 142
anti-NRP Kappa DIQMTQSPSSLSASVGDRVTITCRASQYFSSYLAWYQQKP Antibody
light chain GKAPKLLIYGASSRASGVPSRFSGSGSGTDFTLTISSLQPE SEC10
DFATYYCQQYLGSPPTFGQGTKVEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 143 Human
UniProt MERGLPLLCAVLALVLAPAGAFRNDKCGDTIKIESPGYLT NRP-1 O14786.
SPGYPHSYHPSEKCEWLIQAPDPYQRIMINFNPHFDLEDR Has minor
DCKYDYVEVFDGENENGHFRGKFCGKIAPPPVVSSGPFLF SNP,
IKFVSDYETHGAGFSIRYEIFKRGPECSQNYTTPSGVIKSP V179
GFPEKYPNSLECTYIVFVPKMSEIILEFESFDLEPDSNPPGG
MFCRYDRLEIWDGFPDVGPHIGRYCGQKTPGRIRSSSGIL
SMVFYTDSAIAKEGFSANYSVLQSSVSEDFKCMEALGME
SGEIHSDQITASSQYSTNWSAERSRLNYPENGWTPGEDSY
REWIQVDLGLLRFVTAVGTQGAISKETKKKYYVKTYKID
VSSNGEDWITIKEGNKPVLFQGNTNPTDVVVAVFPKPLIT
RFVRIKPATWETGISMRFEVYGCKITDYPCSGMLGMVSG
LISDSQITSSNQGDRNWMPENIRLVTSRSGWALPPAPHSYI
NEWLQIDLGEEKIVRGIIIQGGKHRENKVFMRKFKIGYSN
NGSDWKMIMDDSKRKAKSFEGNNNYDTPELRTFPALSTR
FIRIYPERATHGGLGLRMELLGCEVEAPTAGPTTPNGNLV
DECDDDQANCHSGTGDDFQLTGGTTVLATEKPTVIDSTI
QSEFPTYGFNCEFGWGSHKTFCHWEHDNHVQLKWSVLT
SKTGPIQDHTGDGNFIYSQADENQKGKVARLVSPVVYSQ
NSAHCMTFWYHMSGSHVGTLRVKLRYQKPEEYDQLVW
MAIGHQGDHWKEGRVLLHKSLKLYQVIFEGEIGKGNLG
GIAVDDISINNHISQEDCAKPADLDKKNPEIKIDETGSTPG
YEGEGEGDKNISRKPGNVLKTLDPILITIIAMSALGVLLGA
VCGVVLYCACWHNGMSERNLSALENYNFELVDGVKLK KDKLNTQSTYSEA 144 SEC3
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQK light
PGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPE chain
DFATYYCQQAWAYLPTFGQGTKVEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN
SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC
145 SEC3 EVQLVESGGGLVQPGGSLRLSCAASGFTISGYGIHWVRQ Heavy
APGKGLEWVAYIYPDSGYTDYADSVKGRFTISADTSKNT Chain
AYLQMNSLRAEDTAVYYCAREDFRNRRRLWYVMDYW
GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK
Sequence CWU 1
1
148126PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 1Gln Val Gln Leu Val Gln Ser Gly Ala
Gly Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly 20 25226PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 2Gln 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 20 25326PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 3Gln Ala 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 20
25426PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 4Gln Val Gln Leu Val Gln Ser Gly Ala
Lys Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly 20 25526PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 5Glu 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 20 25626PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 6Glu 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 20
25726PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 7Gln Val Gln Leu Gln Gln Trp Gly Ala
Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val
Tyr Gly 20 2589PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 8Tyr Thr Phe Arg Ser Tyr Tyr
Met Leu1 599PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 9Tyr Thr Phe Ser Arg Tyr Tyr
Met His1 5109PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 10Tyr Thr Phe Thr Ser Tyr
Tyr Met His1 5119PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 11Phe Thr Phe Ser Ser Tyr
Trp Met Glu1 5129PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 12Phe Thr Phe Ala Ser Tyr
Ala Met Val1 5139PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 13Phe Thr Phe Lys Ser Tyr
Ala Met Val1 5149PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 14Phe Thr Phe Ser Ser Val
Ala Met Val1 5159PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 15Gly Ser Phe Arg Gly Tyr
Tyr Trp Glu1 5169PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 16Gly Ser Phe Val Lys Tyr
Tyr Trp Ser1 51714PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 17Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met Gly1 5 101814PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 18Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met Gly1
5 101914PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 19Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ala1 5 102014PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 20Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser1
5 102114PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 21Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile Gly1 5 102214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 22Trp Ser Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly1
5 102317PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 23Ile Ile Asp Pro Ser Asp
Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln1 5 10
15Gly2417PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 24Ile Ile Asn Pro Leu Gly
Gly Ser Thr Leu Tyr Ala Gln Lys Phe Gln1 5 10
15Gly2517PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 25Ile Ile Asn Pro Gln Gly
Gly Asp Thr Ser Tyr Ala Gln Lys Phe Gln1 5 10
15Gly2617PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 26Arg Ile Lys Arg Asp Gly
Ser Glu Lys Tyr Tyr Val Asp Ser Val Lys1 5 10
15Gly2717PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 27Ile Ile Ser Gly Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly2817PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 28Ile Ile Ser Gly Ser Gly
Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly2917PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 29Ala Ile Ser Gly Ser Gly
Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly3017PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 30Ala Ile Ser Gly Ser Gly
Gly Ala Thr Tyr Tyr Ala Asp Ser Val Glu1 5 10
15Gly3116PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 31Glu Ile Ser His Ser Gly
Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10 153216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 32Asp Ile Trp His Ser Gly Met Thr Asn Tyr Asn Pro Ser Leu
Lys Ser1 5 10 153330PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 33Arg Val Thr Met Thr
Arg Asp Thr Pro Thr Ser Thr Val Tyr Met Glu1 5 10 15Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 303430PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 34Arg Val Thr Met Thr Arg Asp Ala Ser Thr Ser Thr Val
Tyr Met Glu1 5 10 15Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 20 25 303530PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 35Arg Val Thr Met Thr
Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu1 5 10 15Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 303630PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 36Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr Leu Gln1 5 10 15Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 20 25 303730PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 37Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln1 5 10 15Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 303830PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 38Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Gln1 5 10 15Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 20 25 303930PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 39Arg Val Thr Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys1 5 10 15Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 20 25 304030PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 40Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu Lys1 5 10 15Leu Ser Pro Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys 20 25 304114PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 41Ala Arg Gly Ala Arg Arg
Ile Thr Gly Tyr Gly Met Asp Val1 5 104213PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Ala Arg Asp Leu Gly Tyr Tyr Gly Ser Gly Met His Ala1 5
104313PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 43Ala Arg Asp Leu Gly Tyr Tyr Gly Ser
Gly Met His Val1 5 104414PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 44Ala Arg Asp Arg Gly Met Tyr Tyr Ala Ser Gly Phe Gly Pro1
5 104514PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 45Ala Arg Asp Arg Gly Met
Tyr Tyr Ala Ser Gly Phe Asn Pro1 5 104614PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 46Ala Arg Asp Gln Gly Tyr Lys Thr Pro Thr Asp Phe Asp Leu1
5 104720PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 47Ala Lys Asp Pro Gly Tyr
Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn Tyr1 5 10 15Gly Met Asp Val
204814PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 48Ala Arg Ala Arg Pro Tyr Arg Glu Pro
Tyr Gly Met Asp Val1 5 104917PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 49Ala Arg Gly Pro Gly Tyr Asp Ser Ser Gly Tyr Ser Arg Arg
Phe Asp1 5 10 15Pro5011PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 50Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser1 5
105111PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 51Trp Gly Gln Gly Thr Thr Val Ile Val
Ser Ser1 5 105211PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 52Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser1 5 105311PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 53Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser1 5
105423PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 54Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
205523PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 55Glu Ile Val Met Thr Gln Ser Pro Gly
Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
205623PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 56Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
205723PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 57Asp Ile Gln Leu Thr Gln Ser Pro Ser
Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
205823PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 58Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
205911PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 59Arg Ala Ser Gln Gly Ile Ser Ser Trp
Leu Ala1 5 106011PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 60Arg Ala Ser Gln Gly Ile
Ser Arg Trp Leu Ala1 5 106112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 61Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala1 5
106211PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 62Gln Ala Ser Gln Asp Ile Thr Asn Tyr
Leu Asn1 5 106311PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 63Arg Ala Ser Gln Ser Ile
Ser Ser Tyr Leu Asn1 5 106411PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 64Arg Ala Ser Gln Asp Ile Ser Ser Trp Leu Ala1 5
106511PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 65Arg Ala Ser Gln Ser Ile Ser Ser Trp
Leu Ala1 5 106615PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 66Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr1 5 10 15677PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 67Ala Ala Ser Asn Leu Gln Ser1 5687PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 68Ala Ala Ser Ser Leu Gln Ser1 5697PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 69Gly Ala Ser Asn Arg Ala Thr1 5707PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 70Asp Ala Ser Asn Leu Glu Thr1 5717PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 71Gly Ala Ser Ser Leu Gln Ser1 5727PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 72Lys Ala Ser Ser Leu Glu Ser1 57332PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 73Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys 20 25 307432PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 74Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr1 5 10 15Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala
Val Tyr Tyr Cys 20 25 307532PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 75Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr1 5 10 15Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala
Thr Tyr Tyr Cys 20 25 307632PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 76Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Glu Phe Thr1 5 10 15Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala
Thr Tyr Tyr Cys 20 25 30779PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 77Gln Gln Ala Ser Val Phe Pro Phe Thr1 5789PRTArtificial
Sequencesource/note="Description of Artificial Sequence
Synthetic peptide" 78Gln Gln Ala Asn Leu Leu Pro Phe Thr1
5799PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 79Gln Gln Leu Ser Ser Phe Pro Ile Thr1
5809PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 80Gln Gln Ser Asp Val Leu Pro Ile Thr1
5818PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 81Gln Gln Thr Tyr Ser Leu Tyr Thr1
5829PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 82Gln Gln Glu Leu Ala Phe Pro Arg Thr1
5839PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 83Gln Gln Leu Asn Ser Tyr Pro Pro Thr1
58410PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 84Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys1 5 1085121PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 85Gln Val Gln Leu Val
Gln Ser Gly Ala Gly Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Ser Tyr 20 25 30Tyr Met Leu
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asp Pro Ser Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Pro 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 Gly Ala Arg Arg Ile Thr Gly Tyr Gly Met Asp Val Trp
Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12086121PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 86Gln 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 Tyr Thr Phe Arg Ser Tyr 20 25 30Tyr Met Leu
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asp Pro Ser Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Ala 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 Gly Ala Arg Arg Ile Thr Gly Tyr Gly Met Asp Val Trp
Gly 100 105 110Gln Gly Thr Thr Val Ile Val Ser Ser 115
12087120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 87Gln Ala 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 Tyr Thr Phe Ser Arg Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Leu Gly Gly Ser Thr Leu Tyr Ala Gln Lys Phe 50 55 60Gln
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 Asp Leu Gly Tyr Tyr Gly Ser Gly Met His Ala Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12088120PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 88Gln 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 Tyr Thr Phe Ser Arg Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Leu Gly Gly Ser Thr Leu Tyr Ala Gln Lys Phe 50 55 60Gln
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 Asp Leu Gly Tyr Tyr Gly Ser Gly Met His Val Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12089121PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 89Gln 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 Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Gln Gly Gly Asp Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
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 Asp Arg Gly Met Tyr Tyr Ala Ser Gly Phe Gly Pro Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12090121PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 90Gln Val Gln Leu Val
Gln Ser Gly Ala Lys Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Gln Gly Gly Asp Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
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 Asp Arg Gly Met Tyr Tyr Ala Ser Gly Phe Asn Pro Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser 115
12091121PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 91Glu 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 Glu
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg
Ile Lys Arg 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 Gln Gly Tyr Lys Thr Pro Thr Asp Phe Asp Leu Trp
Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser 115
12092127PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 92Glu 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 Ala Ser Tyr 20 25 30Ala Met Val
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ile
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
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 Asp Pro Gly Tyr Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn
Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 12593127PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 93Glu 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 Lys Ser Tyr 20 25 30Ala Met Val Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ile Ile Ser Gly Ser Gly Gly Ala Thr
Tyr Tyr Ala Asp Ser Val 50 55 60Lys 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 Asp Pro Gly Tyr
Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn Tyr 100 105 110Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12594127PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 94Glu 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 Ser Val 20 25 30Ala Met Val
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
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 Asp Pro Gly Tyr Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn
Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 12595127PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 95Glu 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 Ser Val 20 25 30Ala Met Val Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ala Thr
Tyr Tyr Ala Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Pro Gly Tyr
Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn Tyr 100 105 110Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12596127PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 96Glu 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 Ser Val 20 25 30Ala Met Val
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60Glu
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 Asp Pro Gly Tyr Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn
Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 12597120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 97Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Arg Gly Tyr 20 25 30Tyr Trp Glu Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Ser His Ser Gly Ser Thr Asn
Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Arg Pro Tyr Arg
Glu Pro Tyr Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val
Thr Val Ser Ser 115 12098120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 98Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Arg Gly Tyr 20 25 30Tyr Trp Glu Trp Ser Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Ser His Ser Gly Ser Thr Asn
Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Pro Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Arg Pro Tyr Arg
Glu Pro Tyr Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val
Thr Val Ser Ser 115 12099123PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 99Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Val Lys Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Asp Ile Trp His Ser Gly Met Thr Asn
Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Pro Gly Tyr Asp
Ser Ser Gly Tyr Ser Arg Arg Phe Asp Pro 100 105 110Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120100107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 100Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ala Ser Val Phe Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105101107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 101Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ala Asn Leu Leu Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys
100 105102108PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 102Glu Ile Val Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Leu Ser Ser Phe Pro
85 90 95Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105103107PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 103Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr Asn Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Val Leu Pro Ile
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105104106PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 104Asp 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 Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Leu Tyr Thr
85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105105107PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 105Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Glu Leu Ala Phe Pro Arg
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105106107PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 106Asp Ile Gln Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys
Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Pro
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105107448PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 107Gln Val Gln Leu Val
Gln Ser Gly Ala Gly Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Arg Ser Tyr 20 25 30Tyr Met Leu
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asp Pro Ser Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Thr Pro 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 Gly Ala Arg Arg Ile Thr Gly Tyr Gly Met Asp Val Trp
Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445108448PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 108Gln 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 Tyr Thr Phe Arg Ser Tyr 20 25 30Tyr Met Leu
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asp Pro Ser Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
Gly Arg Val Thr Met Thr Arg Asp Ala 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 Gly Ala Arg Arg Ile Thr Gly Tyr Gly Met Asp Val Trp
Gly 100 105 110Gln Gly Thr Thr Val Ile Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445109447PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 109Gln Ala 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 Tyr Thr Phe Ser Arg Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Leu Gly Gly Ser Thr Leu Tyr Ala Gln Lys Phe 50 55 60Gln
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 Asp Leu Gly Tyr Tyr Gly Ser Gly Met His Ala Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445110447PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 110Gln 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 Tyr Thr Phe Ser Arg Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Leu Gly Gly Ser Thr Leu Tyr Ala Gln Lys Phe 50 55 60Gln
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 Asp Leu Gly Tyr Tyr Gly Ser Gly Met His Val Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445111448PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 111Gln 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 Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Gln Gly Gly Asp Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
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 Asp Arg Gly Met Tyr Tyr Ala Ser Gly Phe Gly Pro Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445112448PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 112Gln Val Gln Leu Val
Gln Ser Gly Ala Lys Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met His
Trp Val Arg Gln Val Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Ile
Ile Asn Pro Gln Gly Gly Asp Thr Ser Tyr Ala Gln Lys Phe 50 55 60Gln
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 Asp Arg Gly Met Tyr Tyr Ala Ser Gly Phe Asn Pro Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445113448PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 113Glu 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 Glu
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg
Ile Lys Arg 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 Gln Gly Tyr Lys Thr Pro Thr Asp Phe Asp Leu Trp
Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445114454PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 114Glu 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 Ala Ser Tyr 20 25 30Ala Met Val
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ile
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
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 Asp Pro Gly Tyr Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn
Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Ala 115 120 125Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser 130 135 140Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe145 150 155 160Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr 195 200
205Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu225 230 235 240Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 260 265 270Val Ser Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 290 295 300Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310 315
320Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
325 330 335Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 340 345 350Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn 355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 405 410 415Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 420 425 430Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440
445Ser Leu Ser Leu Gly Lys 450115454PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 115Glu 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 Lys Ser Tyr 20 25 30Ala Met Val Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ile Ile Ser Gly Ser Gly Gly Ala Thr
Tyr Tyr Ala Asp Ser Val 50 55 60Lys 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 Asp Pro Gly Tyr
Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn Tyr 100 105 110Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 130 135
140Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe145 150 155 160Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly 165 170 175Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu 180 185 190Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr 195 200 205Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 210 215 220Val Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu225 230 235 240Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250
255Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn 290 295 300Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp305 310 315 320Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro 325 330 335Ser Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 355 360 365Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375
380Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr385 390 395 400Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Arg 405 410 415Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
Gly Asn Val Phe Ser Cys 420 425 430Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser Leu Gly Lys
450116454PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 116Glu 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 Ser Val 20 25 30Ala Met Val
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
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 Asp Pro Gly Tyr Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn
Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Ala 115 120 125Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser 130 135 140Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe145 150 155 160Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr 195 200
205Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu225 230 235 240Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 260 265 270Val Ser Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 290 295 300Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310 315
320Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
325 330 335Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 340 345 350Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn 355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 405 410 415Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 420 425 430Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440
445Ser Leu Ser Leu Gly Lys 450117454PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 117Glu 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 Ser Val 20 25 30Ala Met Val Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ala Thr
Tyr Tyr Ala Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Ser Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Pro Gly Tyr
Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn Tyr 100 105 110Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser 130 135
140Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe145 150 155 160Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly 165 170 175Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu 180 185 190Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr 195 200 205Thr Cys Asn Val Asp His
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg 210 215 220Val Glu Ser Lys
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu225 230 235 240Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250
255Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn 290 295 300Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp305 310 315 320Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro 325 330 335Ser Ser Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn 355 360 365Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375
380Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr385 390 395 400Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Arg 405 410 415Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
Gly Asn Val Phe Ser Cys 420 425 430Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser Leu Gly Lys
450118454PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 118Glu 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 Ser Val 20 25 30Ala Met Val
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60Glu
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 Asp Pro Gly Tyr Asp Ser Ser Arg Tyr Tyr Tyr Ser Asn
Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Ala 115 120 125Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser 130 135 140Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe145 150 155 160Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr 195 200
205Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu225 230 235 240Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 245 250 255Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp 260 265 270Val Ser Gln Glu Asp Pro Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly 275 280 285Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 290 295 300Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp305 310 315
320Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
325 330 335Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 340 345 350Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn 355 360 365Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 370 375 380Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr385 390 395 400Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg 405 410 415Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys 420 425 430Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 435 440
445Ser Leu Ser Leu Gly Lys 450119447PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 119Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Arg Gly Tyr 20 25 30Tyr Trp Glu Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Ser His Ser Gly Ser Thr Asn
Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Arg Pro Tyr Arg
Glu Pro Tyr Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
260 265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys 435 440 445120447PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 120Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Arg Gly Tyr 20 25 30Tyr Trp Glu Trp Ser Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Ser His Ser Gly Ser Thr Asn
Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Pro Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Ala Arg Pro Tyr Arg
Glu Pro Tyr Gly Met Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
260 265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys 435 440 445121450PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 121Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys
Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser
Phe Val Lys Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Ile 35 40 45Gly Asp Ile Trp His Ser Gly Met Thr Asn
Tyr Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75 80Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Gly Pro Gly Tyr Asp
Ser Ser Gly Tyr Ser Arg Arg Phe Asp Pro 100 105 110Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135
140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220Tyr Gly Pro Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly225 230 235 240Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250
255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
260 265 270Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310
315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415Lys Ser
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425
430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
435 440 445Gly Lys 450122214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 122Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ala Ser Val Phe Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
210123214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 123Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Leu Leu Pro Phe
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210124215PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 124Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Leu Ser Ser Phe Pro 85 90 95Ile Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 215125214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 125Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr Asn Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp
Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Val Leu Pro Ile
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210126213PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 126Asp 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 Ala Ser Gln Ser
Ile Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Thr Tyr Ser Leu Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135
140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
210127214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 127Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Glu Leu Ala Phe Pro Arg
85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210128214PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 128Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Leu Asn Ser Tyr Pro Pro 85 90 95Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
2101292772DNAHomo sapiens 129atggagaggg ggctgccgct cctctgcgcc
gtgctcgccc tcgtcctcgc cccggccggc 60gcttttcgca acgataaatg tggcgatact
ataaaaattg aaagccccgg gtaccttaca 120tctcctggtt atcctcattc
ttatcaccca agtgaaaaat gcgaatggct gattcaggct 180ccggacccat
accagagaat tatgatcaac ttcaaccctc acttcgattt ggaggacaga
240gactgcaagt atgactacgt ggaagtcttc gatggagaaa atgaaaatgg
acattttagg 300ggaaagttct gtggaaagat agcccctcct cctgttgtgt
cttcagggcc atttcttttt 360atcaaatttg tctctgacta cgaaacacat
ggtgcaggat tttccatacg ttatgaaatt 420ttcaagagag gtcctgaatg
ttcccagaac tacacaacac ctagtggagt gataaagtcc 480cccggattcc
ctgaaaaata tcccaacagc cttgaatgca cttatattgt ctttgcgcca
540aagatgtcag agattatcct ggaatttgaa agctttgacc tggagcctga
ctcaaatcct 600ccagggggga tgttctgtcg ctacgaccgg ctagaaatct
gggatggatt ccctgatgtt 660ggccctcaca ttgggcgtta ctgtggacag
aaaacaccag gtcgaatccg atcctcatcg 720ggcattctct ccatggtttt
ttacaccgac agcgcgatag caaaagaagg tttctcagca 780aactacagtg
tcttgcagag cagtgtctca gaagatttca aatgtatgga agctctgggc
840atggaatcag gagaaattca ttctgaccag atcacagctt cttcccagta
tagcaccaac 900tggtctgcag agcgctcccg cctgaactac cctgagaatg
ggtggactcc cggagaggat 960tcctaccgag agtggataca ggtagacttg
ggccttctgc gctttgtcac ggctgtcggg 1020acacagggcg ccatttcaaa
agaaaccaag aagaaatatt atgtcaagac ttacaagatc 1080gacgttagct
ccaacgggga agactggatc accataaaag aaggaaacaa acctgttctc
1140tttcagggaa acaccaaccc cacagatgtt gtggttgcag tattccccaa
accactgata 1200actcgatttg tccgaatcaa gcctgcaact tgggaaactg
gcatatctat gagatttgaa 1260gtatacggtt gcaagataac agattatcct
tgctctggaa tgttgggtat ggtgtctgga 1320cttatttctg actcccagat
cacatcatcc aaccaagggg acagaaactg gatgcctgaa 1380aacatccgcc
tggtaaccag tcgctctggc tgggcacttc cacccgcacc tcattcctac
1440atcaatgagt ggctccaaat agacctgggg gaggagaaga tcgtgagggg
catcatcatt 1500cagggtggga agcaccgaga gaacaaggtg ttcatgagga
agttcaagat cgggtacagc 1560aacaacggct cggactggaa gatgatcatg
gatgacagca aacgcaaggc gaagtctttt 1620gagggcaaca acaactatga
tacacctgag ctgcggactt ttccagctct ctccacgcga 1680ttcatcagga
tctaccccga gagagccact catggcggac tggggctcag aatggagctg
1740ctgggctgtg aagtggaagc ccctacagct ggaccgacca ctcccaacgg
gaacttggtg 1800gatgaatgtg atgacgacca ggccaactgc cacagtggaa
caggtgatga cttccagctc 1860acaggtggca ccactgtgct ggccacagaa
aagcccacgg tcatagacag caccatacaa 1920tcagagtttc caacatatgg
ttttaactgt gaatttggct ggggctctca caagaccttc 1980tgccactggg
aacatgacaa tcacgtgcag ctcaagtgga gtgtgttgac cagcaagacg
2040ggacccattc aggatcacac aggagatggc aacttcatct attcccaagc
tgacgaaaat 2100cagaagggca aagtggctcg cctggtgagc cctgtggttt
attcccagaa ctctgcccac 2160tgcatgacct tctggtatca catgtctggg
tcccacgtcg gcacactcag ggtcaaactg 2220cgctaccaga agccagagga
gtacgatcag ctggtctgga tggccattgg acaccaaggt 2280gaccactgga
aggaagggcg tgtcttgctc cacaagtctc tgaaacttta tcaggtgatt
2340ttcgagggcg aaatcggaaa aggaaacctt ggtgggattg ctgtggatga
cattagtatt 2400aataaccaca tttcacaaga agattgtgca aaaccagcag
acctggataa aaagaaccca 2460gaaattaaaa ttgatgaaac agggagcacg
ccaggatacg aaggtgaagg agaaggtgac 2520aagaacatct ccaggaagcc
aggcaatgtg ttgaagacct tagaccccat cctcatcacc 2580atcatagcca
tgagtgccct gggggtcctc ctgggggctg tctgtggggt cgtgctgtac
2640tgtgcctgtt ggcataatgg gatgtcagaa agaaacttgt ctgccctgga
gaactataac 2700tttgaacttg tggatggtgt gaagttgaaa aaagacaaac
tgaatacaca gagtacttat 2760tcggaggcat ga 2772130923PRTHomo sapiens
130Met Glu Arg Gly Leu Pro Leu Leu Cys Ala Val Leu Ala Leu Val Leu1
5 10 15Ala Pro Ala Gly Ala Phe Arg Asn Asp Lys Cys Gly Asp Thr Ile
Lys 20 25 30Ile Glu Ser Pro Gly Tyr Leu Thr Ser Pro Gly Tyr Pro His
Ser Tyr 35 40 45His Pro Ser Glu Lys Cys Glu Trp Leu Ile Gln Ala Pro
Asp Pro Tyr 50 55 60Gln Arg Ile Met Ile Asn Phe Asn Pro His Phe Asp
Leu Glu Asp Arg65 70 75 80Asp Cys Lys Tyr Asp Tyr Val Glu Val Phe
Asp Gly Glu Asn Glu Asn 85 90 95Gly His Phe Arg Gly Lys Phe Cys Gly
Lys Ile Ala Pro Pro Pro Val 100 105 110Val Ser Ser Gly Pro Phe Leu
Phe Ile Lys Phe Val Ser Asp Tyr Glu 115 120 125Thr His Gly Ala Gly
Phe Ser Ile Arg Tyr Glu Ile Phe Lys Arg Gly 130 135 140Pro Glu Cys
Ser Gln Asn Tyr Thr Thr Pro Ser Gly Val Ile Lys Ser145 150 155
160Pro Gly Phe Pro Glu Lys Tyr Pro Asn Ser Leu Glu Cys Thr Tyr Ile
165 170 175Val Phe Ala Pro Lys Met Ser Glu Ile Ile Leu Glu Phe Glu
Ser Phe 180 185 190Asp Leu Glu Pro Asp Ser Asn Pro Pro Gly Gly Met
Phe Cys Arg Tyr 195 200 205Asp Arg Leu Glu Ile Trp Asp Gly Phe Pro
Asp Val Gly Pro His Ile 210 215 220Gly Arg Tyr Cys Gly Gln Lys Thr
Pro Gly Arg Ile Arg Ser Ser Ser225 230 235
240Gly Ile Leu Ser Met Val Phe Tyr Thr Asp Ser Ala Ile Ala Lys Glu
245 250 255Gly Phe Ser Ala Asn Tyr Ser Val Leu Gln Ser Ser Val Ser
Glu Asp 260 265 270Phe Lys Cys Met Glu Ala Leu Gly Met Glu Ser Gly
Glu Ile His Ser 275 280 285Asp Gln Ile Thr Ala Ser Ser Gln Tyr Ser
Thr Asn Trp Ser Ala Glu 290 295 300Arg Ser Arg Leu Asn Tyr Pro Glu
Asn Gly Trp Thr Pro Gly Glu Asp305 310 315 320Ser Tyr Arg Glu Trp
Ile Gln Val Asp Leu Gly Leu Leu Arg Phe Val 325 330 335Thr Ala Val
Gly Thr Gln Gly Ala Ile Ser Lys Glu Thr Lys Lys Lys 340 345 350Tyr
Tyr Val Lys Thr Tyr Lys Ile Asp Val Ser Ser Asn Gly Glu Asp 355 360
365Trp Ile Thr Ile Lys Glu Gly Asn Lys Pro Val Leu Phe Gln Gly Asn
370 375 380Thr Asn Pro Thr Asp Val Val Val Ala Val Phe Pro Lys Pro
Leu Ile385 390 395 400Thr Arg Phe Val Arg Ile Lys Pro Ala Thr Trp
Glu Thr Gly Ile Ser 405 410 415Met Arg Phe Glu Val Tyr Gly Cys Lys
Ile Thr Asp Tyr Pro Cys Ser 420 425 430Gly Met Leu Gly Met Val Ser
Gly Leu Ile Ser Asp Ser Gln Ile Thr 435 440 445Ser Ser Asn Gln Gly
Asp Arg Asn Trp Met Pro Glu Asn Ile Arg Leu 450 455 460Val Thr Ser
Arg Ser Gly Trp Ala Leu Pro Pro Ala Pro His Ser Tyr465 470 475
480Ile Asn Glu Trp Leu Gln Ile Asp Leu Gly Glu Glu Lys Ile Val Arg
485 490 495Gly Ile Ile Ile Gln Gly Gly Lys His Arg Glu Asn Lys Val
Phe Met 500 505 510Arg Lys Phe Lys Ile Gly Tyr Ser Asn Asn Gly Ser
Asp Trp Lys Met 515 520 525Ile Met Asp Asp Ser Lys Arg Lys Ala Lys
Ser Phe Glu Gly Asn Asn 530 535 540Asn Tyr Asp Thr Pro Glu Leu Arg
Thr Phe Pro Ala Leu Ser Thr Arg545 550 555 560Phe Ile Arg Ile Tyr
Pro Glu Arg Ala Thr His Gly Gly Leu Gly Leu 565 570 575Arg Met Glu
Leu Leu Gly Cys Glu Val Glu Ala Pro Thr Ala Gly Pro 580 585 590Thr
Thr Pro Asn Gly Asn Leu Val Asp Glu Cys Asp Asp Asp Gln Ala 595 600
605Asn Cys His Ser Gly Thr Gly Asp Asp Phe Gln Leu Thr Gly Gly Thr
610 615 620Thr Val Leu Ala Thr Glu Lys Pro Thr Val Ile Asp Ser Thr
Ile Gln625 630 635 640Ser Glu Phe Pro Thr Tyr Gly Phe Asn Cys Glu
Phe Gly Trp Gly Ser 645 650 655His Lys Thr Phe Cys His Trp Glu His
Asp Asn His Val Gln Leu Lys 660 665 670Trp Ser Val Leu Thr Ser Lys
Thr Gly Pro Ile Gln Asp His Thr Gly 675 680 685Asp Gly Asn Phe Ile
Tyr Ser Gln Ala Asp Glu Asn Gln Lys Gly Lys 690 695 700Val Ala Arg
Leu Val Ser Pro Val Val Tyr Ser Gln Asn Ser Ala His705 710 715
720Cys Met Thr Phe Trp Tyr His Met Ser Gly Ser His Val Gly Thr Leu
725 730 735Arg Val Lys Leu Arg Tyr Gln Lys Pro Glu Glu Tyr Asp Gln
Leu Val 740 745 750Trp Met Ala Ile Gly His Gln Gly Asp His Trp Lys
Glu Gly Arg Val 755 760 765Leu Leu His Lys Ser Leu Lys Leu Tyr Gln
Val Ile Phe Glu Gly Glu 770 775 780Ile Gly Lys Gly Asn Leu Gly Gly
Ile Ala Val Asp Asp Ile Ser Ile785 790 795 800Asn Asn His Ile Ser
Gln Glu Asp Cys Ala Lys Pro Ala Asp Leu Asp 805 810 815Lys Lys Asn
Pro Glu Ile Lys Ile Asp Glu Thr Gly Ser Thr Pro Gly 820 825 830Tyr
Glu Gly Glu Gly Glu Gly Asp Lys Asn Ile Ser Arg Lys Pro Gly 835 840
845Asn Val Leu Lys Thr Leu Asp Pro Ile Leu Ile Thr Ile Ile Ala Met
850 855 860Ser Ala Leu Gly Val Leu Leu Gly Ala Val Cys Gly Val Val
Leu Tyr865 870 875 880Cys Ala Cys Trp His Asn Gly Met Ser Glu Arg
Asn Leu Ser Ala Leu 885 890 895Glu Asn Tyr Asn Phe Glu Leu Val Asp
Gly Val Lys Leu Lys Lys Asp 900 905 910Lys Leu Asn Thr Gln Ser Thr
Tyr Ser Glu Ala 915 9201312772DNAMacaca fascicularis 131atggagaagg
ggttgccgct cctctgcgcc gcgctcgccc tcgccctcgc cccggccggc 60gcttttcgca
acgataaatg tggcgatact ataaaaattg aaagccccgg gtaccttaca
120tctcctggtt atcctcattc ttatcaccca agtgaaaaat gtgaatggct
gattcaggct 180ccggacccat accagagaat tatgatcaac ttcaaccctc
acttcgattt ggaggacaga 240gattgcaagt atgactacgt ggaagtcttc
gatggagaaa atgaaaatgg acgtttatgg 300ggaaagttct gtggaaagat
agcccctcct cctgttgtgt cttcagggca atttcttttt 360atcaaatttg
tctctgacta cgaaacacac ggtgcaggat tttccatacg ttatgaaatt
420ttcaagagag gtcctgaatg ttcccagaac tacacaacac ctagtggagt
gataaagtcc 480cccggattcc ctgaaaaata tcccaacagc cttgaatgca
cttatattgt ctttgcacca 540aagatgtcag agattatcct ggaatttgaa
agctttgacc tggagcctga ctcaaatcct 600ccagggggga tgttctgtcg
ctacgaccgg ctggaaatct gggatggatt ccctgacgtt 660ggccctcaca
ttgggcgtta ctgtggacag aaaacaccag gtcgaatccg atcctcatcg
720ggcattctct ccatggtttt ttacaccgac agcgcaatag caaaagaagg
tttctcagca 780aactacagtg tcttgcagag cagtgtctca gaagatttca
aatgtatgga agctgtgggc 840atggaatcag gagaaattca ttctgaccag
atcacagctt cttcccagta cagcaccaac 900tggtctgcag agcgctcccg
cctgaactat cctgagaatg ggtggactcc cggagaagat 960tcctaccgag
agtggataca ggtggacttg ggccttctac gcttcgttac ggctgtcggg
1020acacagggcg ccatttcaaa agaaaccaag aagaaatatt atgtcaagac
ttacaaaatt 1080gacattagct ccaacgggga agactggatc accataaaag
aaggaaacaa acctgttctc 1140tttcagggaa acaccaaccc cacagacgtt
gtggttgcag tattccccaa gccactgata 1200actcgatttg tccgaatcaa
gcctgcaact tgggaaactg gcatatctct gagatttgaa 1260gtatatggtt
gcaagataac agattatcct tgctccggaa tgttgggtat ggtgtctgga
1320cttatttctg actcccagat cacatcatcc aaccaagggg acagaaactg
gatgcctgaa 1380aacatccgcc tggtaaccag tcgctccggc tgggcactgc
cacccgcacc tcattcctac 1440gtcaatgagt ggctccaaat agacctgggg
gaggagaaga tcgtgagggg catcatcatt 1500cagggtggga agcaccgaga
gaacaaggta ttcatgagga agttcaagat cgggtacagc 1560aacaacggct
ccgactggaa gatgatcatg gacgacagca aacgcaaggc aaagtctttt
1620gagggcaaca acaactatga cacacctgag ctgcggactt ttccagctct
ctccacgcga 1680ttcatcagga tctaccccga gagagccact catggcggac
tggggctccg aatggagctg 1740ctgggctgtg aagtggaagc ccctacagct
ggaccgacca ctcccaacgg gaacccggtg 1800gatgaatgtg atgacgacca
ggccaactgc cacagtggaa caggtgatga cttccagctc 1860acaggtggca
ccactgtgct ggccacagaa aagcccacgg tcatagacag caccatacaa
1920tcagagtttc ctacatatgg ttttaactgt gaatttggct ggggctctca
caagaccttc 1980tgccactggg aacatgacaa tcacgtgcag ctcaagtgga
gtgtgttgac cagcaagacg 2040ggacccattc aggatcacac aggagatggc
aacttcatct attcccaagc tgatgaaaat 2100cagaagggca aagtggctcg
cctggtgagc cctgtggttt attcccagaa ctctgcccac 2160tgcatgacct
tctggtatca catgtctggg tcccacgtcg gcacactcag ggtcaaactg
2220cgctaccaga agccagagga gtacgatcag ctggtctgga tggccattgg
acaccaaggt 2280gaccactgga aggaagggcg tgtcttgctt cacaagtctc
tgaaacttta tcaggtgatt 2340ttcgagggcg aaatcggaaa aggaaacctt
ggtgggattg ctgtggatga cattagtatc 2400aataaccaca tttcacaaga
agattgtgca aaaccagcag acctggataa aaagaaccca 2460gaaattaaaa
ttgatgaaac agggagcaca ccaggatatg aaggtgaagg agaaggtgac
2520aagaacatct ccaggaaacc aggcaatgtg ttgaagacct tagaccccat
cctcatcacc 2580atcatagcca tgagcgccct gggggtcctc ctgggggctg
tgtgcggggt cgtgctgtac 2640tgtgcctgtt ggcataatgg gatgtcagaa
agaaacttgt ctgccctgga gaactataac 2700tttgaacttg tggacggtgt
gaagttgaaa aaagacaaac tgaatacaca gagtacttat 2760tcggaggcat ga
2772132923PRTMacaca fascicularis 132Met Glu Lys Gly Leu Pro Leu Leu
Cys Ala Ala Leu Ala Leu Ala Leu1 5 10 15Ala Pro Ala Gly Ala Phe Arg
Asn Asp Lys Cys Gly Asp Thr Ile Lys 20 25 30Ile Glu Ser Pro Gly Tyr
Leu Thr Ser Pro Gly Tyr Pro His Ser Tyr 35 40 45His Pro Ser Glu Lys
Cys Glu Trp Leu Ile Gln Ala Pro Asp Pro Tyr 50 55 60Gln Arg Ile Met
Ile Asn Phe Asn Pro His Phe Asp Leu Glu Asp Arg65 70 75 80Asp Cys
Lys Tyr Asp Tyr Val Glu Val Phe Asp Gly Glu Asn Glu Asn 85 90 95Gly
Arg Leu Trp Gly Lys Phe Cys Gly Lys Ile Ala Pro Pro Pro Val 100 105
110Val Ser Ser Gly Gln Phe Leu Phe Ile Lys Phe Val Ser Asp Tyr Glu
115 120 125Thr His Gly Ala Gly Phe Ser Ile Arg Tyr Glu Ile Phe Lys
Arg Gly 130 135 140Pro Glu Cys Ser Gln Asn Tyr Thr Thr Pro Ser Gly
Val Ile Lys Ser145 150 155 160Pro Gly Phe Pro Glu Lys Tyr Pro Asn
Ser Leu Glu Cys Thr Tyr Ile 165 170 175Val Phe Ala Pro Lys Met Ser
Glu Ile Ile Leu Glu Phe Glu Ser Phe 180 185 190Asp Leu Glu Pro Asp
Ser Asn Pro Pro Gly Gly Met Phe Cys Arg Tyr 195 200 205Asp Arg Leu
Glu Ile Trp Asp Gly Phe Pro Asp Val Gly Pro His Ile 210 215 220Gly
Arg Tyr Cys Gly Gln Lys Thr Pro Gly Arg Ile Arg Ser Ser Ser225 230
235 240Gly Ile Leu Ser Met Val Phe Tyr Thr Asp Ser Ala Ile Ala Lys
Glu 245 250 255Gly Phe Ser Ala Asn Tyr Ser Val Leu Gln Ser Ser Val
Ser Glu Asp 260 265 270Phe Lys Cys Met Glu Ala Val Gly Met Glu Ser
Gly Glu Ile His Ser 275 280 285Asp Gln Ile Thr Ala Ser Ser Gln Tyr
Ser Thr Asn Trp Ser Ala Glu 290 295 300Arg Ser Arg Leu Asn Tyr Pro
Glu Asn Gly Trp Thr Pro Gly Glu Asp305 310 315 320Ser Tyr Arg Glu
Trp Ile Gln Val Asp Leu Gly Leu Leu Arg Phe Val 325 330 335Thr Ala
Val Gly Thr Gln Gly Ala Ile Ser Lys Glu Thr Lys Lys Lys 340 345
350Tyr Tyr Val Lys Thr Tyr Lys Ile Asp Ile Ser Ser Asn Gly Glu Asp
355 360 365Trp Ile Thr Ile Lys Glu Gly Asn Lys Pro Val Leu Phe Gln
Gly Asn 370 375 380Thr Asn Pro Thr Asp Val Val Val Ala Val Phe Pro
Lys Pro Leu Ile385 390 395 400Thr Arg Phe Val Arg Ile Lys Pro Ala
Thr Trp Glu Thr Gly Ile Ser 405 410 415Leu Arg Phe Glu Val Tyr Gly
Cys Lys Ile Thr Asp Tyr Pro Cys Ser 420 425 430Gly Met Leu Gly Met
Val Ser Gly Leu Ile Ser Asp Ser Gln Ile Thr 435 440 445Ser Ser Asn
Gln Gly Asp Arg Asn Trp Met Pro Glu Asn Ile Arg Leu 450 455 460Val
Thr Ser Arg Ser Gly Trp Ala Leu Pro Pro Ala Pro His Ser Tyr465 470
475 480Val Asn Glu Trp Leu Gln Ile Asp Leu Gly Glu Glu Lys Ile Val
Arg 485 490 495Gly Ile Ile Ile Gln Gly Gly Lys His Arg Glu Asn Lys
Val Phe Met 500 505 510Arg Lys Phe Lys Ile Gly Tyr Ser Asn Asn Gly
Ser Asp Trp Lys Met 515 520 525Ile Met Asp Asp Ser Lys Arg Lys Ala
Lys Ser Phe Glu Gly Asn Asn 530 535 540Asn Tyr Asp Thr Pro Glu Leu
Arg Thr Phe Pro Ala Leu Ser Thr Arg545 550 555 560Phe Ile Arg Ile
Tyr Pro Glu Arg Ala Thr His Gly Gly Leu Gly Leu 565 570 575Arg Met
Glu Leu Leu Gly Cys Glu Val Glu Ala Pro Thr Ala Gly Pro 580 585
590Thr Thr Pro Asn Gly Asn Pro Val Asp Glu Cys Asp Asp Asp Gln Ala
595 600 605Asn Cys His Ser Gly Thr Gly Asp Asp Phe Gln Leu Thr Gly
Gly Thr 610 615 620Thr Val Leu Ala Thr Glu Lys Pro Thr Val Ile Asp
Ser Thr Ile Gln625 630 635 640Ser Glu Phe Pro Thr Tyr Gly Phe Asn
Cys Glu Phe Gly Trp Gly Ser 645 650 655His Lys Thr Phe Cys His Trp
Glu His Asp Asn His Val Gln Leu Lys 660 665 670Trp Ser Val Leu Thr
Ser Lys Thr Gly Pro Ile Gln Asp His Thr Gly 675 680 685Asp Gly Asn
Phe Ile Tyr Ser Gln Ala Asp Glu Asn Gln Lys Gly Lys 690 695 700Val
Ala Arg Leu Val Ser Pro Val Val Tyr Ser Gln Asn Ser Ala His705 710
715 720Cys Met Thr Phe Trp Tyr His Met Ser Gly Ser His Val Gly Thr
Leu 725 730 735Arg Val Lys Leu Arg Tyr Gln Lys Pro Glu Glu Tyr Asp
Gln Leu Val 740 745 750Trp Met Ala Ile Gly His Gln Gly Asp His Trp
Lys Glu Gly Arg Val 755 760 765Leu Leu His Lys Ser Leu Lys Leu Tyr
Gln Val Ile Phe Glu Gly Glu 770 775 780Ile Gly Lys Gly Asn Leu Gly
Gly Ile Ala Val Asp Asp Ile Ser Ile785 790 795 800Asn Asn His Ile
Ser Gln Glu Asp Cys Ala Lys Pro Ala Asp Leu Asp 805 810 815Lys Lys
Asn Pro Glu Ile Lys Ile Asp Glu Thr Gly Ser Thr Pro Gly 820 825
830Tyr Glu Gly Glu Gly Glu Gly Asp Lys Asn Ile Ser Arg Lys Pro Gly
835 840 845Asn Val Leu Lys Thr Leu Asp Pro Ile Leu Ile Thr Ile Ile
Ala Met 850 855 860Ser Ala Leu Gly Val Leu Leu Gly Ala Val Cys Gly
Val Val Leu Tyr865 870 875 880Cys Ala Cys Trp His Asn Gly Met Ser
Glu Arg Asn Leu Ser Ala Leu 885 890 895Glu Asn Tyr Asn Phe Glu Leu
Val Asp Gly Val Lys Leu Lys Lys Asp 900 905 910Lys Leu Asn Thr Gln
Ser Thr Tyr Ser Glu Ala 915 9201332772DNAMus musculus 133atggagaggg
ggctgccgtt gctgtgcgcc acgctcgccc ttgccctcgc cctggcgggc 60gctttccgca
gcgacaaatg tggcgggacc ataaaaatcg aaaacccagg gtacctcaca
120tctcccggtt accctcattc ttaccatcca agtgagaagt gtgaatggct
aatccaagct 180ccggaaccct accagagaat catgatcaac ttcaacccac
atttcgattt ggaggacaga 240gactgcaagt atgactacgt ggaagtaatc
gatggggaga atgaaggcgg ccgcctgtgg 300gggaagttct gtgggaagat
tgcaccttct cctgtggtgt cttcagggcc ctttctcttc 360atcaaatttg
tctctgacta tgagacacat ggggcagggt tttccatccg ctatgaaatc
420ttcaagagag ggcccgaatg ttctcagaac tatacagcac ctactggagt
gataaagtcc 480cctgggttcc ctgaaaaata ccccaacagc ttggagtgca
cctacatcat ctttgcacca 540aagatgtctg agataatcct ggagtttgaa
agttttgacc tggagcaaga ctcgaatcct 600cccggaggaa tgttctgtcg
ctatgaccgg ctggagatct gggatggatt ccctgaagtt 660ggccctcaca
ttgggcgtta ttgtgggcag aaaactcctg gccggatccg ctcctcttca
720ggcgttctat ccatggtctt ttacactgac agcgcaatag caaaagaagg
tttctcagcc 780aactacagtg tgctacagag cagcatctct gaagatttta
agtgtatgga ggctctgggc 840atggaatctg gagagatcca ttctgatcag
atcactgcat cttcacagta tggtaccaac 900tggtctgtag agcgctcccg
cctgaactac cctgaaaatg ggtggactcc aggagaagac 960tcctacaagg
agtggatcca ggtggacttg ggcctcctgc gattcgttac tgctgtaggg
1020acacagggtg ccatttccaa ggaaaccaag aagaaatatt atgtcaagac
ttacagagta 1080gacatcagct ccaacggaga ggactggatc tccctgaaag
agggaaataa agccattatc 1140tttcagggaa acaccaaccc cacagatgtt
gtcttaggag ttttctccaa accactgata 1200actcgatttg tccgaatcaa
acctgtatcc tgggaaactg gtatatctat gagatttgaa 1260gtttatggct
gcaagataac agattatcct tgctctggaa tgttgggcat ggtgtctgga
1320cttatttcag actcccagat tacagcatcc aatcaagccg acaggaattg
gatgccagaa 1380aacatccgtc tggtgaccag tcgtaccggc tgggcactgc
caccctcacc ccacccatac 1440accaatgaat ggctccaagt ggacctggga
gatgagaaga tagtaagagg tgtcatcatt 1500cagggtggga agcaccgaga
aaacaaggtg ttcatgagga agttcaagat cgcctatagt 1560aacaatggct
ctgactggaa aactatcatg gatgacagca agcgcaaggc taagtcgttc
1620gaaggcaaca acaactatga cacacctgag cttcggacgt tttcacctct
ctccacaagg 1680ttcatcagga tctaccctga gagagccaca cacagtgggc
ttgggctgag gatggagcta 1740ctgggctgtg aagtggaagc acctacagct
ggaccaacca cacccaatgg gaacccagtg 1800gatgagtgtg acgacgacca
ggccaactgc cacagtggca caggtgatga cttccagctc 1860acaggaggca
ccactgtcct ggccacagag aagccaacca ttatagacag caccatccaa
1920tcagagttcc cgacatacgg ttttaactgc gagtttggct ggggctctca
caagacattc 1980tgccactggg agcatgacag ccatgcacag ctcaggtgga
gtgtgctgac cagcaagaca 2040gggccgattc aggaccatac aggagatggc
aacttcatct attcccaagc tgatgaaaat 2100cagaaaggca aagtagcccg
cctggtgagc cctgtggtct attcccagag ctctgcccac 2160tgtatgacct
tctggtatca catgtccggc tctcatgtgg gtacactgag ggtcaaacta
2220cgctaccaga agccagagga atatgatcaa ctggtctgga tggtggttgg
gcaccaagga 2280gaccactgga aagaaggacg tgtcttgctg cacaaatctc
tgaaactata tcaggttatt 2340tttgaaggtg aaatcggaaa aggaaacctt
ggtggaattg
ctgtggatga tatcagtatt 2400aacaaccata tttctcagga agactgtgca
aaaccaacag acctagataa aaagaacaca 2460gaaattaaaa ttgatgaaac
agggagcact ccaggatatg aaggagaagg ggaaggtgac 2520aagaacatct
ccaggaagcc aggcaatgtg cttaagaccc tggatcccat cctgatcacc
2580atcatagcca tgagtgccct gggagtactc ctgggtgcag tctgtggagt
tgtgctgtac 2640tgtgcctgtt ggcacaatgg gatgtcagaa aggaacctat
ctgccctgga gaactataac 2700tttgaacttg tggatggtgt aaagttgaaa
aaagataaac tgaacccaca gagtaattac 2760tcagaggcgt ga 2772134923PRTMus
musculus 134Met Glu Arg Gly Leu Pro Leu Leu Cys Ala Thr Leu Ala Leu
Ala Leu1 5 10 15Ala Leu Ala Gly Ala Phe Arg Ser Asp Lys Cys Gly Gly
Thr Ile Lys 20 25 30Ile Glu Asn Pro Gly Tyr Leu Thr Ser Pro Gly Tyr
Pro His Ser Tyr 35 40 45His Pro Ser Glu Lys Cys Glu Trp Leu Ile Gln
Ala Pro Glu Pro Tyr 50 55 60Gln Arg Ile Met Ile Asn Phe Asn Pro His
Phe Asp Leu Glu Asp Arg65 70 75 80Asp Cys Lys Tyr Asp Tyr Val Glu
Val Ile Asp Gly Glu Asn Glu Gly 85 90 95Gly Arg Leu Trp Gly Lys Phe
Cys Gly Lys Ile Ala Pro Ser Pro Val 100 105 110Val Ser Ser Gly Pro
Phe Leu Phe Ile Lys Phe Val Ser Asp Tyr Glu 115 120 125Thr His Gly
Ala Gly Phe Ser Ile Arg Tyr Glu Ile Phe Lys Arg Gly 130 135 140Pro
Glu Cys Ser Gln Asn Tyr Thr Ala Pro Thr Gly Val Ile Lys Ser145 150
155 160Pro Gly Phe Pro Glu Lys Tyr Pro Asn Ser Leu Glu Cys Thr Tyr
Ile 165 170 175Ile Phe Ala Pro Lys Met Ser Glu Ile Ile Leu Glu Phe
Glu Ser Phe 180 185 190Asp Leu Glu Gln Asp Ser Asn Pro Pro Gly Gly
Met Phe Cys Arg Tyr 195 200 205Asp Arg Leu Glu Ile Trp Asp Gly Phe
Pro Glu Val Gly Pro His Ile 210 215 220Gly Arg Tyr Cys Gly Gln Lys
Thr Pro Gly Arg Ile Arg Ser Ser Ser225 230 235 240Gly Val Leu Ser
Met Val Phe Tyr Thr Asp Ser Ala Ile Ala Lys Glu 245 250 255Gly Phe
Ser Ala Asn Tyr Ser Val Leu Gln Ser Ser Ile Ser Glu Asp 260 265
270Phe Lys Cys Met Glu Ala Leu Gly Met Glu Ser Gly Glu Ile His Ser
275 280 285Asp Gln Ile Thr Ala Ser Ser Gln Tyr Gly Thr Asn Trp Ser
Val Glu 290 295 300Arg Ser Arg Leu Asn Tyr Pro Glu Asn Gly Trp Thr
Pro Gly Glu Asp305 310 315 320Ser Tyr Lys Glu Trp Ile Gln Val Asp
Leu Gly Leu Leu Arg Phe Val 325 330 335Thr Ala Val Gly Thr Gln Gly
Ala Ile Ser Lys Glu Thr Lys Lys Lys 340 345 350Tyr Tyr Val Lys Thr
Tyr Arg Val Asp Ile Ser Ser Asn Gly Glu Asp 355 360 365Trp Ile Ser
Leu Lys Glu Gly Asn Lys Ala Ile Ile Phe Gln Gly Asn 370 375 380Thr
Asn Pro Thr Asp Val Val Leu Gly Val Phe Ser Lys Pro Leu Ile385 390
395 400Thr Arg Phe Val Arg Ile Lys Pro Val Ser Trp Glu Thr Gly Ile
Ser 405 410 415Met Arg Phe Glu Val Tyr Gly Cys Lys Ile Thr Asp Tyr
Pro Cys Ser 420 425 430Gly Met Leu Gly Met Val Ser Gly Leu Ile Ser
Asp Ser Gln Ile Thr 435 440 445Ala Ser Asn Gln Ala Asp Arg Asn Trp
Met Pro Glu Asn Ile Arg Leu 450 455 460Val Thr Ser Arg Thr Gly Trp
Ala Leu Pro Pro Ser Pro His Pro Tyr465 470 475 480Thr Asn Glu Trp
Leu Gln Val Asp Leu Gly Asp Glu Lys Ile Val Arg 485 490 495Gly Val
Ile Ile Gln Gly Gly Lys His Arg Glu Asn Lys Val Phe Met 500 505
510Arg Lys Phe Lys Ile Ala Tyr Ser Asn Asn Gly Ser Asp Trp Lys Thr
515 520 525Ile Met Asp Asp Ser Lys Arg Lys Ala Lys Ser Phe Glu Gly
Asn Asn 530 535 540Asn Tyr Asp Thr Pro Glu Leu Arg Thr Phe Ser Pro
Leu Ser Thr Arg545 550 555 560Phe Ile Arg Ile Tyr Pro Glu Arg Ala
Thr His Ser Gly Leu Gly Leu 565 570 575Arg Met Glu Leu Leu Gly Cys
Glu Val Glu Ala Pro Thr Ala Gly Pro 580 585 590Thr Thr Pro Asn Gly
Asn Pro Val Asp Glu Cys Asp Asp Asp Gln Ala 595 600 605Asn Cys His
Ser Gly Thr Gly Asp Asp Phe Gln Leu Thr Gly Gly Thr 610 615 620Thr
Val Leu Ala Thr Glu Lys Pro Thr Ile Ile Asp Ser Thr Ile Gln625 630
635 640Ser Glu Phe Pro Thr Tyr Gly Phe Asn Cys Glu Phe Gly Trp Gly
Ser 645 650 655His Lys Thr Phe Cys His Trp Glu His Asp Ser His Ala
Gln Leu Arg 660 665 670Trp Ser Val Leu Thr Ser Lys Thr Gly Pro Ile
Gln Asp His Thr Gly 675 680 685Asp Gly Asn Phe Ile Tyr Ser Gln Ala
Asp Glu Asn Gln Lys Gly Lys 690 695 700Val Ala Arg Leu Val Ser Pro
Val Val Tyr Ser Gln Ser Ser Ala His705 710 715 720Cys Met Thr Phe
Trp Tyr His Met Ser Gly Ser His Val Gly Thr Leu 725 730 735Arg Val
Lys Leu Arg Tyr Gln Lys Pro Glu Glu Tyr Asp Gln Leu Val 740 745
750Trp Met Val Val Gly His Gln Gly Asp His Trp Lys Glu Gly Arg Val
755 760 765Leu Leu His Lys Ser Leu Lys Leu Tyr Gln Val Ile Phe Glu
Gly Glu 770 775 780Ile Gly Lys Gly Asn Leu Gly Gly Ile Ala Val Asp
Asp Ile Ser Ile785 790 795 800Asn Asn His Ile Ser Gln Glu Asp Cys
Ala Lys Pro Thr Asp Leu Asp 805 810 815Lys Lys Asn Thr Glu Ile Lys
Ile Asp Glu Thr Gly Ser Thr Pro Gly 820 825 830Tyr Glu Gly Glu Gly
Glu Gly Asp Lys Asn Ile Ser Arg Lys Pro Gly 835 840 845Asn Val Leu
Lys Thr Leu Asp Pro Ile Leu Ile Thr Ile Ile Ala Met 850 855 860Ser
Ala Leu Gly Val Leu Leu Gly Ala Val Cys Gly Val Val Leu Tyr865 870
875 880Cys Ala Cys Trp His Asn Gly Met Ser Glu Arg Asn Leu Ser Ala
Leu 885 890 895Glu Asn Tyr Asn Phe Glu Leu Val Asp Gly Val Lys Leu
Lys Lys Asp 900 905 910Lys Leu Asn Pro Gln Ser Asn Tyr Ser Glu Ala
915 920135922PRTRattus norvegicus 135Met Glu Arg Gly Leu Pro Leu
Leu Cys Ala Thr Leu Ala Leu Ala Leu1 5 10 15Ala Leu Ala Gly Ala Phe
Arg Ser Asp Lys Cys Gly Gly Thr Ile Lys 20 25 30Ile Glu Asn Pro Gly
Tyr Leu Thr Ser Pro Gly Tyr Pro His Ser Tyr 35 40 45His Pro Ser Glu
Lys Cys Glu Trp Leu Ile Gln Ala Pro Glu Pro Tyr 50 55 60Gln Arg Ile
Met Ile Asn Phe Asn Pro His Phe Asp Leu Glu Asp Arg65 70 75 80Asp
Cys Lys Tyr Asp Tyr Val Glu Val Ile Asp Gly Glu Asn Glu Gly 85 90
95Gly Arg Leu Trp Gly Lys Phe Cys Gly Lys Ile Ala Pro Ser Pro Val
100 105 110Val Ser Ser Gly Pro Phe Leu Phe Ile Lys Phe Val Ser Asp
Tyr Glu 115 120 125Thr His Gly Ala Gly Phe Ser Ile Arg Tyr Glu Ile
Phe Lys Arg Gly 130 135 140Pro Glu Cys Ser Gln Asn Tyr Thr Ala Pro
Thr Gly Val Ile Lys Ser145 150 155 160Pro Gly Phe Pro Glu Lys Tyr
Pro Asn Ser Leu Glu Cys Thr Tyr Ile 165 170 175Ile Phe Ala Pro Lys
Met Ser Glu Ile Ile Leu Glu Phe Glu Ser Phe 180 185 190Asp Leu Glu
Gln Asp Ser Asn Pro Pro Gly Gly Val Phe Cys Arg Tyr 195 200 205Asp
Arg Leu Glu Ile Trp Asp Gly Phe Pro Glu Val Gly Pro His Ile 210 215
220Gly Arg Tyr Cys Gly Gln Lys Thr Pro Gly Arg Ile Arg Ser Ser
Ser225 230 235 240Gly Ile Leu Ser Met Val Phe Tyr Thr Asp Ser Ala
Ile Ala Lys Glu 245 250 255Gly Phe Ser Ala Asn Tyr Ser Val Leu Gln
Ser Ser Ile Ser Glu Asp 260 265 270Phe Lys Cys Met Glu Ala Leu Gly
Met Glu Ser Gly Glu Ile His Ser 275 280 285Asp Gln Ile Thr Ala Ser
Ser Gln Tyr Gly Thr Asn Trp Ser Val Glu 290 295 300Arg Ser Arg Leu
Asn Tyr Pro Glu Asn Gly Trp Thr Pro Gly Glu Asp305 310 315 320Ser
Tyr Arg Glu Trp Ile Gln Val Asp Leu Gly Leu Leu Arg Phe Val 325 330
335Thr Ala Val Gly Thr Gln Gly Ala Ile Ser Lys Glu Thr Lys Lys Lys
340 345 350Tyr Tyr Val Lys Thr Tyr Arg Val Asp Ile Ser Ser Asn Gly
Glu Asp 355 360 365Trp Ile Thr Leu Lys Glu Gly Asn Lys Ala Ile Ile
Phe Gln Gly Asn 370 375 380Thr Asn Pro Thr Asp Val Val Phe Gly Val
Phe Pro Lys Pro Leu Ile385 390 395 400Thr Arg Phe Val Arg Ile Lys
Pro Ala Ser Trp Glu Thr Gly Ile Ser 405 410 415Met Arg Phe Glu Val
Tyr Gly Cys Lys Ile Thr Asp Tyr Pro Cys Ser 420 425 430Gly Met Leu
Gly Met Val Ser Gly Leu Ile Ser Asp Ser Gln Ile Thr 435 440 445Ala
Ser Asn Gln Gly Asp Arg Asn Trp Met Pro Glu Asn Ile Arg Leu 450 455
460Val Thr Ser Arg Thr Gly Trp Ala Leu Pro Pro Ser Pro His Pro
Tyr465 470 475 480Ile Asn Glu Trp Leu Gln Val Asp Leu Gly Asp Glu
Lys Ile Val Arg 485 490 495Gly Val Ile Ile Gln Gly Gly Lys His Arg
Glu Asn Lys Val Phe Met 500 505 510Arg Lys Phe Lys Ile Ala Tyr Ser
Asn Asn Gly Ser Asp Trp Lys Met 515 520 525Ile Met Asp Asp Ser Lys
Arg Lys Ala Lys Ser Phe Glu Gly Asn Asn 530 535 540Asn Tyr Asp Thr
Pro Glu Leu Arg Ala Phe Thr Pro Leu Ser Thr Arg545 550 555 560Phe
Ile Arg Ile Tyr Pro Glu Arg Ala Thr His Ser Gly Leu Gly Leu 565 570
575Arg Met Glu Leu Leu Gly Cys Glu Val Glu Val Pro Thr Ala Gly Pro
580 585 590Thr Thr Pro Asn Gly Asn Pro Val Asp Glu Cys Asp Asp Asp
Gln Ala 595 600 605Asn Cys His Ser Gly Thr Gly Asp Asp Phe Gln Leu
Thr Gly Gly Thr 610 615 620Thr Val Leu Ala Thr Glu Lys Pro Thr Ile
Ile Asp Ser Thr Ile Gln625 630 635 640Ser Glu Phe Pro Thr Tyr Gly
Phe Asn Cys Glu Phe Gly Trp Gly Ser 645 650 655His Lys Thr Phe Cys
His Trp Glu His Asp Ser His Ala Gln Leu Arg 660 665 670Trp Arg Val
Leu Thr Ser Lys Thr Gly Pro Ile Gln Asp His Thr Gly 675 680 685Asp
Gly Asn Phe Ile Tyr Ser Gln Ala Asp Glu Asn Gln Lys Gly Lys 690 695
700Val Ala Arg Leu Val Ser Pro Val Val Tyr Ser Gln Ser Ser Ala
His705 710 715 720Cys Met Thr Phe Trp Tyr His Met Ser Gly Ser His
Val Gly Thr Leu 725 730 735Arg Val Lys Leu His Tyr Gln Lys Pro Glu
Glu Tyr Asp Gln Leu Val 740 745 750Trp Met Val Val Gly His Gln Gly
Asp His Trp Lys Glu Gly Arg Val 755 760 765Leu Leu His Lys Ser Leu
Lys Leu Tyr Gln Val Ile Phe Glu Gly Glu 770 775 780Ile Gly Lys Gly
Asn Leu Gly Gly Ile Ala Val Asp Asp Ile Ser Ile785 790 795 800Asn
Asn His Ile Pro Gln Glu Asp Cys Ala Lys Pro Thr Asp Leu Asp 805 810
815Lys Lys Asn Thr Glu Ile Lys Ile Asp Glu Thr Gly Ser Thr Pro Gly
820 825 830Tyr Glu Glu Gly Lys Gly Asp Lys Asn Ile Ser Arg Lys Pro
Gly Asn 835 840 845Val Leu Lys Thr Leu Asp Pro Ile Leu Ile Thr Ile
Ile Ala Met Ser 850 855 860Ala Leu Gly Val Leu Leu Gly Ala Val Cys
Gly Val Val Leu Tyr Cys865 870 875 880Ala Cys Trp His Asn Gly Met
Ser Glu Arg Asn Leu Ser Ala Leu Glu 885 890 895Asn Tyr Asn Phe Glu
Leu Val Asp Gly Val Lys Leu Lys Lys Asp Lys 900 905 910Leu Asn Pro
Gln Ser Asn Tyr Ser Glu Ala 915 92013617PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"VARIANT(1)..(1)/replace="Ala"VARIANT(8)..(8)/replace="Ala"VARIANT-
(16)..(16)/replace="Glu"MISC_FEATURE(1)..(17)/note="Variant
residues given in the sequence have no preference with respect to
those in the annotations for variant positions" 136Ile Ile Ser Gly
Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10
15Gly1379PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide"VARIANT(4)..(4)/replace="Lys"
or
"Ser"VARIANT(6)..(6)/replace="Val"MISC_FEATURE(1)..(9)/note="Variant
residues given in the sequence have no preference with respect to
those in the annotations for variant positions" 137Phe Thr Phe Ala
Ser Tyr Ala Met Val1 513813PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"VARIANT(13)..(13)/replace="Val"MISC_FEATURE(1)..(13)/note="Varian-
t residues given in the sequence have no preference with respect to
those in the annotations for variant positions" 138Ala Arg Asp Leu
Gly Tyr Tyr Gly Ser Gly Met His Ala1 5 1013914PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"VARIANT(13)..(13)/replace="Asn"MISC_FEATURE(1)..(14)/note="Varian-
t residues given in the sequence have no preference with respect to
those in the annotations for variant positions" 139Ala Arg Asp Arg
Gly Met Tyr Tyr Ala Ser Gly Phe Gly Pro1 5 101405PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 140Gly Gly Gly Gly Ser1 5141453PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 141Glu 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 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Gln Ile Ser Pro Ala Gly Gly Tyr Thr
Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Gly Glu Leu Pro
Tyr Tyr Arg Met Ser Lys Val Met Asp Val 100 105 110Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135
140Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val145 150 155 160Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe 165 170 175Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val 180 185 190Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu225 230 235 240Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250
255Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265
270Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser 290 295 300Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu305 310 315 320Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 325 330 335Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr385 390
395 400Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu 405 410 415Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser 420 425 430Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser 435 440 445Leu Ser Pro Gly Lys
450142214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 142Asp 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 Ala Ser Gln Tyr Phe Ser Ser Tyr 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Gly
Ala Ser Ser Arg Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Gly Ser Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210143923PRTHomo sapiens 143Met Glu Arg
Gly Leu Pro Leu Leu Cys Ala Val Leu Ala Leu Val Leu1 5 10 15Ala Pro
Ala Gly Ala Phe Arg Asn Asp Lys Cys Gly Asp Thr Ile Lys 20 25 30Ile
Glu Ser Pro Gly Tyr Leu Thr Ser Pro Gly Tyr Pro His Ser Tyr 35 40
45His Pro Ser Glu Lys Cys Glu Trp Leu Ile Gln Ala Pro Asp Pro Tyr
50 55 60Gln Arg Ile Met Ile Asn Phe Asn Pro His Phe Asp Leu Glu Asp
Arg65 70 75 80Asp Cys Lys Tyr Asp Tyr Val Glu Val Phe Asp Gly Glu
Asn Glu Asn 85 90 95Gly His Phe Arg Gly Lys Phe Cys Gly Lys Ile Ala
Pro Pro Pro Val 100 105 110Val Ser Ser Gly Pro Phe Leu Phe Ile Lys
Phe Val Ser Asp Tyr Glu 115 120 125Thr His Gly Ala Gly Phe Ser Ile
Arg Tyr Glu Ile Phe Lys Arg Gly 130 135 140Pro Glu Cys Ser Gln Asn
Tyr Thr Thr Pro Ser Gly Val Ile Lys Ser145 150 155 160Pro Gly Phe
Pro Glu Lys Tyr Pro Asn Ser Leu Glu Cys Thr Tyr Ile 165 170 175Val
Phe Val Pro Lys Met Ser Glu Ile Ile Leu Glu Phe Glu Ser Phe 180 185
190Asp Leu Glu Pro Asp Ser Asn Pro Pro Gly Gly Met Phe Cys Arg Tyr
195 200 205Asp Arg Leu Glu Ile Trp Asp Gly Phe Pro Asp Val Gly Pro
His Ile 210 215 220Gly Arg Tyr Cys Gly Gln Lys Thr Pro Gly Arg Ile
Arg Ser Ser Ser225 230 235 240Gly Ile Leu Ser Met Val Phe Tyr Thr
Asp Ser Ala Ile Ala Lys Glu 245 250 255Gly Phe Ser Ala Asn Tyr Ser
Val Leu Gln Ser Ser Val Ser Glu Asp 260 265 270Phe Lys Cys Met Glu
Ala Leu Gly Met Glu Ser Gly Glu Ile His Ser 275 280 285Asp Gln Ile
Thr Ala Ser Ser Gln Tyr Ser Thr Asn Trp Ser Ala Glu 290 295 300Arg
Ser Arg Leu Asn Tyr Pro Glu Asn Gly Trp Thr Pro Gly Glu Asp305 310
315 320Ser Tyr Arg Glu Trp Ile Gln Val Asp Leu Gly Leu Leu Arg Phe
Val 325 330 335Thr Ala Val Gly Thr Gln Gly Ala Ile Ser Lys Glu Thr
Lys Lys Lys 340 345 350Tyr Tyr Val Lys Thr Tyr Lys Ile Asp Val Ser
Ser Asn Gly Glu Asp 355 360 365Trp Ile Thr Ile Lys Glu Gly Asn Lys
Pro Val Leu Phe Gln Gly Asn 370 375 380Thr Asn Pro Thr Asp Val Val
Val Ala Val Phe Pro Lys Pro Leu Ile385 390 395 400Thr Arg Phe Val
Arg Ile Lys Pro Ala Thr Trp Glu Thr Gly Ile Ser 405 410 415Met Arg
Phe Glu Val Tyr Gly Cys Lys Ile Thr Asp Tyr Pro Cys Ser 420 425
430Gly Met Leu Gly Met Val Ser Gly Leu Ile Ser Asp Ser Gln Ile Thr
435 440 445Ser Ser Asn Gln Gly Asp Arg Asn Trp Met Pro Glu Asn Ile
Arg Leu 450 455 460Val Thr Ser Arg Ser Gly Trp Ala Leu Pro Pro Ala
Pro His Ser Tyr465 470 475 480Ile Asn Glu Trp Leu Gln Ile Asp Leu
Gly Glu Glu Lys Ile Val Arg 485 490 495Gly Ile Ile Ile Gln Gly Gly
Lys His Arg Glu Asn Lys Val Phe Met 500 505 510Arg Lys Phe Lys Ile
Gly Tyr Ser Asn Asn Gly Ser Asp Trp Lys Met 515 520 525Ile Met Asp
Asp Ser Lys Arg Lys Ala Lys Ser Phe Glu Gly Asn Asn 530 535 540Asn
Tyr Asp Thr Pro Glu Leu Arg Thr Phe Pro Ala Leu Ser Thr Arg545 550
555 560Phe Ile Arg Ile Tyr Pro Glu Arg Ala Thr His Gly Gly Leu Gly
Leu 565 570 575Arg Met Glu Leu Leu Gly Cys Glu Val Glu Ala Pro Thr
Ala Gly Pro 580 585 590Thr Thr Pro Asn Gly Asn Leu Val Asp Glu Cys
Asp Asp Asp Gln Ala 595 600 605Asn Cys His Ser Gly Thr Gly Asp Asp
Phe Gln Leu Thr Gly Gly Thr 610 615 620Thr Val Leu Ala Thr Glu Lys
Pro Thr Val Ile Asp Ser Thr Ile Gln625 630 635 640Ser Glu Phe Pro
Thr Tyr Gly Phe Asn Cys Glu Phe Gly Trp Gly Ser 645 650 655His Lys
Thr Phe Cys His Trp Glu His Asp Asn His Val Gln Leu Lys 660 665
670Trp Ser Val Leu Thr Ser Lys Thr Gly Pro Ile Gln Asp His Thr Gly
675 680 685Asp Gly Asn Phe Ile Tyr Ser Gln Ala Asp Glu Asn Gln Lys
Gly Lys 690 695 700Val Ala Arg Leu Val Ser Pro Val Val Tyr Ser Gln
Asn Ser Ala His705 710 715 720Cys Met Thr Phe Trp Tyr His Met Ser
Gly Ser His Val Gly Thr Leu 725 730 735Arg Val Lys Leu Arg Tyr Gln
Lys Pro Glu Glu Tyr Asp Gln Leu Val 740 745 750Trp Met Ala Ile Gly
His Gln Gly Asp His Trp Lys Glu Gly Arg Val 755 760 765Leu Leu His
Lys Ser Leu Lys Leu Tyr Gln Val Ile Phe Glu Gly Glu 770 775 780Ile
Gly Lys Gly Asn Leu Gly Gly Ile Ala Val Asp Asp Ile Ser Ile785 790
795 800Asn Asn His Ile Ser Gln Glu Asp Cys Ala Lys Pro Ala Asp Leu
Asp 805 810 815Lys Lys Asn Pro Glu Ile Lys Ile Asp Glu Thr Gly Ser
Thr Pro Gly 820 825 830Tyr Glu Gly Glu Gly Glu Gly Asp Lys Asn Ile
Ser Arg Lys Pro Gly 835 840 845Asn Val Leu Lys Thr Leu Asp Pro Ile
Leu Ile Thr Ile Ile Ala Met 850 855 860Ser Ala Leu Gly Val Leu Leu
Gly Ala Val Cys Gly Val Val Leu Tyr865 870 875 880Cys Ala Cys Trp
His Asn Gly Met Ser Glu Arg Asn Leu Ser Ala Leu 885 890 895Glu Asn
Tyr Asn Phe Glu Leu Val Asp Gly Val Lys Leu Lys Lys Asp 900 905
910Lys Leu Asn Thr Gln Ser Thr Tyr Ser Glu Ala 915
920144214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 144Asp 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 Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Trp Ala Tyr Leu Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210145454PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 145Glu 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
Ile Ser Gly Tyr 20 25 30Gly Ile His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ala Tyr Ile Tyr Pro Asp Ser Gly Tyr Thr
Asp Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Glu Asp Phe Arg
Asn Arg Arg Arg Leu Trp Tyr Val Met Asp 100 105 110Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135
140Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro145 150 155 160Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr 165 170 175Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val 180 185 190Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro 210 215 220Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu225 230 235 240Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250
255Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly 275 280 285Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn 290 295 300Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp305 310 315 320Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360 365Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375
380Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr385 390 395 400Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys 405 410 415Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 420 425 430Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu 435 440 445Ser Leu Ser Pro Gly Lys
45014615PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 146Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile Tyr1 5 10 151474PRTHomo sapiens
147Glu Leu Leu Gly11484PRTHomo sapiens 148Glu Phe Leu Gly1
* * * * *
References