U.S. patent application number 15/682206 was filed with the patent office on 2019-01-10 for fully human anti-c-x-c chemokine receptor 3 (cxcr3) antibodies.
The applicant listed for this patent is Sorrento Therapeutics, Inc.. Invention is credited to Dingqiu Huang, Barbara A. Swanson, Heyue Zhou.
Application Number | 20190008955 15/682206 |
Document ID | / |
Family ID | 55347733 |
Filed Date | 2019-01-10 |
United States Patent
Application |
20190008955 |
Kind Code |
A1 |
Swanson; Barbara A. ; et
al. |
January 10, 2019 |
FULLY HUMAN ANTI-C-X-C CHEMOKINE RECEPTOR 3 (CXCR3) ANTIBODIES
Abstract
There is disclosed compositions and methods relating to or
derived from anti-CXCR3 antibodies. More specifically, there is
disclosed fully human antibodies that bind CXCR3, CXCR3-binding
fragments and derivatives of such antibodies, and CXCR3-binding
polypeptides comprising such fragments. Further still, there is
disclosed nucleic acids encoding such antibodies, antibody
fragments and derivatives and polypeptides, cells comprising such
polynucleotides, methods of making such antibodies, antibody
fragments and derivatives and polypeptides, and methods of using
such antibodies, antibody fragments and derivatives and
polypeptides, including methods of treating or diagnosing subjects
having CXCR3 related disorders or conditions, including various
inflammatory disorders and various cancers.
Inventors: |
Swanson; Barbara A.;
(Encinitas, CA) ; Huang; Dingqiu; (San Diego,
CA) ; Zhou; Heyue; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sorrento Therapeutics, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
55347733 |
Appl. No.: |
15/682206 |
Filed: |
August 21, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14821559 |
Aug 7, 2015 |
9765145 |
|
|
15682206 |
|
|
|
|
62041020 |
Aug 22, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/16 20180101; A61P
11/00 20180101; C07K 2317/92 20130101; C07K 16/2809 20130101; C07K
2317/21 20130101; A61P 29/00 20180101; A61P 35/04 20180101; A61P
17/06 20180101; A61P 37/06 20180101; A61P 1/04 20180101; C07K
2317/76 20130101; A61P 35/00 20180101; A61P 37/00 20180101; A61P
35/02 20180101; A61P 33/06 20180101; A61P 25/00 20180101; A61P 3/10
20180101; A61K 39/395 20130101; A61P 9/10 20180101; C07K 16/2866
20130101; A61P 19/02 20180101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28 |
Claims
1.-7. (canceled)
8. A method for treating a subject having cancer, comprising
administering an effective amount of an anti-C-X-C chemokine
receptor-3 (CXCR3) polypeptide to a subject in need thereof,
wherein the anti-CXCR3 polypeptide is selected from the group
consisting of a recombinant fully human anti-CXCR3 antibody that
binds to CXCR3 a recombinant fully human anti-CXCR3 antibody Fab
fragment that binds to CXCR3, and a recombinant anti-CXCR3 single
chain antibody that binds to CXCR3, wherein the anti-CXCR3
polypeptide comprises a heavy chain variable domain sequence that
is at least 95% identical to the amino acid sequence set forth in
SEQ ID NO. 17, and comprises a light chain variable domain
comprising an amino acid sequence that is at least 95% identical to
the amino acid sequence set forth in SEQ ID NO. 18.
9.-11. (canceled)
12. The method of claim 8, wherein the cancer is a CXCR3 positive
cancer.
13. The method of claim 8, wherein the cancer is selected from the
group consisting of carcinoma, leukemia, lymphoma, prostate cancer,
non-small cell lung cancer, breast cancer, endometrial cancer,
ovarian cancer, gastric cancer, head and neck cancer, melanoma,
osteosarcoma, intestinal and colon cancer, and metastatic
cancer.
14.-18. (canceled)
19. A method for treating a subject having cancer, comprising
administering an effective amount of a recombinant fully human
anti-CXCR3 antibody, or an antigen-binding fragment thereof, that
binds to CXCR3, comprising a heavy chain variable domain comprising
complementarity determining regions (CDRs) as set forth in the
heavy chain variable domain amino acid sequence of SEQ ID NO: 17;
and comprising a light chain variable domain comprising CDRs as set
forth in the light chain variable domain amino acid sequence of SEQ
ID NO: 18 to the subject having cancer.
20. The method of claim 19, wherein the cancer is selected from the
group consisting of carcinoma, leukemia, lymphoma, prostate cancer,
non-small cell lung cancer, breast cancer, endometrial cancer,
ovarian cancer, gastric cancer, head and neck cancer, melanoma,
osteosarcoma, intestinal and colon cancer, and metastatic
cancer.
21. The method of claim 19, wherein the recombinant fully human
anti-CXCR3 antibody, or antigen-binding fragment thereof, is
classified as an isotype selected from the group consisting of:
IgG, IgM, IgD, IgA, and IgE.
22. The method of claim 21, wherein the antibody is an IgG1 or an
IgG4.
23. The method of claim 19, wherein the recombinant fully human
antigen binding fragment is a Fab fragment or a single chain
antibody.
24. The method of claim 19, wherein the recombinant fully human
anti-CXCR3 antibody, or an antigen-binding fragment thereof,
comprises a heavy chain variable domain comprising the amino acid
sequence as set forth in SEQ ID NO: 17; and comprises a light chain
variable domain comprising the amino acid sequence as set forth in
SEQ ID NO: 18.
25. The method of claim 19, wherein the recombinant fully human
anti-CXCR3 antibody, or antigen-binding fragment thereof, has a
binding affinity of at least 1.times.10.sup.-6 M.
26. The method of claim 19, wherein the recombinant fully human
anti-CXCR3 antibody, or antigen-binding fragment thereof, is an
Fab, an Fab', an F(ab')2, an Fv, a domain antibody (dAb), a
single-chain antibody (scFv), a diabody, a triabody or a
tetrabody.
27. The method of claim 8, wherein the recombinant fully human
anti-CXCR3 antibody, or recombinant fully human anti-CXCR3 antibody
Fab fragment, is classified as an isotype selected from the group
consisting of: IgG, IgM, IgD, IgA, and IgE.
28. The method of claim 27, wherein the recombinant fully human
anti-CXCR3 antibody or recombinant fully human anti-CXCR3 antibody
Fab fragment is an IgG1 or an IgG4.
29. The method of claim 8, wherein the recombinant fully human
anti-CXCR3 antibody, or recombinant fully human anti-CXCR3 antibody
Fab fragment, has a binding affinity of at least 1.times.10.sup.-6
M.
30. The method of claim 8, wherein the recombinant fully human
anti-CXCR3 antibody, or recombinant fully human anti-CXCR3 antibody
Fab fragment, is an Fab, an Fab', an F(ab')2, an Fv, a domain
antibody (dAb), a single-chain antibody (scFv), a diabody, a
triabody or a tetrabody.
Description
PRIORITY APPLICATION
[0001] This patent application claims priority from pending U.S.
provisional patent application Ser. No. 62/041,020 filed 22 Aug.
2015.
TECHNICAL FIELD
[0002] The present disclosure provides compositions and methods
relating to or derived from anti-CXCR3 antibodies. More
specifically, the present disclosure provides human antibodies that
bind CXCR3, CXCR3-binding fragments and derivatives of such
fragments, and CXCR3-binding polypeptides comprising such
fragments. Further still, the present disclosure provides nucleic
acids encoding such antibodies, antibody fragments and derivatives
and polypeptides, cells comprising such polynucleotides, methods of
making such antibodies, antibody fragments and derivatives and
polypeptides, and methods of using such antibodies, antibody
fragments and derivatives and polypeptides, including methods of
treating or diagnosing subjects having CXCR3 related disorders or
conditions, including various inflammatory disorders and various
cancers.
BACKGROUND
[0003] C-X-C chemokine receptor-3 (CXCR3) is expressed on certain
leukocytes, such as activated T cells and NK cells. The CXCR3
receptor binds ligands, such as Interferon Gamma-inducible 10 kD
Protein (IP-10), Monokine Induced by Gamma interferon (MIG; Mig),
Interferon-inducible T-cell Alpha Chemoattractant (1-TAC) and B
cell-attracting chemokine-1 (BCA-1). Certain forms of CXCR3 also
bind platelet factor-4 (PF-4, Lasagni et al., J. Exp. Med.
197:1537-1549, 2003). The expression of some of the CXCR3 ligands
(IP-10, MIG and I-TAC), is induced in tissues by inteferons or
Tumor Necrosis Factor (TNF), potent mediators of inflammation
(Farber, J. M. J. Leukoc. Biol. 61:246-257, 2007; Piali, et al.
Eur. J. Immunol. 28:961-972, 1998; and Cole et al. J. Exp. Med.
187:2009-2021, 1998). Because of these findings, it has been
postulated that during inflammation, expression of ligands for
CXCR3 is upregulated, resulting in recruitment of CXCR3+
lymphocytes into the inflamed tissue. The infiltrating CXCR3+
lymphocytes can contribute to adverse pathological effects of
inflammation. Inhibiting the activities of CXCR3, therefore, can
have beneficial anti-inflammatory effects. Therefore, there is a
need for therapeutic agents that inhibit CXCR3 function.
[0004] The interaction between chemokines and their receptors is an
important step in the control of leukocyte migration. Chemokines
also mediate a variety of effects independent of chemotaxis,
including induction and enhancement of cell-associated cytokine
responses.
[0005] The human cell surface protein CD183 is a G protein-coupled
receptor with selectivity for three chemokines including IP10
(interferon-.gamma.-inducible 10 kDa protein). Mig (monokine
induced by interferon-.gamma.) and I-TAC (interferon-inducible T
cell .alpha.-chemoattractant). These three chemokines belong to the
structural subfamily of "CXC" chemokines, in which a single amino
acid residue separates the first two of four highly conserved Cys
residues. Historically, CD183 is the third CXC chemokine receptor
discovered and, therefore, CD183 is commonly designated as "CXCR3."
Binding of chemokines to CXCR3 induces cellular responses that are
involved in leukocyte traffic, most notably integrin activation,
cytoskeletal changes and chemotactic migration. CXCR3 is expressed
on effector/memory T cells and/or in T cells present in many types
of inflamed tissues (e.g., T-helper 1 cells or Th1 cells and
CD8.sup.+ Tc1 cells). In addition, IP10, Mig and I-TAC are commonly
produced by local cells in inflammatory lesions, suggesting that
CXCR3 and its chemokines participate in the recruitment of white
blood cells to sites of inflammation. Therefore. CXCR3 is a target
for the development of antibodies and antagonists, which may be
used in the treatment and diagnosis of diverse inflammatory and
immune diseases and disorders, such as rheumatoid arthritis,
multiple sclerosis, Crohn's disease, inflammatory bowel disease,
chronic obstructive pulmonary disease, psoriasis, type 1 diabetes
and transplant rejection. Because CXCR3 is expressed on a subset of
B-cell lymphomas, CXCR3 may also be a target for treating and
diagnosing lymphomas and leukemias.
[0006] Therefore, there is a need in the art for an improved
anti-CXCR3 antibodies that can be used as therapeutic agents.
SUMMARY
[0007] The present disclosure provides a fully human antibody of an
IgG class that binds to an CXCR3 epitope with a binding affinity of
at least 10.sup.-6M, which has a heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3,
SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.
13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ
ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO.
31, SEQ ID NO. 33, and combinations thereof, and that has a light
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10,
SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID
NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28,
SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID
NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and combinations thereof.
Preferably, the fully human antibody has both a heavy chain and a
light chain wherein the antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ ID
NO. 4 (called 32D12 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called H1
herein), SEQ ID NO. 7/SEQ ID NO. 8 (called B7 herein), SEQ ID NO.
9/SEQ ID NO. 10 (called C12 herein), SEQ ID NO. 11/SEQ ID NO. 12
(called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called E1
herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQ ID
NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO.
20 (called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein). SEQ ID
NO. 25/SEQ ID NO.26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO.
28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12
herein), SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO.
31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34
(called 2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7
herein), SEQ ID NO. 31/SEQ ID NO. 36 (called 3A8 herein), SEQ ID
NO. 31/SEQ ID NO. 37 (called 4D5 herein), and combinations
thereof.
[0008] The present disclosure provides a fully human antibody Fab
fragment, having a variable domain region from a heavy chain and a
variable domain region from a light chain, wherein the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17. SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof,
and that has a light chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ
ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO.
16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ
ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and
combinations thereof. Preferably, the fully human antibody Fab
fragment has both a heavy chain variable domain region and a light
chain variable domain region wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4,
SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.
9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID
NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18,
SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID
NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34,
SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID
NO. 31/SEQ ID NO. 37, and combinations thereof.
[0009] The present disclosure provides a single chain human
antibody, having a variable domain region from a heavy chain and a
variable domain region from a light chain and a peptide linker
connection the heavy chain and light chain variable domain regions,
wherein the heavy chain variable domain sequence that is at least
95% identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.
7, SEQ ID NO.9, SEQ ID NO. II, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID
NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25,
SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and
combinations thereof, and that has a light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36,
SEQ ID NO. 37, and combinations thereof. Preferably, the fully
human single chain antibody has both a heavy chain variable domain
region and a light chain variable domain region, wherein the single
chain fully human antibody has a heavy chain/light chain variable
domain sequence selected from the group consisting of SEQ ID NO.
1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.
6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID
NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO.
15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID
NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24,
SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID
NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO.
31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID
NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37,
and combinations thereof.
[0010] The present disclosure further provides a method for
treating a broad spectrum of mammalian cancers, comprising
administering an effective amount of an anti-CXCR3 polypeptide,
wherein the anti-CXCR3 polypeptide is selected from the group
consisting of a fully human antibody of an IgG class that binds to
a CXCR3 epitope with a binding affinity of at least 10.sup.-6M, a
fully human antibody Fab fragment, having a variable domain region
from a heavy chain and a variable domain region from a light chain,
a single chain human antibody, having a variable domain region from
a heavy chain and a variable domain region from a light chain and a
peptide linker connection the heavy chain and light chain variable
domain regions, and combinations thereof;
[0011] wherein the fully human antibody has a heavy chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 1, SEQ
ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11,
SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID
NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29,
SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof, and that
has a light chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ
ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37,
combinations thereof:
[0012] wherein the fully human antibody Fab fragment has the heavy
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,
SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID
NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27,
SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations
thereof, and that has the light chain variable domain sequence that
is at least 95% identical to the amino acid sequences selected from
the group consisting of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6,
SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24,
SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID
NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37,
and combinations thereof; and
[0013] wherein the single chain human antibody has the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 1, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof,
and that has the light chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ
ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO.
16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ
ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and
combinations thereof.
[0014] Preferably, the fully human antibody has both a heavy chain
and a light chain wherein the antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
SEQ ID NO. 1/SEQ ID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ
ID NO. 4 (called 32D12 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called
H1 herein). SEQ ID NO. 7/SEQ ID NO. 8 (called B7 herein), SEQ ID
NO. 9/SEQ ID NO. 10 (called C12 herein), SEQ ID NO. 11/SEQ ID NO.
12 (called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called E1
herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQ ID
NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO.
20 (called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein). SEQ ID
NO. 25/SEQ ID NO.26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO.
28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12
herein), SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO.
31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34
(called 2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7
herein), SEQ ID NO. 31/SEQ ID NO. 36 (called 3A8 herein). SEQ ID
NO. 31/SEQ ID NO. 37 (called 4D5 herein), and combinations thereof.
Preferably, the fully human antibody Fab fragment has both a heavy
chain variable domain region and a light chain variable domain
region wherein the antibody has a heavy chain/light chain variable
domain sequence selected from the group consisting of SEQ ID NO.
1/SEQ ID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4
(called 32D12 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called H1
herein), SEQ ID NO. 7/SEQ ID NO. 8 (called B7 herein), SEQ ID NO.
9/SEQ ID NO. 10 (called C12 herein), SEQ ID NO. 11/SEQ ID NO. 12
(called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called E1
herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQ ID
NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO.
20 (called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein), SEQ ID
NO. 25/SEQ ID NO.26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO.
28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12
herein), SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO.
31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34
(called 2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7
herein). SEQ ID NO. 31/SEQ ID NO. 36 (called 3A8 herein). SEQ ID
NO. 31/SEQ ID NO. 37 (called 4D5 herein), and combinations thereof.
Preferably, the fully human single chain antibody has both a heavy
chain variable domain region and a light chain variable domain
region, wherein the single chain fully human antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4,
SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.
9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID
NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18,
SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID
NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34,
SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID
NO. 31/SEQ ID NO. 37, and combinations thereof.
[0015] Preferably, the broad spectrum of mammalian cancers to be
treated the cancer is a CXCR3-positive cancer. Preferably, the
broad spectrum of mammalian cancers to be treated is selected from
the group consisting of leukemias, lymphomas, cancinomas, prostate
cancer, non-small cell lung cancer, breast cancer, endometrial
cancer, ovarian cancer, gastric cancers, head and neck cancers,
melanoma, osteosarcoma, intestinal and colon cancer, and various
metastatic cancers.
[0016] The present disclosure further provides a method for
treating an inflammatory disorders, comprising administering an
effective amount of an anti-CXCR3 polypeptide, wherein the
anti-CXCR3 polypeptide is selected from the group consisting of a
fully human antibody of an IgG class that binds to a CXCR3 epitope
with a binding affinity of at least 10.sup.-6M, a fully human
antibody Fab fragment, having a variable domain region from a heavy
chain and a variable domain region from a light chain, a single
chain human antibody, having a variable domain region from a heavy
chain and a variable domain region from a light chain and a peptide
linker connection the heavy chain and light chain variable domain
regions, and combinations thereof;
[0017] wherein the fully human antibody has a heavy chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 1, SEQ
ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11,
SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID
NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29,
SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof, and that
has a light chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ
ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and
combinations thereof;
[0018] wherein the fully human antibody Fab fragment has the heavy
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,
SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID
NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27,
SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations
thereof, and that has the light chain variable domain sequence that
is at least 95% identical to the amino acid sequences selected from
the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6,
SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24,
SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID
NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37,
and combinations thereof; and
[0019] wherein the single chain human antibody has the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof,
and that has the light chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ
ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO.
16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ
ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and
combinations thereof.
[0020] Preferably, the fully human antibody has both a heavy chain
and a light chain wherein the antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
SEQ ID NO. 1/SEQ ID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ
ID NO. 4 (called 32D12 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called
H1 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called B7 herein), SEQ ID
NO. 9/SEQ ID NO. 10 (called C12 herein), SEQ ID NO. 11/SEQ ID NO.
12 (called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called E1
herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQ ID
NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO.
20 (called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein), SEQ ID
NO. 25/SEQ ID NO.26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO.
28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12
herein), SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO.
31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34
(called 2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7
herein), SEQ ID NO. 31/SEQ ID NO. 36 (called 3A8 herein), SEQ ID
NO. 31/SEQ ID NO. 37 (called 4D5 herein), and combinations thereof.
Preferably, the fully human antibody Fab fragment has both a heavy
chain variable domain region and a light chain variable domain
region wherein the antibody has a heavy chain/light chain variable
domain sequence selected from the group consisting of SEQ ID NO.
1/SEQ ID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ ID NO. 4
(called 32D12 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called H1
herein), SEQ ID NO. 7/SEQ ID NO. 8 (called B7 herein), SEQ ID NO.
9/SEQ ID NO. 10 (called C12 herein), SEQ ID NO. 11/SEQ ID NO. 12
(called D12 herein). SEQ ID NO. 13/SEQ ID NO. 14 (called E1
herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQ ID
NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO.
20 (called 3A5 herein). SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein), SEQ ID
NO. 25/SEQ ID NO.26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO.
28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12
herein). SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO.
31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34
(called 2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7
herein), SEQ ID NO. 31/SEQ ID NO. 36 (called 3A8 herein), SEQ ID
NO. 31/SEQ ID NO. 37 (called 4D5 herein), and combinations thereof.
Preferably, the fully human single chain antibody has both a heavy
chain variable domain region and a light chain variable domain
region, wherein the single chain fully human antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4,
SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.
9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID
NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18,
SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID
NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34,
SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID
NO. 31/SEQ ID NO. 37, and combinations thereof.
[0021] Preferably, the inflammatory disorder to be treated is
selected from the group consisting of rheumatoid arthritis,
multiple sclerosis, Crohn's disease, Graves disease, Sjogren's
syndrome, inflammatory bowel disease, chronic obstructive pulmonary
disease, psoriasis, type 1 diabetes, transplant rejection chronic
hepatitis C, malaria, and atherosclerosis.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows binding of the anti-CXCR3 antibody 32B12 to
CHO-CXCR3 cells.
DETAILED DESCRIPTION
[0023] The present disclosure provides a fully human antibody of an
IgG class that binds to a CXCR3 epitope with a binding affinity of
10.sup.-6M or less, that has a heavy chain variable domain sequence
that is at least 95% identical to the amino acid sequences selected
from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO.
5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID
NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23,
SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID
NO. 33, and combinations thereof, and that has a light chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ
ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO.
20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ
ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO.
35, SEQ ID NO. 36, SEQ ID NO. 37, and combinations thereof.
Preferably, the fully human antibody has both a heavy chain and a
light chain wherein the antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2 (called 32B12 herein), SEQ ID NO. 3/SEQ ID
NO. 4 (called 32D12 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called H1
herein), SEQ ID NO. 7/SEQ ID NO. 8 (called B7 herein), SEQ ID NO.
9/SEQ ID NO. 10 (called C12 herein), SEQ ID NO. 11/SEQ ID NO. 12
(called D12 herein), SEQ ID NO. 13/SEQ ID NO. 14 (called E1
herein), SEQ ID NO. 15/SEQ ID NO. 16 (called G12 herein), SEQ ID
NO. 17/SEQ ID NO. 18 (called 3A3 herein), SEQ ID NO. 19/SEQ ID NO.
20 (called 3A5 herein), SEQ ID NO. 21/SEQ ID NO. 22 (called 3A11
herein), SEQ ID NO. 23/SEQ ID NO. 24 (called 3B12 herein). SEQ ID
NO. 25/SEQ ID NO.26 (called 3C11 herein), SEQ ID NO. 27/SEQ ID NO.
28 (called 4C3 herein), SEQ ID NO. 29/SEQ ID NO. 30 (called B12
herein), SEQ ID NO. 31/SEQ ID NO. 32 (called C4 herein), SEQ ID NO.
31/SEQ ID NO. 33 (called F7 herein), SEQ ID NO. 31/SEQ ID NO. 34
(called 2A3 herein), SEQ ID NO. 31/SEQ ID NO. 35 (called 3A7
herein), SEQ ID NO. 31/SEQ ID NO. 36 (called 3A8 herein), SEQ ID
NO. 31/SEQ ID NO. 37 (called 4D5 herein), and combinations
thereof.
[0024] The present disclosure provides a fully human antibody Fab
fragment, having a variable domain region from a heavy chain and a
variable domain region from a light chain, wherein the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17. SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof,
and that has a light chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ
ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO.
16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ
ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and
combinations thereof. Preferably, the fully human antibody Fab
fragment has both a heavy chain variable domain region and a light
chain variable domain region wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4,
SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.
9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID
NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18,
SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID
NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34,
SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID
NO. 31/SEQ ID NO. 37, and combinations thereof.
[0025] The present disclosure provides a single chain human
antibody, having a variable domain region from a heavy chain and a
variable domain region from a light chain and a peptide linker
connection the heavy chain and light chain variable domain regions,
wherein the heavy chain variable domain sequence that is at least
95% identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.
7, SEQ ID NO.9, SEQ ID NO. II, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID
NO. 17, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25,
SEQ ID NO. 27, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and
combinations thereof, and that has a light chain variable domain
sequence that is at least 95% identical to the amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4,
SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID
NO. 14, SEQ ID NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22,
SEQ ID NO. 24, SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID
NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36,
SEQ ID NO. 37, and combinations thereof. Preferably, the fully
human single chain antibody has both a heavy chain variable domain
region and a light chain variable domain region, wherein the single
chain fully human antibody has a heavy chain/light chain variable
domain sequence selected from the group consisting of SEQ ID NO.
1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.
6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID
NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO.
15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID
NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID NO. 24,
SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28, SEQ ID
NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO.
31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO. 31/SEQ ID
NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID NO. 37,
and combinations thereof.
[0026] The present disclosure further provides a method for
treating a broad spectrum of mammalian cancers or inflammatory
diseases or autoimmune diseases, comprising administering an
effective amount of an anti-CXCR3 polypeptide, wherein the
anti-CXCR3 polypeptide is selected from the group consisting of a
fully human antibody of an IgG class that binds to a CXCR3 epitope
with a binding affinity of at least 10.sup.-6M, a fully human
antibody Fab fragment, having a variable domain region from a heavy
chain and a variable domain region from a light chain, a single
chain human antibody, having a variable domain region from a heavy
chain and a variable domain region from a light chain and a peptide
linker connection the heavy chain and light chain variable domain
regions, and combinations thereof;
[0027] wherein the fully human antibody has a heavy chain variable
domain sequence that is at least 95% identical to the amino acid
sequences selected from the group consisting of SEQ ID NO. 1, SEQ
ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11,
SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 19, SEQ ID
NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ ID NO. 29,
SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof, and that
has a light chain variable domain sequence that is at least 95%
identical to the amino acid sequences selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ
ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ ID NO.
26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ
ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and
combinations thereof;
[0028] wherein the fully human antibody Fab fragment has the heavy
chain variable domain sequence that is at least 95% identical to
the amino acid sequences selected from the group consisting of SEQ
ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,
SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID
NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27,
SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations
thereof, and that has the light chain variable domain sequence that
is at least 95% identical to the amino acid sequences selected from
the group consisting of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6,
SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID
NO. 16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24,
SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID
NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37,
and combinations thereof; and
[0029] wherein the single chain human antibody has the heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequences selected from the group consisting of SEQ ID
NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ
ID NO. 1, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO.
19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 27, SEQ
ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 33, and combinations thereof,
and that has the light chain variable domain sequence that is at
least 95% identical to the amino acid sequences selected from the
group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ
ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO.
16, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 24, SEQ
ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.
33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, and
combinations thereof.
[0030] Preferably, the fully human antibody has both a heavy chain
and a light chain wherein the antibody has a heavy chain/light
chain variable domain sequence selected from the group consisting
of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO.
5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO.
10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ
ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18, SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID
NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO.
31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID
NO. 37, and combinations thereof. Preferably, the fully human
antibody Fab fragment has both a heavy chain variable domain region
and a light chain variable domain region wherein the antibody has a
heavy chain/light chain variable domain sequence selected from the
group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID
NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID
NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ
ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO.
18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ
ID NO. 23/SEQ ID NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO.
27/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID
NO. 32, SEQ ID NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34,
SEQ ID NO. 31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID
NO. 31/SEQ ID NO. 37, and combinations thereof. Preferably, the
fully human single chain antibody has both a heavy chain variable
domain region and a light chain variable domain region, wherein the
single chain fully human antibody has a heavy chain/light chain
variable domain sequence selected from the group consisting of SEQ
ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ
ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10,
SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID
NO. 15/SEQ ID NO. 16, SEQ ID NO. 17/SEQ ID NO. 18. SEQ ID NO.
19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 23/SEQ ID
NO. 24, SEQ ID NO. 25/SEQ ID NO. 26, SEQ ID NO. 27/SEQ ID NO. 28,
SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID
NO. 31/SEQ ID NO. 33, SEQ ID NO. 31/SEQ ID NO. 34, SEQ ID NO.
31/SEQ ID NO. 35, SEQ ID NO. 31/SEQ ID NO. 36, SEQ ID NO. 31/SEQ ID
NO. 37, and combinations thereof.
[0031] Preferably, the broad spectrum of mammalian cancers to be
treated the cancer is a CXCR3-positive cancer. Preferably, the
broad spectrum of mammalian cancers to be treated is selected from
the group consisting of leukemias, lymphomas, cancinomas, prostate
cancer, non-small cell lung cancer, breast cancer, endometrial
cancer, ovarian cancer, gastric cancers, head and neck cancers,
melanoma, osteosarcoma, intestinal and colon cancer, and various
metastatic cancers. Preferably, the inflammatory disorder to be
treated is selected from the group consisting of rheumatoid
arthritis, multiple sclerosis, Crohn's disease, Graves' disease.
Sjogren's syndrome, inflammatory bowel disease, chronic obstructive
pulmonary disease, psoriasis, type 1 diabetes, transplant rejection
chronic hepatitis C, malaria, and atherosclerosis.
[0032] An "antigen binding protein" is a protein comprising a
portion that binds to an antigen and, optionally, a scaffold or
framework portion that allows the antigen binding portion to adopt
a conformation that promotes binding of the antigen binding protein
to the antigen. Examples of antigen binding proteins include
antibodies, antibody fragments (e.g., an antigen binding portion of
an antibody), antibody derivatives, and antibody analogs. The
antigen binding protein can comprise, for example, an alternative
protein scaffold or artificial scaffold with grafted CDRs or CDR
derivatives. Such scaffolds include, but are not limited to,
antibody-derived scaffolds comprising mutations introduced to, for
example, stabilize the three-dimensional structure of the antigen
binding protein as well as wholly synthetic scaffolds comprising,
for example, a biocompatible polymer. See, for example, Korndorfer
et al., 2003, Proteins: Structure, Function, and Bioinformatics,
Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog.
20:639-654. In addition, peptide antibody mimetics ("PA Ms") can be
used, as well as scaffolds based on antibody mimetics utilizing
fibronection components as a scaffold.
[0033] An antigen binding protein can have, for example, the
structure of a naturally occurring immunoglobulin. An
"immunoglobulin" is a tetrameric molecule. In a naturally occurring
immunoglobulin, each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa or lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Within light and heavy chains, the variable and constant regions
are joined by a "J" region of about 12 or more amino acids, with
the heavy chain also including a "D" region of about 10 more amino
acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,
2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its
entirety for all purposes). The variable regions of each
light/heavy chain pair form the antibody binding site such that an
intact immunoglobulin has two binding sites.
[0034] The variable regions of naturally occurring immunoglobulin
chains exhibit the same general structure of relatively conserved
framework regions (FR) joined by three hypervariable regions, also
called complementarity determining regions or CDRs. From N-terminus
to C-terminus, both light and heavy chains comprise the domains
FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino
acids to each domain is in accordance with the definitions of Kabat
et al. in Sequences of Proteins of Immunological Interest, 5.sup.th
Ed., US Dept. of Health and Human Services, PHS, NIH, NIH
Publication no. 91-3242, 1991. Other numbering systems for the
amino acids in immunoglobulin chains include IMGT.RTM.
(international ImMunoGeneTics information system; Lefranc et al,
Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and
Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).
[0035] Antibodies can be obtained from sources such as serum or
plasma that contain immunoglobulins having varied antigenic
specificity. If such antibodies are subjected to affinity
purification, they can be enriched for a particular antigenic
specificity. Such enriched preparations of antibodies usually are
made of less than about 10% antibody having specific binding
activity for the particular antigen. Subjecting these preparations
to several rounds of affinity purification can increase the
proportion of antibody having specific binding activity for the
antigen. Antibodies prepared in this manner are often referred to
as "monospecific." Monospecfic antibody preparations can be made up
of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, 97%, 99%, or 99.9% antibody having specific binding activity
for the particular antigen.
[0036] An "antibody" refers to an intact immunoglobulin or to an
antigen binding portion thereof that competes with the intact
antibody for specific binding, unless otherwise specified. Antigen
binding portions may be produced by recombinant DNA techniques or
by enzymatic or chemical cleavage of intact antibodies. Antigen
binding portions include, inter alia, Fab, Fab', F(ab').sub.2, Fv,
domain antibodies (dAbs), and complementarity determining region
(CDR) fragments, single-chain antibodies (scFv), chimeric
antibodies, diabodies, triabodies, tetrabodies, and polypeptides
that contain at least a portion of an immunoglobulin that is
sufficient to confer specific antigen binding to the
polypeptide.
[0037] A Fab fragment is a monovalent fragment having the V.sub.L,
V.sub.H, C.sub.L and C.sub.H1 domains; a F(ab').sub.2 fragment is a
bivalent fragment having two Fab fragments linked by a disulfide
bridge at the hinge region; a Fd fragment has the V.sub.H and
C.sub.H1 domains; an Fv fragment has the V.sub.L and V.sub.H
domains of a single arm of an antibody; and a dAb fragment has a
V.sub.H domain, a V.sub.L domain, or an antigen-binding fragment of
a V.sub.H or VL domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US
Patent Applications 2002/02512; 2004/0202995; 2004/0038291;
2004/0009507; 2003/0039958, and Ward et al., Nature 341:544-546,
1989).
[0038] A single-chain antibody (scFv) is an antibody in which a
V.sub.L and a V.sub.H region are joined via a linker (e.g., a
synthetic sequence of amino acid residues) to form a continuous
protein chain wherein the linker is long enough to allow the
protein chain to fold back on itself and form a monovalent antigen
binding site (Bird et al., 1988, Science 242:423-26 and Huston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). Diabodies are
bivalent antibodies comprising two polypeptide chains, wherein each
polypeptide chain comprises V.sub.H and V.sub.L domains joined by a
linker that is too short to allow for pairing between two domains
on the same chain, thus allowing each domain to pair with a
complementary domain on another polypeptide chain (Holliger et al.,
Proc. Natl. Acad. Sci. USA 90:6444-48, 1993 and Poljak et al.,
Structure 2:1121-23, 1994). If the two polypeptide chains of a
diabody are identical, then a diabody resulting from their pairing
will have two identical antigen binding sites. Polypeptide chains
having different sequences can be used to make a diabody with two
different antigen binding sites. Similarly, tribodies and
tetrabodies are antibodies comprising three and four polypeptide
chains, respectively, and forming three and four antigen binding
sites, respectively, which can be the same or different.
[0039] Complementarity determining regions (CDRs) and framework
regions (FR) of a given antibody may be identified using the system
described by Kabat et al. supra; Lefranc et al., supra and/or
Honegger and Pluckthun, supra. One or more CDRs may be incorporated
into a molecule either covalently or noncovalently to make it an
antigen binding protein. An antigen binding protein may incorporate
the CDR(s) as part of a larger polypeptide chain, may covalently
link the CDR(s) to another polypeptide chain, or may incorporate
the CDR(s) noncovalently. The CDRs permit the antigen binding
protein to specifically bind to a particular antigen of
interest.
[0040] An antigen binding protein may have one or more binding
sites. If there is more than one binding site, the binding sites
may be identical to one another or may be different. For example, a
naturally occurring human immunoglobulin typically has two
identical binding sites, while a "bispecific" or "bifunctional"
antibody has two different binding sites.
[0041] The term "human antibody" includes all antibodies that have
one or more variable and constant regions derived from human
immunoglobulin sequences. In one embodiment, all of the variable
and constant domains are derived from human immunoglobulin
sequences (a fully human antibody). These antibodies may be
prepared in a variety of ways, examples of which are described
below, including through the immunization with an antigen of
interest of a mouse that is genetically modified to express
antibodies derived from human heavy and/or light chain-encoding
genes.
[0042] A humanized antibody has a sequence that differs from the
sequence of an antibody derived from a non-human species by one or
more amino acid substitutions, deletions, and/or additions, such
that the humanized antibody is less likely to induce an immune
response, and/or induces a less severe immune response, as compared
to the non-human species antibody, when it is administered to a
human subject. In one embodiment, certain amino acids in the
framework and constant domains of the heavy and/or light chains of
the non-human species antibody are mutated to produce the humanized
antibody. In another embodiment, the constant domain(s) from a
human antibody are fused to the variable domain(s) of a non-human
species. In another embodiment, one or more amino acid residues in
one or more CDR sequences of a non-human antibody are changed to
reduce the likely immunogenicity of the non-human antibody when it
is administered to a human subject, wherein the changed amino acid
residues either are not critical for immunospecific binding of the
antibody to its antigen, or the changes to the amino acid sequence
that are made are conservative changes, such that the binding of
the humanized antibody to the antigen is not significantly worse
than the binding of the non-human antibody to the antigen. Examples
of how to make humanized antibodies may be found in U.S. Pat. No.
6,054,297, 5,886,152 and 5,877,293.
[0043] The term "chimeric antibody" refers to an antibody that
contains one or more regions from one antibody and one or more
regions from one or more other antibodies. In one embodiment, one
or more of the CDRs are derived from a human anti-CXCR3 antibody.
In another embodiment, all of the CDRs are derived from a human
anti-CXCR3 antibody. In another embodiment, the CDRs from more than
one human anti-CXCR3 antibodies are mixed and matched in a chimeric
antibody. For instance, a chimeric antibody may comprise a CDR1
from the light chain of a first human anti-PAR-2 antibody, a CDR2
and a CDR3 from the light chain of a second human anti-CXCR3
antibody, and the CDRs from the heavy chain from a third anti-CXCR3
antibody. Other combinations are possible.
[0044] Further, the framework regions may be derived from one of
the same anti-CXCR3 antibodies, from one or more different
antibodies, such as a human antibody, or from a humanized antibody.
In one example of a chimeric antibody, a portion of the heavy
and/or light chain is identical with, homologous to, or derived
from an antibody from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is/are identical with, homologous to, or derived from an
antibody (-ies) from another species or belonging to another
antibody class or subclass. Also included are fragments of such
antibodies that exhibit the desired biological activity (i.e., the
ability to specifically bind CXCR3).
[0045] A "neutralizing antibody" or an "inhibitory antibody" is an
antibody that inhibits the activation of CXCR3 when an excess of
the anti-CXCR3 antibody reduces the amount of activation by at
least about 20% using an assay such as those described herein in
the Examples. In various embodiments, the antigen binding protein
reduces the amount of amount of activation of CXCR3 by at least
30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and
99.9%.
[0046] Fragments or analogs of antibodies can be readily prepared
by those of ordinary skill in the art following the teachings of
this specification and using techniques known in the art. Preferred
amino- and carboxy-termini of fragments or analogs occur near
boundaries of functional domains. Structural and functional domains
can be identified by comparison of the nucleotide and/or amino acid
sequence data to public or proprietary sequence databases.
Computerized comparison methods can be used to identify sequence
motifs or predicted protein conformation domains that occur in
other proteins of known structure and/or function. Methods to
identify protein sequences that fold into a known three-dimensional
structure are known. See, Bowie et al., 1991, Science 253:164.
[0047] A "CDR grafted antibody" is an antibody comprising one or
more CDRs derived from an antibody of a particular species or
isotype and the framework of another antibody of the same or
different species or isotype.
[0048] A "multi-specific antibody" is an antibody that recognizes
more than one epitope on one or more antigens. A subclass of this
type of antibody is a "bi-specific antibody" which recognizes two
distinct epitopes on the same or different antigens.
[0049] An antigen binding protein "specifically binds" to an
antigen (e.g., human CXCR3) if it binds to the antigen with a
dissociation constant of 1 nanomolar or less.
[0050] An "antigen binding domain," "antigen binding region," or
"antigen binding site" is a portion of an antigen binding protein
that contains amino acid residues (or other moieties) that interact
with an antigen and contribute to the antigen binding protein's
specificity and affinity for the antigen. For an antibody that
specifically binds to its antigen, this will include at least part
of at least one of its CDR domains.
[0051] An "epitope" is the portion of a molecule that is bound by
an antigen binding protein (e.g., by an antibody). An epitope can
comprise non-contiguous portions of the molecule (e.g., in a
polypeptide, amino acid residues that are not contiguous in the
polypeptide's primary sequence but that, in the context of the
polypeptide's tertiary and quaternary structure, are near enough to
each other to be bound by an antigen binding protein).
[0052] The "percent identity" of two polynucleotide or two
polypeptide sequences is determined by comparing the sequences
using the GAP computer program (a part of the GCG Wisconsin
Package, version 10.3 (Accelrys, San Diego. Calif.)) using its
default parameters.
[0053] The terms "polynucleotide," "oligonucleotide" and "nucleic
acid" are used interchangeably throughout and include DNA molecules
(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of
the DNA or RNA generated using nucleotide analogs (e.g., peptide
nucleic acids and non-naturally occurring nucleotide analogs), and
hybrids thereof. The nucleic acid molecule can be single-stranded
or double-stranded. In one embodiment, the nucleic acid molecules
of the invention comprise a contiguous open reading frame encoding
an antibody, or a fragment, derivative, mutein, or variant
thereof.
[0054] Two single-stranded polynucleotides are "the complement" of
each other if their sequences can be aligned in an anti-parallel
orientation such that every nucleotide in one polynucleotide is
opposite its complementary nucleotide in the other polynucleotide,
without the introduction of gaps, and without unpaired nucleotides
at the 5' or the 3' end of either sequence. A polynucleotide is
"complementary" to another polynucleotide if the two
polynucleotides can hybridize to one another under moderately
stringent conditions. Thus, a polynucleotide can be complementary
to another polynucleotide without being its complement.
[0055] A "vector" is a nucleic acid that can be used to introduce
another nucleic acid linked to it into a cell. One type of vector
is a "plasmid." which refers to a linear or circular double
stranded DNA molecule into which additional nucleic acid segments
can be ligated. Another type of vector is a viral vector (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), wherein additional DNA segments can be
introduced into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors comprising a bacterial origin
of replication and episomal mammalian vectors). Other vectors
(e.g., non-episomal mammalian vectors) are integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. An "expression
vector" is a type of vector that can direct the expression of a
chosen polynucleotide.
[0056] A nucleotide sequence is "operably linked" to a regulatory
sequence if the regulatory sequence affects the expression (e.g.,
the level, timing, or location of expression) of the nucleotide
sequence. A "regulatory sequence" is a nucleic acid that affects
the expression (e.g., the level, timing, or location of expression)
of a nucleic acid to which it is operably linked. The regulatory
sequence can, for example, exert its effects directly on the
regulated nucleic acid, or through the action of one or more other
molecules (e.g., polypeptides that bind to the regulatory sequence
and/or the nucleic acid). Examples of regulatory sequences include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Further examples of regulatory sequences
are described in, for example, Goeddel, 1990, Gene Expression
Technology: Methods in Enzymology 185. Academic Press, San Diego,
Calif. and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.
[0057] A "host cell" is a cell that can be used to express a
nucleic acid, e.g., a nucleic acid of the invention. A host cell
can be a prokaryote, for example, E. coli, or it can be a
eukaryote, for example, a single-celled eukaryote (e.g., a yeast or
other fungus), a plant cell (e.g., a tobacco or tomato plant cell),
an animal cell (e.g., a human cell, a monkey cell, a hamster cell,
a rat cell, a mouse cell, or an insect cell) or a hybridoma.
Examples of host cells include the COS-7 line of monkey kidney
cells (ATCC CRL 1651) (see Gluzman et al., 1981. Cell 23:175), L
cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary
(CHO) cells or their derivatives such as Veggie CHO and related
cell lines which grow in serum-free media (see Rasmussen et al.,
1998, Cytotechnology 28:31) or CHO strain DX-BI 1, which is
deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci.
USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the
CV1/EBNA cell line derived from the African green monkey kidney
cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J.
10:2821), human embryonic kidney cells such as 293,293 EBNA or MSR
293, human epidermal A431 cells, human Colo205 cells, other
transformed primate cell lines, normal diploid cells, cell strains
derived from in vitro culture of primary tissue, primary explants,
HL-60, U937, HaK or Jurkat cells. Typically, a host cell is a
cultured cell that can be transformed or transfected with a
polypeptide-encoding nucleic acid, which can then be expressed in
the host cell. The phrase "recombinant host cell" can be used to
denote a host cell that has been transformed or transfected with a
nucleic acid to be expressed. A host cell also can be a cell that
comprises the nucleic acid but does not express it at a desired
level unless a regulatory sequence is introduced into the host cell
such that it becomes operably linked with the nucleic acid. It is
understood that the term host cell refers not only to the
particular subject cell but also to the progeny or potential
progeny of such a cell. Because certain modifications may occur in
succeeding generations due to, e.g., mutation or environmental
influence, such progeny may not, in fact, be identical to the
parent cell, but are still included within the scope of the term as
used herein.
[0058] Preferably, the mammalian cancer to be treated is selected
from the group consisting of ovarian, colon, breast or hepatic
carcinoma cell lines, myelomas, neuroblastic-derived CNS tumors,
monocytic leukemias, B-cell derived leukemia's, T-cell derived
leukemias, B-cell derived lymphomas, T-cell derived lymphomas, mast
cell derived tumors, and combinations thereof.
[0059] Polypeptides of the present disclosure can be produced using
any standard methods known in the art. In one example, the
polypeptides are produced by recombinant DNA methods by inserting a
nucleic acid sequence (e.g., a cDNA) encoding the polypeptide into
a recombinant expression vector and expressing the DNA sequence
under conditions promoting expression.
[0060] Nucleic acids encoding any of the various polypeptides
disclosed herein may be synthesized chemically. Codon usage may be
selected so as to improve expression in a cell. Such codon usage
will depend on the cell type selected. Specialized codon usage
patterns have been developed for E. coli and other bacteria, as
well as mammalian cells, plant cells, yeast cells and insect cells.
See for example: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003
100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002
(1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9;
Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al.
Yeast. 1991 7(7):657-78.
[0061] General techniques for nucleic acid manipulation are
described for example in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press,
2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular
Biology (Green Publishing and Wiley-Interscience: New York, 1987)
and periodic updates, herein incorporated by reference. The DNA
encoding the polypeptide is operably linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, viral, or insect genes. Such regulatory elements include
a transcriptional promoter, an optional operator sequence to
control transcription, a sequence encoding suitable mRNA ribosomal
binding sites, and sequences that control the termination of
transcription and translation. The ability to replicate in a host,
usually conferred by an origin of replication, and a selection gene
to facilitate recognition of transformants is additionally
incorporated.
[0062] The recombinant DNA can also include any type of protein tag
sequence that may be useful for purifying the protein. Examples of
protein tags include but are not limited to a histidine tag, a FLAG
tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and
expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts can be found in Cloning Vectors: A
Laboratory Manual, (Elsevier, N.Y., 1985).
[0063] The expression construct is introduced into the host cell
using a method appropriate to the host cell. A variety of methods
for introducing nucleic acids into host cells are known in the art,
including, but not limited to, electroporation; transfection
employing calcium chloride, rubidium chloride, calcium phosphate,
DEAE-dextran, or other substances; microprojectile bombardment;
lipofection; and infection (where the vector is an infectious
agent). Suitable host cells include prokaryotes, yeast, mammalian
cells, or bacterial cells.
[0064] Suitable bacteria include gram negative or gram positive
organisms, for example, E. coli or Bacillus spp. Yeast, preferably
from the Saccharomyces species, such as S. cerevisiae, may also be
used for production of polypeptides. Various mammalian or insect
cell culture systems can also be employed to express recombinant
proteins. Baculovirus systems for production of heterologous
proteins in insect cells are reviewed by Luckow and Summers,
(Bio/Technology, 6:47, 1988). Examples of suitable mammalian host
cell lines include endothelial cells, COS-7 monkey kidney cells,
CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human
embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines.
Purified polypeptides are prepared by culturing suitable
host/vector systems to express the recombinant proteins. For many
applications, the small size of many of the polypeptides disclosed
herein would make expression in E. coli as the preferred method for
expression. The protein is then purified from culture media or cell
extracts.
[0065] Proteins disclosed herein can also be produced using
cell-translation systems. For such purposes the nucleic acids
encoding the polypeptide must be modified to allow in vitro
transcription to produce mRNA and to allow cell-free translation of
the mRNA in the particular cell-free system being utilized
(eukaryotic such as a mammalian or yeast cell-free translation
system or prokaryotic such as a bacterial cell-free translation
system.
[0066] CXCR3-binding polypeptides can also be produced by chemical
synthesis (e.g., by the methods described in Solid Phase Peptide
Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.).
Modifications to the protein can also be produced by chemical
synthesis.
[0067] The polypeptides of the present disclosure can be purified
by isolation/purification methods for proteins generally known in
the field of protein chemistry. Non-limiting examples include
extraction, recrystallization, salting out (e.g., with ammonium
sulfate or sodium sulfate), centrifugation, dialysis,
ultrafiltration, adsorption chromatography, ion exchange
chromatography, hydrophobic chromatography, normal phase
chromatography, reversed-phase chromatography, gel filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, countercurrent distribution or any combinations of
these. After purification, polypeptides may be exchanged into
different buffers and/or concentrated by any of a variety of
methods known to the art, including, but not limited to, filtration
and dialysis.
[0068] The purified polypeptide is preferably at least 85% pure,
more preferably at least 95% pure, and most preferably at least 98%
pure. Regardless of the exact numerical value of the purity, the
polypeptide is sufficiently pure for use as a pharmaceutical
product.
Post-Translational Modifications of Polypeptides
[0069] In certain embodiments, the binding polypeptides of the
invention may further comprise post-translational modifications.
Exemplary post-translational protein modifications include
phosphorylation, acetylation, methylation, ADP-ribosylation,
ubiquitination, glycosylation, carbonylation, sumoylation,
biotinylation or addition of a polypeptide side chain or of a
hydrophobic group. As a result, the modified soluble polypeptides
may contain non-amino acid elements, such as lipids, poly- or
mono-saccharide, and phosphates. A preferred form of glycosylation
is sialylation, which conjugates one or more sialic acid moieties
to the polypeptide. Sialic acid moieties improve solubility and
serum half-life while also reducing the possible immunogeneticity
of the protein. See Raju et al. Biochemistry. 2001 31;
40(30):8868-76.
[0070] In one specific embodiment, modified forms of the subject
soluble polypeptides comprise linking the subject soluble
polypeptides to nonproteinaceous polymers. In one specific
embodiment, the polymer is polyethylene glycol ("PEG"),
polypropylene glycol, or polyoxyalkylenes, in the manner as set
forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337. Examples of the modified polypeptide
include PEGylated antibodies.
[0071] PEG is a water soluble polymer that is commercially
available or can be prepared by ring-opening polymerization of
ethylene glycol according to methods well known in the art (Sandler
and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,
pages 138-161). The term "PEG" is used broadly to encompass any
polyethylene glycol molecule, without regard to size or to
modification at an end of the PEG, and can be represented by the
formula: X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH (1),
where n is 20 to 2300 and X is H or a terminal modification, e.g.,
a C.sub.1-4 alkyl. In one embodiment, the PEG of the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or
CH.sub.3 ("methoxy PEG"). A PEG can contain further chemical groups
which are necessary for binding reactions; which results from the
chemical synthesis of the molecule; or which is a spacer for
optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked
together. PEGs with more than one PEG chain are called multiarmed
or branched PEGs. Branched PEGs can be prepared, for example, by
the addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEG are described in, for example, EP-A 0 473 084
and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEG
side-chains (PEG2) linked via the primary amino groups of a lysine
(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).
[0072] Although PEG is well-known, this is the first demonstration
that a pegylated.sup.10Fn3 polypeptide can be pegylated and retain
ligand binding activity. In a preferred embodiment, the
pegylated.sup.10 n3 polypeptide is produced by site-directed
pegylation, particularly by conjugation of PEG to a cysteine moiety
at the N- or C-terminus. Accordingly, the present disclosure
provides a target-binding .sup.10Fn3 polypeptide with improved
pharmacokinetic properties, the polypeptide comprising: a
.sup.10Fn3 domain having from about 80 to about 150 amino acids,
wherein at least one of the loops of said .sup.10Fn3 domain
participate in target binding; and a covalently bound PEG moiety,
wherein said .sup.10Fn3 polypeptide binds to the target with a
K.sub.D of less than 100 nM and has a clearance rate of less than
30 mL/hr/kg in a mammal. The PEG moiety may be attached to the
.sup.10Fn3 polypeptide by site directed pegylation, such as by
attachment to a Cys residue, where the Cys residue may be
positioned at the N-terminus of the .sup.0Fn3 polypeptide or
between the N-terminus and the most N-terminal beta or beta-like
strand or at the C-terminus of the .sup.10Fn3 polypeptide or
between the C-terminus and the most C-terminal beta or beta-like
strand. A Cys residue may be situated at other positions as well,
particularly any of the loops that do not participate in target
binding. A PEG moiety may also be attached by other chemistry,
including by conjugation to amines.
[0073] PEG conjugation to peptides or proteins generally involves
the activation of PEG and coupling of the activated
PEG-intermediates directly to target proteins/peptides or to a
linker, which is subsequently activated and coupled to target
proteins/peptides (see Abuchowski et al., J. Biol. Chem., 252, 3571
(1977) and J. Biol. Chem., 252, 3582 (1977), Zalipsky, et al., and
Harris et. al., in: Poly(ethylene glycol) Chemistry: Biotechnical
and Biomedical Applications; (J. M. Harris ed.) Plenum Press: New
York, 1992; Chap. 21 and 22). It is noted that a binding
polypeptide containing a PEG molecule is also known as a conjugated
protein, whereas the protein lacking an attached PEG molecule can
be referred to as unconjugated.
[0074] A variety of molecular mass forms of PEG can be selected,
e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to
2300), for conjugating to CXCR3-binding polypeptides. The number of
repeating units "n" in the PEG is approximated for the molecular
mass described in Daltons. It is preferred that the combined
molecular mass of PEG on an activated linker is suitable for
pharmaceutical use. Thus, in one embodiment, the molecular mass of
the PEG molecules does not exceed 100,000 Da. For example, if three
PEG molecules are attached to a linker, where each PEG molecule has
the same molecular mass of 12,000 Da (each n is about 270), then
the total molecular mass of PEG on the linker is about 36,000 Da
(total n is about 820). The molecular masses of the PEG attached to
the linker can also be different, e.g., of three molecules on a
linker two PEG molecules can be 5,000 Da each (each n is about 110)
and one PEG molecule can be 12,000 Da (n is about 270).
[0075] In a specific embodiment of the disclosure an CXCR3 binding
polypeptide is covalently linked to one poly(ethylene glycol) group
of the formula:
--CO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR, with the
--CO (i.e. carbonyl) of the poly(ethylene glycol) group forming an
amide bond with one of the amino groups of the binding polypeptide;
R being lower alkyl; x being 2 or 3; m being from about 450 to
about 950; and n and m being chosen so that the molecular weight of
the conjugate minus the binding polypeptide is from about 10 to 40
kDa. In one embodiment, a binding polypeptide's 6-amino group of a
lysine is the available (free) amino group.
[0076] The above conjugates may be more specifically presented by
formula (II):
P--NHCO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR (II),
wherein P is the group of a binding polypeptide as described
herein, (i.e. without the amino group or amino groups which form an
amide linkage with the carbonyl shown in formula (II); and wherein
R is lower alkyl; x is 2 or 3; m is from about 450 to about 950 and
is chosen so that the molecular weight of the conjugate minus the
binding polypeptide is from about 10 to about 40 kDa. As used
herein, the given ranges of "m" have an orientational meaning. The
ranges of "m" are determined in any case, and exactly, by the
molecular weight of the PEG group.
[0077] One skilled in the art can select a suitable molecular mass
for PEG. e.g., based on how the pegylated binding polypeptide will
be used therapeutically, the desired dosage, circulation time,
resistance to proteolysis, immunogenicity, and other
considerations. For a discussion of PEG and its use to enhance the
properties of proteins, see Katre, Advanced Drug Delivery Reviews
10: 91-114 (1993).
[0078] In one embodiment, PEG molecules may be activated to react
with amino groups on a binding polypeptide, such as with lysines
(Bencham et al., Anal. Biochem., 131, 25 (1983); Veronese et al.,
Appl. Biochem., 11, 141 (1985); Zalipsky et al., Polymeric Drugs
and Drug Delivery Systems, adrs 9-110 ACS Symposium Series 469
(1999); Zalipsky et al., Europ. Polym. J., 19, 1177-1183 (1983);
Delgado et al., Biotechnology and Applied Biochemistry, 12, 119-128
(1990)).
[0079] In one specific embodiment, carbonate esters of PEG are used
to form the PEG-binding polypeptide conjugates.
N,N'-disuccinimidylcarbonate (DSC) may be used in the reaction with
PEG to form active mixed PEG-succinimidyl carbonate that may be
subsequently reacted with a nucleophilic group of a linker or an
amino group of a binding polypeptide (see U.S. Pat. Nos. 5,281,698
and 5,932,462). In a similar type of reaction,
1,1'-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may be
reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed
carbonate (U.S. Pat. No. 5,382,657), respectively.
[0080] Pegylation of a .sup.10Fn3 polypeptide can be performed
according to the methods of the state of the art, for example by
reaction of the binding polypeptide with electrophilically active
PEGs (supplier: Shearwater Corp., USA, www.shearwatercorp.com).
Preferred PEG reagents of the present invention are, e.g.,
N-hydroxysuccinimidyl propionates (PEG-SPA), butanoates (PEG-SBA),
PEG-succinimidyl propionate or branched N-hydroxysuccinimides such
as mPEG2-NHS (Monfardini et al., Bioconjugate Chem. 6 (1995)
62-69). Such methods may be used to pegylated at an f-amino group
of a binding polypeptide lysine or the N-terminal amino group of
the binding polypeptide.
[0081] In another embodiment, PEG molecules may be coupled to
sulfhydryl groups on a binding polypeptide (Sartore et al., Appl.
Biochem. Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon.
Chem., 7, 363-368 (1996); Goodson et al., Bio/Technology (1990) 8,
343; U.S. Pat. No. 5,766,897). U.S. Pat. Nos. 6,610,281 and
5,766,897 describes exemplary reactive PEG species that may be
coupled to sulfhydryl groups.
[0082] In some embodiments where PEG molecules are conjugated to
cysteine residues on a binding polypeptide, the cysteine residues
are native to the binding polypeptide, whereas in other
embodiments, one or more cysteine residues are engineered into the
binding polypeptide. Mutations may be introduced into a binding
polypeptide coding sequence to generate cysteine residues. This
might be achieved, for example, by mutating one or more amino acid
residues to cysteine. Preferred amino acids for mutating to a
cysteine residue include serine, threonine, alanine and other
hydrophilic residues. Preferably, the residue to be mutated to
cysteine is a surface-exposed residue. Algorithms are well-known in
the art for predicting surface accessibility of residues based on
primary sequence or a protein. Alternatively, surface residues may
be predicted by comparing the amino acid sequences of binding
polypeptides, given that the crystal structure of the framework
based on which binding polypeptides are designed and evolved has
been solved (see Himanen et al., Nature. (2001) 20-27;
414(6866):933-8) and thus the surface-exposed residues identified.
In one embodiment, cysteine residues are introduced into binding
polypeptides at or near the N- and/or C-terminus, or within loop
regions.
[0083] In some embodiments, the pegylated binding polypeptide
comprises a PEG molecule covalently attached to the alpha amino
group of the N-terminal amino acid. Site specific N-terminal
reductive amination is described in Pepinsky et al., (2001) JPET,
297, 1059, and U.S. Pat. No. 5,824,784. The use of a PEG-aldehyde
for the reductive amination of a protein utilizing other available
nucleophilic amino groups is described in U.S. Pat. No. 4,002,531,
in Wieder et al., (1979) J. Biol. Chem. 254, 12579, and in Chamow
et al., (1994) Bioconjugate Chem. 5, 133.
[0084] In another embodiment, pegylated binding polypeptide
comprises one or more PEG molecules covalently attached to a
linker, which in turn is attached to the alpha amino group of the
amino acid residue at the N-terminus of the binding polypeptide.
Such an approach is disclosed in U.S. Patent Publication
2002/0044921 and in WO094/01451.
[0085] In one embodiment, a binding polypeptide is pegylated at the
C-terminus. In a specific embodiment, a protein is pegylated at the
C-terminus by the introduction of C-terminal azido-methionine and
the subsequent conjugation of a methyl-PEG-triarylphosphine
compound via the Staudinger reaction. This C-terminal conjugation
method is described in Cazalis et al., Bioconjug. Chem. 2004;
15(5):1005-1009.
[0086] Monopegylation of a binding polypeptide can also be produced
according to the general methods described in WO 94/01451. WO
94/01451 describes a method for preparing a recombinant polypeptide
with a modified terminal amino acid alpha-carbon reactive group.
The steps of the method involve forming the recombinant polypeptide
and protecting it with one or more biologically added protecting
groups at the N-terminal alpha-amine and C-terminal alpha-carboxyl.
The polypeptide can then be reacted with chemical protecting agents
to selectively protect reactive side chain groups and thereby
prevent side chain groups from being modified. The polypeptide is
then cleaved with a cleavage reagent specific for the biological
protecting group to form an unprotected terminal amino acid
alpha-carbon reactive group. The unprotected terminal amino acid
alpha-carbon reactive group is modified with a chemical modifying
agent. The side chain protected terminally modified single copy
polypeptide is then deprotected at the side chain groups to form a
terminally modified recombinant single copy polypeptide. The number
and sequence of steps in the method can be varied to achieve
selective modification at the N- and/or C-terminal amino acid of
the polypeptide.
[0087] The ratio of a binding polypeptide to activated PEG in the
conjugation reaction can be from about 1:0.5 to 1:50, between from
about 1:1 to 1:30, or from about 1:5 to 1:15. Various aqueous
buffers can be used in the present method to catalyze the covalent
addition of PEG to the binding polypeptide. In one embodiment, the
pH of a buffer used is from about 7.0 to 9.0. In another
embodiment, the pH is in a slightly basic range, e.g., from about
7.5 to 8.5. Buffers having a pKa close to neutral pH range may be
used, e.g., phosphate buffer.
[0088] Conventional separation and purification techniques known in
the art can be used to purify PEGylated binding polypeptide, such
as size exclusion (e.g. gel filtration) and ion exchange
chromatography. Products may also be separated using SDS-PAGE.
Products that may be separated include mono-, di-, tri- poly- and
un-pegylated binding polypeptide, as well as free PEG. The
percentage of mono-PEG conjugates can be controlled by pooling
broader fractions around the elution peak to increase the
percentage of mono-PEG in the composition. About ninety-percent
mono-PEG conjugates represents a good balance of yield and
activity. Compositions in which, for example, at least ninety-two
percent or at least ninety-six percent of the conjugates are
mono-PEG species may be desired. In an embodiment of this invention
the percentage of mono-PEG conjugates is from ninety percent to
ninety-six percent.
[0089] In one embodiment, PEGylated binding polypeptide of the
invention contain one, two or more PEG moieties. In one embodiment,
the PEG moiety(ies) are bound to an amino acid residue which is on
the surface of the protein and/or away from the surface that
contacts the target ligand. In one embodiment, the combined or
total molecular mass of PEG in PEG-binding polypeptide is from
about 3,000 Da to 60,000 Da, optionally from about 10,000 Da to
36,000 Da. In a one embodiment, the PEG in pegylated binding
polypeptide is a substantially linear, straight-chain PEG.
[0090] In one embodiment of the invention, the PEG in pegylated
binding polypeptide is not hydrolyzed from the pegylated amino acid
residue using a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH
6.5) over 8 to 16 hours at room temperature, and is thus stable. In
one embodiment, greater than 80% of the composition is stable
mono-PEG-binding polypeptide, more preferably at least 90%, and
most preferably at least 95%.
[0091] In another embodiment, the pegylated binding polypeptides of
the invention will preferably retain at least 25%, 50%, 60%, 70%,
80%, 85%, 90%, 95% or 100% of the biological activity associated
with the unmodified protein. In one embodiment, biological activity
refers to its ability to bind to CXCR3, as assessed by KD, k.sub.on
or k.sub.off. In one specific embodiment, the pegylated binding
polypeptide protein shows an increase in binding to CXCR3 relative
to unpegylated binding polypeptide.
[0092] The serum clearance rate of PEG-modified polypeptide may be
decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even
90%, relative to the clearance rate of the unmodified binding
polypeptide. The PEG-modified polypeptide may have a half-life
(t.sub.1/2) which is enhanced relative to the half-life of the
unmodified protein. The half-life of PEG-binding polypeptide may be
enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by
1000% relative to the half-life of the unmodified binding
polypeptide. In some embodiments, the protein half-life is
determined in vitro, such as in a buffered saline solution or in
serum. In other embodiments, the protein half-life is an in vivo
half-life, such as the half-life of the protein in the serum or
other bodily fluid of an animal.
Therapeutic Formulations and Modes of Administration
[0093] The present disclosure features methods for treating
conditions or preventing pre-conditions which respond to an
inhibition of CXCR3 biological activity. Preferred examples are
conditions that are characterized by inflammation or cellular
hyperproliferation. Techniques and dosages for administration vary
depending on the type of specific polypeptide and the specific
condition being treated but can be readily determined by the
skilled artisan. In general, regulatory agencies require that a
protein reagent to be used as a therapeutic is formulated so as to
have acceptably low levels of pyrogens. Accordingly, therapeutic
formulations will generally be distinguished from other
formulations in that they are substantially pyrogen free, or at
least contain no more than acceptable levels of pyrogen as
determined by the appropriate regulatory agency (e.g., FDA).
[0094] Therapeutic compositions of the present disclosure may be
administered with a pharmaceutically acceptable diluent, carrier,
or excipient, in unit dosage form. Administration may be parenteral
(e.g., intravenous, subcutaneous), oral, or topical, as
non-limiting examples. In addition, any gene therapy technique,
using nucleic acids encoding the polypeptides of the invention, may
be employed, such as naked DNA delivery, recombinant genes and
vectors, cell-based delivery, including ex vivo manipulation of
patients' cells, and the like.
[0095] The composition can be in the form of a pill, tablet,
capsule, liquid, or sustained release tablet for oral
administration; or a liquid for intravenous, subcutaneous or
parenteral administration; gel, lotion, ointment, cream, or a
polymer or other sustained release vehicle for local
administration.
[0096] Methods well known in the art for making formulations are
found, for example, in "Remington: The Science and Practice of
Pharmacy" (20th ed., ed. A. R. Gennaro A R., 2000. Lippincott
Williams & Wilkins, Philadelphia, Pa.). Formulations for
parenteral administration may, for example, contain excipients,
sterile water, saline, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, or hydrogenated napthalenes.
Biocompatible, biodegradable lactide polymer, lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers may be
used to control the release of the compounds. Nanoparticulate
formulations (e.g., biodegradable nanoparticles, solid lipid
nanoparticles, liposomes) may be used to control the
biodistribution of the compounds. Other potentially useful
parenteral delivery systems include ethylene-vinyl acetate
copolymer particles, osmotic pumps, implantable infusion systems,
and liposomes. The concentration of the compound in the formulation
varies depending upon a number of factors, including the dosage of
the drug to be administered, and the route of administration.
[0097] The polypeptide may be optionally administered as a
pharmaceutically acceptable salt, such as non-toxic acid addition
salts or metal complexes that are commonly used in the
pharmaceutical industry. Examples of acid addition salts include
organic acids such as acetic, lactic, pamoic, maleic, citric,
malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic,
tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic
acids or the like; polymeric acids such as tannic acid,
carboxymethyl cellulose, or the like; and inorganic acid such as
hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid,
or the like. Metal complexes include zinc, iron, and the like. In
one example, the polypeptide is formulated in the presence of
sodium acetate to increase thermal stability.
[0098] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose and sorbitol), lubricating
agents, glidants, and anti-adhesives (e.g., magnesium stearate,
zinc stearate, stearic acid, silicas, hydrogenated vegetable oils,
or talc).
[0099] Formulations for oral use may also be provided as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent, or as soft gelatin capsules
wherein the active ingredient is mixed with water or an oil
medium.
[0100] A therapeutically effective dose refers to a dose that
produces the therapeutic effects for which it is administered. The
exact dose will depend on the disorder to be treated, and may be
ascertained by one skilled in the art using known techniques. In
general, the polypeptide is administered at about 0.01 .mu.g/kg to
about 50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per
day, most preferably 0.1 mg/kg to about 20 mg/kg per day. The
polypeptide may be given daily (e.g., once, twice, three times, or
four times daily) or preferably less frequently (e.g., weekly,
every two weeks, every three weeks, monthly, or quarterly). In
addition, as is known in the art, adjustments for age as well as
the body weight, general health, sex, diet, time of administration,
drug interaction, and the severity of the disease may be necessary,
and will be ascertainable with routine experimentation by those
skilled in the art.
Exemplary Uses
[0101] The CXCR3 binding proteins described herein and their
related variants are useful in a number of therapeutic and
diagnostic applications. These include the inhibition of the
biological activity of CXCR3 by competing for or blocking the
binding to a CXCR3 as well as the delivery of cytotoxic or imaging
moieties to cells, preferably cells expressing CXCR3. The small
size and stable structure of these molecules can be particularly
valuable with respect to manufacturing of the drug, rapid clearance
from the body for certain applications where rapid clearance is
desired or formulation into novel delivery systems that are
suitable or improved using a molecule with such
characteristics.
[0102] On the basis of their efficacy as inhibitors of CXCR3
biological activity, the polypeptides of this disclosure are
effective against a number of cancer conditions as well as
complications arising from cancer, such as pleural effusion and
ascites, colorectal cancers, leukemias, lymphomas, head and neck
cancers, small cell lung cancer, non-small cell lung cancer (NSCLC)
and pancreatic cancer. Non-limiting examples of cancers include
bladder, blood, bone, brain, breast, cartilage, colon kidney,
liver, lung, lymph node, nervous tissue, ovary, pancreatic,
prostate, skeletal muscle, skin, spinal cord, spleen, stomach,
testes, thymus, thyroid, trachea, urogenital tract, ureter,
urethra, uterus, or vaginal cancer.
[0103] A CXCR3 binding polypeptide can be administered alone or in
combination with one or more additional therapies such as
chemotherapy radiotherapy, immunotherapy, surgical intervention, or
any combination of these. Long-term therapy is equally possible as
is adjuvant therapy in the context of other treatment strategies,
as described above. Preferably, in breast cancer the combination is
with Herceptin or other Her-2 therapies.
[0104] In certain embodiments of such methods, one or more
polypeptide therapeutic agents can be administered, together
(simultaneously) or at different times (sequentially). In addition,
polypeptide therapeutic agents can be administered with another
type of compounds for treating cancer or for inhibiting
angiogenesis.
[0105] In certain embodiments, the subject anti-CXCR3 antibodies
agents of the invention can be used alone. Alternatively, the
subject agents may be used in combination with other conventional
anti-cancer therapeutic approaches directed to treatment or
prevention of proliferative disorders (e.g., tumor). For example,
such methods can be used in prophylactic cancer prevention,
prevention of cancer recurrence and metastases after surgery, and
as an adjuvant of other conventional cancer therapy. The present
disclosure recognizes that the effectiveness of conventional cancer
therapies (e.g., chemotherapy, radiation therapy, phototherapy,
immunotherapy, and surgery) can be enhanced through the use of a
subject polypeptide therapeutic agent.
[0106] A wide array of conventional compounds has been shown to
have anti-neoplastic activities. These compounds have been used as
pharmaceutical agents in chemotherapy to shrink solid tumors,
prevent metastases and further growth, or decrease the number of
malignant cells in leukemic or bone marrow malignancies. Although
chemotherapy has been effective in treating various types of
malignancies, many anti-neoplastic compounds induce undesirable
side effects. It has been shown that when two or more different
treatments are combined, the treatments may work synergistically
and allow reduction of dosage of each of the treatments, thereby
reducing the detrimental side effects exerted by each compound at
higher dosages. In other instances, malignancies that are
refractory to a treatment may respond to a combination therapy of
two or more different treatments.
[0107] When a polypeptide therapeutic agent of the present
invention is administered in combination with another conventional
anti-neoplastic agent, either concomitantly or sequentially, such
therapeutic agent may be found to enhance the therapeutic effect of
the anti-neoplastic agent or overcome cellular resistance to such
anti-neoplastic agent. This allows decrease of dosage of an
anti-neoplastic agent, thereby reducing the undesirable side
effects, or restores the effectiveness of an anti-neoplastic agent
in resistant cells.
[0108] Pharmaceutical compounds that may be used for combinatory
anti-tumor therapy include, merely to illustrate:
aminoglutethimide, amsacrine, anastrozole, asparaginase, beg,
bicalutamide, bleomycin, buserelin, busulfan, campothecin,
capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,
cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol,
diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,
estramustine, etoposide, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,
ifosfamide, imatinib, interferon, irinotecan, ironotecan,
letrozole, leucovorin, leuprolide, levamisole, lomustine,
mechlorethamine, medroxyprogesterone, megestrol, melphalan,
mercaptopurine, mesna, methotrexate, mitomycin, mitotane,
mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,
paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,
procarbazine, raltitrexed, rituximab, streptozocin, suramin,
tamoxifen, temozolomide, teniposide, testosterone, thioguanine,
thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin,
vinblastine, vincristine, vindesine, and vinorelbine.
[0109] Certain chemotherapeutic anti-tumor compounds may be
categorized by their mechanism of action into, for example,
following groups: anti-metabolites/anti-cancer agents, such as
pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine,
gemcitabine and cytarabine) and purine analogs, folate antagonists
and related inhibitors (mercaptopurine, thioguanine, pentostatin
and 2-chlorodeoxyadenosine (cladribine));
antiproliferative/antimitotic agents including natural products
such as vinca alkaloids (vinblastine, vincristine, and
vinorelbine), microtubule disruptors such as taxane (paclitaxel,
docetaxel), vincristin, vinblastin, nocodazole, epothilones and
navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA
damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin,
busulfan, camptothecin, carboplatin, chlorambucil, cisplatin,
cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,
epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,
procarbazine, taxol, taxotere, teniposide,
triethylenethiophosphoramide and etoposide (VP16)); antibiotics
such as dactinomycin (actinomycin D), daunorubicin, doxorubicin
(adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins,
plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase
which systemically metabolizes L-asparagine and deprives cells
which do not have the capacity to synthesize their own asparagine);
antiplatelet agents; antiproliferative/antimitotic alkylating
agents such as nitrogen mustards (mechlorethamine, cyclophosphamide
and analogs, melphalan, chlorambucil), ethylenimines and
methylmelamines (hexamethylmelamine and thiotepa), alkyl
sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,
streptozocin), trazenes-dacarbazinine (DTIC);
antiproliferative/antimitotic antimetabolites such as folic acid
analogs (methotrexate); platinum coordination complexes (cisplatin,
carboplatin), procarbazine, hydroxyurea, mitotane,
aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen,
goserelin, bicalutamide, nilutamide) and aromatase inhibitors
(letrozole, anastrozole); anticoagulants (heparin, synthetic
heparin salts and other inhibitors of thrombin); fibrinolytic
agents (such as tissue plasminogen activator, streptokinase and
urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel,
abciximab; antimigratory agents; antisecretory agents (breveldin);
immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus
(rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic
compounds (TNP-470, genistein) and growth factor inhibitors (e.g.,
VEGF inhibitors, fibroblast growth factor (FGF) inhibitors);
angiotensin receptor blocker, nitric oxide donors; anti-sense
oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors
and differentiation inducers (tretinoin); mTOR inhibitors,
topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,
camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,
etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),
corticosteroids (cortisone, dexamethasone, hydrocortisone,
methylpednisolone, prednisone, and prenisolone); growth factor
signal transduction kinase inhibitors; mitochondrial dysfunction
inducers and caspase activators; and chromatin disruptors.
[0110] Depending on the nature of the combinatory therapy,
administration of the polypeptide therapeutic agents may be
continued while the other therapy is being administered and/or
thereafter. Administration of the polypeptide therapeutic agents
may be made in a single dose, or in multiple doses. In some
instances, administration of the polypeptide therapeutic agents is
commenced at least several days prior to the conventional therapy,
while in other instances, administration is begun either
immediately before or at the time of the administration of the
conventional therapy.
[0111] In one example of a diagnostic application, a biological
sample, such as serum or a tissue biopsy, from a patient suspected
of having a condition characterized by inappropriate angiogenesis
is contacted with a detectably labeled polypeptide of the
disclosure to detect levels of CXCR3. The levels of CXCR3 detected
are then compared to levels of CXCR3 detected in a normal sample
also contacted with the labeled polypeptide. An increase of at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in the levels
of the CXCR3 may be considered a diagnostic indicator.
[0112] In certain embodiments, the CXCR3 binding polypeptides are
further attached to a label that is able to be detected (e.g., the
label can be a radioisotope, fluorescent compound, enzyme or enzyme
co-factor). The active moiety may be a radioactive agent, such as:
radioactive heavy metals such as iron chelates, radioactive
chelates of gadolinium or manganese, positron emitters of oxygen,
nitrogen, iron, carbon, or gallium, .sup.43K, .sup.52Fe, .sup.57Co,
.sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.123I, .sup.125I, .sup.131I,
.sup.132, or .sup.99Tc. A binding agent affixed to such a moiety
may be used as an imaging agent and is administered in an amount
effective for diagnostic use in a mammal such as a human and the
localization and accumulation of the imaging agent is then
detected. The localization and accumulation of the imaging agent
may be detected by radioscintigraphy, nuclear magnetic resonance
imaging, computed tomography or positron emission tomography.
Immunoscintigraphy using CXCR3 binding polypeptides directed at
CXCR3 may be used to detect and/or diagnose cancers and
vasculature. For example, any of the binding polypeptide against a
CXCR3 marker labeled with .sup.99Technetium, .sup.111Indium, or
.sup.125Iodine may be effectively used for such imaging. As will be
evident to the skilled artisan, the amount of radioisotope to be
administered is dependent upon the radioisotope. Those having
ordinary skill in the art can readily formulate the amount of the
imaging agent to be administered based upon the specific activity
and energy of a given radionuclide used as the active moiety.
Typically 0.1-100 millicuries per dose of imaging agent, preferably
1-10 millicuries, most often 2-5 millicuries are administered.
Thus, compositions according to the present invention useful as
imaging agents comprising a targeting moiety conjugated to a
radioactive moiety comprise 0.1-100 millicuries, in some
embodiments preferably 1-10 millicuries, in some embodiments
preferably 2-5 millicuries, in some embodiments more preferably 1-5
millicuries.
[0113] The CXCR3 binding polypeptides can also be used to deliver
additional therapeutic agents (including but not limited to drug
compounds, chemotherapeutic compounds, and radiotherapeutic
compounds) to a cell or tissue expressing CXCR3. In one example,
the CXCR3 binding polypeptide is fused to a chemotherapeutic agent
for targeted delivery of the chemotherapeutic agent to a tumor cell
or tissue expressing CXCR3.
[0114] The CXCR3 binding polypeptides are useful in a variety of
applications, including research, diagnostic and therapeutic
applications. For instance, they can be used to isolate and/or
purify receptor or portions thereof, and to study receptor
structure (e.g., conformation) and function.
[0115] In certain aspects, the various binding polypeptides can be
used to detect or measure the expression of CXCR3, for example, on
endothelial cells (e.g., venous endothelial cells), or on cells
transfected with a CXCR3 gene. Thus, they also have utility in
applications such as cell sorting and imaging (e.g., flow
cytometry, and fluorescence activated cell sorting), for diagnostic
or research purposes.
[0116] In certain embodiments, the binding polypeptides of
fragments thereof can be labeled or unlabeled for diagnostic
purposes. Typically, diagnostic assays entail detecting the
formation of a complex resulting from the binding of a binding
polypeptide to CXCR3. The binding polypeptides or fragments can be
directly labeled, similar to antibodies. A variety of labels can be
employed, including, but not limited to, radionuclides,
fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme
inhibitors and ligands (e.g., biotin, haptens). Numerous
appropriate immunoassays are known to the skilled artisan (see, for
example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and
4,098,876). When unlabeled, the binding polypeptides can be used in
assays, such as agglutination assays. Unlabeled binding
polypeptides can also be used in combination with another (one or
more) suitable reagent which can be used to detect the binding
polypeptide, such as a labeled antibody reactive with the binding
polypeptide or other suitable reagent (e.g., labeled protein
A).
[0117] In one embodiment, the binding polypeptides of the present
invention can be utilized in enzyme immunoassays, wherein the
subject polypeptides are conjugated to an enzyme. When a biological
sample comprising a CXCR3 protein is combined with the subject
binding polypeptides, binding occurs between the binding
polypeptides and the CXCR3 protein. In one embodiment, a sample
containing cells expressing a CXCR3 protein (e.g., endothelial
cells) is combined with the subject antibodies, and binding occurs
between the binding polypeptides and cells bearing a CXCR3 protein
recognized by the binding polypeptide. These bound cells can be
separated from unbound reagents and the presence of the binding
polypeptide-enzyme conjugate specifically bound to the cells can be
determined, for example, by contacting the sample with a substrate
of the enzyme which produces a color or other detectable change
when acted on by the enzyme. In another embodiment, the subject
binding polypeptides can be unlabeled, and a second, labeled
polypeptide (e.g., an antibody) can be added which recognizes the
subject binding polypeptide.
[0118] In certain aspects, kits for use in detecting the presence
of a CXCR3 protein in a biological sample can also be prepared.
Such kits will include a CXCR3 binding polypeptide which binds to a
CXCR3 protein or portion of said receptor, as well as one or more
ancillary reagents suitable for detecting the presence of a complex
between the binding polypeptide and the receptor protein or
portions thereof. The polypeptide compositions of the present
invention can be provided in lyophilized form, either alone or in
combination with additional antibodies specific for other epitopes.
The binding polypeptides and/or antibodies, which can be labeled or
unlabeled, can be included in the kits with adjunct ingredients
(e.g., buffers, such as Tris, phosphate and carbonate, stabilizers,
excipients, biocides and/or inert proteins, e.g., bovine serum
albumin). For example, the binding polypeptides and/or antibodies
can be provided as a lyophilized mixture with the adjunct
ingredients, or the adjunct ingredients can be separately provided
for combination by the user. Generally these adjunct materials will
be present in less than about 5% weight based on the amount of
active binding polypeptide or antibody, and usually will be present
in a total amount of at least about 0.001% weight based on
polypeptide or antibody concentration. Where a second antibody
capable of binding to the binding polypeptide is employed, such
antibody can be provided in the kit, for instance in a separate
vial or container. The second antibody, if present, is typically
labeled, and can be formulated in an analogous manner with the
antibody formulations described above.
[0119] Similarly, the present disclosure also provides a method of
detecting and/or quantitating expression of CXCR3, wherein a
composition comprising a cell or fraction thereof (e.g., membrane
fraction) is contacted with a binding polypeptide which binds to a
CXCR3 or portion of the receptor under conditions appropriate for
binding thereto, and the binding is monitored. Detection of the
binding polypeptide, indicative of the formation of a complex
between binding polypeptide and CXCR3 or a portion thereof,
indicates the presence of the receptor. Binding of a polypeptide to
the cell can be determined by standard methods, such as those
described in the working examples. The method can be used to detect
expression of CXCR3 on cells from an individual. Optionally, a
quantitative expression of CXCR3 on the surface of endothelial
cells can be evaluated, for instance, by flow cytometry, and the
staining intensity can be correlated with disease susceptibility,
progression or risk.
[0120] The present disclosure also provides a method of detecting
the susceptibility of a mammal to certain diseases. To illustrate,
the method can be used to detect the susceptibility of a mammal to
diseases which progress based on the amount of CXCR3 present on
cells and/or the number of CXCR3-positive cells in a mammal.
[0121] Polypeptide sequences are indicated using standard one- or
three-letter abbreviations. Unless otherwise indicated, each
polypeptide sequence has amino termini at the left and a carboxy
termini at the right; each single-stranded nucleic acid sequence,
and the top strand of each double-stranded nucleic acid sequence,
has a 5' termini at the left and a 3' termini at the right. A
particular polypeptide sequence also can be described by explaining
how it differs from a reference sequence.
[0122] The following terms, unless otherwise indicated, shall be
understood to have the following meanings:
[0123] The terms "CXCR3 inhibitor" and "CXCR3 antagonist" are used
interchangeably. Each is a molecule that detectably inhibits at
least one function of CXCR3. Conversely, an "CXCR3 agonist" is a
molecule that detectably increases at least one function of CXCR3.
The inhibition caused by an CXCR3 inhibitor need not be complete so
long as it is detectable using an assay. Any assay of a function of
CXCR3 can be used, examples of which are provided herein. Examples
of functions of CXCR3 that can be inhibited by a CXCR3 inhibitor,
or increased by a CXCR3 agonist, include cancer cell growth or
apoptosis (programmed cell death), and so on. Examples of types of
CXCR3 inhibitors and CXCR3 agonists include, but are not limited
to, CXCR3 binding polypeptides such as antigen binding proteins
(e.g., CXCR3 inhibiting antigen binding proteins), antibodies,
antibody fragments, and antibody derivatives.
[0124] The terms "peptide." "polypeptide" and "protein" each refers
to a molecule comprising two or more amino acid residues joined to
each other by peptide bonds. These terms encompass, e.g., native
and artificial proteins, protein fragments and polypeptide analogs
(such as muteins, variants, and fusion proteins) of a protein
sequence as well as post-translationally, or otherwise covalently
or non-covalently, modified proteins. A peptide, polypeptide, or
protein may be monomeric or polymeric.
[0125] A "variant" of a polypeptide (for example, an antibody)
comprises an amino acid sequence wherein one or more amino acid
residues are inserted into, deleted from and/or substituted into
the amino acid sequence relative to another polypeptide sequence.
Disclosed variants include, for example, fusion proteins.
[0126] A "derivative" of a polypeptide is a polypeptide (e.g., an
antibody) that has been chemically modified, e.g., via conjugation
to another chemical moiety (such as, for example, polyethylene
glycol or albumin, e.g., human serum albumin), phosphorylation, and
glycosylation. Unless otherwise indicated, the term "antibody"
includes, in addition to antibodies comprising two full-length
heavy chains and two full-length light chains, derivatives,
variants, fragments, and muteins thereof, examples of which are
described below.
[0127] An "antigen binding protein" is a protein comprising a
portion that binds to an antigen and, optionally, a scaffold or
framework portion that allows the antigen binding portion to adopt
a conformation that promotes binding of the antigen binding protein
to the antigen. Examples of antigen binding proteins include
antibodies, antibody fragments (e.g., an antigen binding portion of
an antibody), antibody derivatives, and antibody analogs. The
antigen binding protein can comprise, for example, an alternative
protein scaffold or artificial scaffold with grafted CDRs or CDR
derivatives. Such scaffolds include, but are not limited to,
antibody-derived scaffolds comprising mutations introduced to, for
example, stabilize the three-dimensional structure of the antigen
binding protein as well as wholly synthetic scaffolds comprising,
for example, a biocompatible polymer. See, for example, Korndorfer
et al., 2003, Proteins: Structure, Function, and Bioinformatics,
Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog.
20:639-654. In addition, peptide antibody mimetics ("PAMs") can be
used, as well as scaffolds based on antibody mimetics utilizing
fibronection components as a scaffold.
[0128] An antigen binding protein can have, for example, the
structure of a naturally occurring immunoglobulin. An
"immunoglobulin" is a tetrameric molecule. In a naturally occurring
immunoglobulin, each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa or lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Preferably, the anti-CXCR3 antibodies disclosed herein are
characterized by their variable domain region sequences in the
heavy V.sub.H and light V.sub.L amino acid sequences. The preferred
antibody is A6 which is a kappa IgG antibody. Within light and
heavy chains, the variable and constant regions are joined by a "J"
region of about 12 or more amino acids, with the heavy chain also
including a "D" region of about 10 more amino acids. See generally,
Fundamental Immunology Ch. 7 (Paul. W., ed., 2nd ed. Raven Press,
N.Y. (1989)). The variable regions of each light/heavy chain pair
form the antibody binding site such that an intact immunoglobulin
has two binding sites.
[0129] A "multi-specific antibody" is an antibody that recognizes
more than one epitope on one or more antigens. A subclass of this
type of antibody is a "bi-specific antibody" which recognizes two
distinct epitopes on the same or different antigens.
[0130] An antigen binding protein "specifically binds" to an
antigen (e.g., human CXCR3) if it binds to the antigen with a
dissociation constant of 1 nanomolar or less.
[0131] An "antigen binding domain, "antigen binding region," or
"antigen binding site" is a portion of an antigen binding protein
that contains amino acid residues (or other moieties) that interact
with an antigen and contribute to the antigen binding protein's
specificity and affinity for the antigen. For an antibody that
specifically binds to its antigen, this will include at least part
of at least one of its CDR domains.
[0132] An "epitope" is the portion of a molecule that is bound by
an antigen binding protein (e.g., by an antibody). An epitope can
comprise non-contiguous portions of the molecule (e.g., in a
polypeptide, amino acid residues that are not contiguous in the
polypeptide's primary sequence but that, in the context of the
polypeptide's tertiary and quaternary structure, are near enough to
each other to be bound by an antigen binding protein).
[0133] The "percent homology" of two polynucleotide or two
polypeptide sequences is determined by comparing the sequences
using the GAP computer program (a part of the GCG Wisconsin
Package, version 10.3 (Accelrys, San Diego, Calif.)) using its
default parameters.
[0134] A "host cell" is a cell that can be used to express a
nucleic acid. A host cell can be a prokaryote, for example, E.
coli, or it can be a eukaryote, for example, a single-celled
eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a
tobacco or tomato plant cell), an animal cell (e.g., a human cell,
a monkey cell, a hamster cell, a rat cell, a mouse cell, or an
insect cell) or a hybridoma. Examples of host cells include the
COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,
1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163),
Chinese hamster ovary (CHO) cells or their derivatives such as
Veggie CHO and related cell lines which grow in serum-free media
(Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain
DX-B11, which is deficient in DHFR (Urlaub et al., 1980, Proc.
Natl. Acad. Sci. USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10)
cell lines, the CV1/EBNA cell line derived from the African green
monkey kidney cell line CV1 (ATCC CCL 70) (McMahan et al., 1991,
EMBO J. 10:2821), human embryonic kidney cells such as 293,293 EBNA
or MSR 293, human epidermal A431 cells, human Colo205 cells, other
transformed primate cell lines, normal diploid cells, cell strains
derived from in vitro culture of primary tissue, primary explants.
HL-60, U937, HaK or Jurkat cells. Typically, a host cell is a
cultured cell that can be transformed or transfected with a
polypeptide-encoding nucleic acid, which can then be expressed in
the host cell. The phrase "recombinant host cell" can be used to
denote a host cell that has been transformed or transfected with a
nucleic acid to be expressed. A host cell also can be a cell that
comprises the nucleic acid but does not express it at a desired
level unless a regulatory sequence is introduced into the host cell
such that it becomes operably linked with the nucleic acid. It is
understood that the term host cell refers not only to the
particular subject cell but also to the progeny or potential
progeny of such a cell. Because certain modifications may occur in
succeeding generations due to, e.g., mutation or environmental
influence, such progeny may not, in fact, be identical to the
parent cell, but are still included within the scope of the term as
used herein.
Antigen Binding Proteins
[0135] Antigen binding proteins (e.g., antibodies, antibody
fragments, antibody derivatives, antibody muteins, and antibody
variants) are polypeptides that bind to CXCR3, (preferably, human
CXCR3). Antigen binding proteins include antigen binding proteins
that inhibit a biological activity of CXCR3.
[0136] Oligomers that contain one or more antigen binding proteins
may be employed as CXCR3 antagonists. Oligomers may be in the form
of covalently-linked or non-covalently-linked dimers, trimers, or
higher oligomers. Oligomers comprising two or more antigen binding
protein are contemplated for use, with one example being a
homodimer. Other oligomers include heterodimers, homotrimers,
heterotrimers, homotetramers, heterotetramers, etc.
[0137] One embodiment is directed to oligomers comprising multiple
antigen binding proteins joined via covalent or non-covalent
interactions between peptide moieties fused to the antigen binding
proteins. Such peptides may be peptide linkers (spacers), or
peptides that have the property of promoting oligomerization.
Leucine zippers and certain polypeptides derived from antibodies
are among the peptides that can promote oligomerization of antigen
binding proteins attached thereto, as described in more detail
below.
[0138] In particular embodiments, the oligomers comprise from two
to four antigen binding proteins. The antigen binding proteins of
the oligomer may be in any form, such as any of the forms described
above, e.g., variants or fragments. Preferably, the oligomers
comprise antigen binding proteins that have CXCR3 binding
activity.
[0139] In one embodiment, an oligomer is prepared using
polypeptides derived from immunoglobulins. Preparation of Fusion
Proteins Comprising Certain Heterologous Polypeptides Fused to
Various Portions of antibody-derived polypeptides (including the Fc
domain) has been described, e.g., by Ashkenazi et al., 1991, Proc.
Natl. Acad. Sci. USA 88:10535; Byrn et al., 1990, Nature 344:677;
and Hollenbaugh et al., 1992 "Construction of Immunoglobulin Fusion
Proteins", in Current Protocols in Immunology, Suppl. 4, pages
10.19.1-10.19.11.
[0140] One embodiment is directed to a dimer comprising two fusion
proteins created by fusing a CXCR3 binding fragment of an
anti-CXCR3 antibody to the Fc region of an antibody. The dimer can
be made by, for example, inserting a gene fusion encoding the
fusion protein into an appropriate expression vector, expressing
the gene fusion in host cells transformed with the recombinant
expression vector, and allowing the expressed fusion protein to
assemble much like antibody molecules, whereupon interchain
disulfide bonds form between the Fc moieties to yield the
dimer.
[0141] The term "Fc polypeptide" includes native and mutein forms
of polypeptides derived from the Fc region of an antibody.
Truncated forms of such polypeptides containing the hinge region
that promotes dimerization also are included. Fusion proteins
comprising Fc moieties (and oligomers formed therefrom) offer the
advantage of facile purification by affinity chromatography over
Protein A or Protein G columns.
[0142] Another method for preparing oligomeric antigen binding
proteins involves use of a leucine zipper. Leucine zipper domains
are peptides that promote oligomerization of the proteins in which
they are found. Leucine zippers were originally identified in
several DNA-binding proteins (Landschulz et al., 1988, Science
240:1759), and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurring
peptides and derivatives thereof that dimerize or trimerize.
Examples of leucine zipper domains suitable for producing soluble
oligomeric proteins are described in WO 94/10308, and the leucine
zipper derived from lung surfactant protein D (SPD) described in
Hoppe et al., 1994, FEBS Letters 344:191. The use of a modified
leucine zipper that allows for stable trimerization of a
heterologous protein fused thereto is described in Fanslow et al.,
1994, Semin. Immunol. 6:267-78. In one approach, recombinant fusion
proteins comprising an anti-CXCR3 antibody fragment or derivative
fused to a leucine zipper peptide are expressed in suitable host
cells, and the soluble oligomeric anti-CXCR3 antibody fragments or
derivatives that form are recovered from the culture
supernatant.
[0143] Antigen-binding fragments of antigen binding proteins of the
invention may be produced by conventional techniques. Examples of
such fragments include, but are not limited to, Fab and
F(ab').sub.2 fragments.
[0144] The present disclosure provides monoclonal antibodies that
bind to CXCR3. Monoclonal antibodies may be produced using any
technique known in the art, e.g., by immortalizing spleen cells
harvested from the transgenic animal after completion of the
immunization schedule. The spleen cells can be immortalized using
any technique known in the art, e.g., by fusing them with myeloma
cells to produce hybridomas. Myeloma cells for use in
hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency, and enzyme
deficiencies that render them incapable of growing in certain
selective media which support the growth of only the desired fused
cells (hybridomas). Examples of suitable cell lines for use in
mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4
1, Sp210-Ag14, FO, NSO/U, MPC-1, MPC11-X45-GTG 1.7 and S194/5XX0
Bul; examples of cell lines used in rat fusions include R210.RCY3,
Y3-Ag 1.2.3, IR983F and 48210. Other cell lines useful for cell
fusions are U-266, GMI 500-GRG2, LICR-LON-HMy2 and UC729-6.
[0145] Antigen binding proteins directed against CXCR3 can be used,
for example, in assays to detect the presence of CXCR3
polypeptides, either in vitro or in vivo. The antigen binding
proteins also may be employed in purifying CXCR3 proteins by
immunoaffinity chromatography. Blocking antigen binding proteins
can be used in the methods disclosed herein. Such antigen binding
proteins that function as CXCR3 antagonists may be employed in
treating any CXCR3-induced condition, including but not limited to
various cancers.
[0146] Antigen binding proteins may be employed in an in vitro
procedure, or administered in vivo to inhibit CXCR3-induced
biological activity. Disorders caused or exacerbated (directly or
indirectly) by the proteolytic activation of CXCR3, examples of
which are provided herein, thus may be treated. In one embodiment,
the present invention provides a therapeutic method comprising in
vivo administration of a CXCR3 blocking antigen binding protein to
a mammal in need thereof in an amount effective for reducing a
CXCR3-induced biological activity.
[0147] Antigen binding proteins include fully human monoclonal
antibodies that inhibit a biological activity of CXCR3.
[0148] Antigen binding proteins may be prepared by any of a number
of conventional techniques. For example, they may be purified from
cells that naturally express them (e.g., an antibody can be
purified from a hybridoma that produces it), or produced in
recombinant expression systems, using any technique known in the
art. See, for example, Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Kennet et al. (eds.). Plenum
Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow
and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1988).
[0149] Any expression system known in the art can be used to make
the recombinant polypeptides of the invention. In general, host
cells are transformed with a recombinant expression vector that
comprises DNA encoding a desired polypeptide. Among the host cells
that may be employed are prokaryotes, yeast or higher eukaryotic
cells. Prokaryotes include gram negative or gram positive
organisms, for example E. coli or bacilli. Higher eukaryotic cells
include insect cells and established cell lines of mammalian
origin. Examples of suitable mammalian host cell lines include the
COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,
1981, Cell 23:175), L cells, 293 cells. C127 cells, 3T3 cells (ATCC
CCL 163). Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC
CRL 10) cell lines, and the CV1/EBNA cell line derived from the
African green monkey kidney cell line CV1 (ATCC CCL 70) as
described by McMahan et al., 1991, EMBO J. 10: 2821. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described by Pouwels et al.
(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).
[0150] The transformed cells can be cultured under conditions that
promote expression of the polypeptide, and the polypeptide
recovered by conventional protein purification procedures. One such
purification procedure includes the use of affinity chromatography,
e.g., over a matrix having all or a portion (e.g., the
extracellular domain) of CXCR3 bound thereto. Polypeptides
contemplated for use herein include substantially homogeneous
recombinant mammalian anti-CXCR3 antibody polypeptides
substantially free of contaminating endogenous materials.
[0151] Antigen binding proteins may be prepared, and screened for
desired properties, by any of a number of known techniques. Certain
of the techniques involve isolating a nucleic acid encoding a
polypeptide chain (or portion thereof) of an antigen binding
protein of interest (e.g., an anti-CXCR3 antibody), and
manipulating the nucleic acid through recombinant DNA technology.
The nucleic acid may be fused to another nucleic acid of interest,
or altered (e.g., by mutagenesis or other conventional techniques)
to add, delete, or substitute one or more amino acid residues, for
example.
[0152] Single chain antibodies may be formed by linking heavy and
light chain variable domain (Fv region) fragments via an amino acid
bridge (short peptide linker), resulting in a single polypeptide
chain. Such single-chain Fvs (scFvs) have been prepared by fusing
DNA encoding a peptide linker between DNAs encoding the two
variable domain polypeptides (V.sub.L and V.sub.H). The resulting
polypeptides can fold back on themselves to form antigen-binding
monomers, or they can form multimers (e.g., dimers, trimers, or
tetramers), depending on the length of a flexible linker between
the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423;
Kortt et al., 2001, Biomol. Eng. 18:95-108). By combining different
V.sub.L and V.sub.H-comprising polypeptides, one can form
multimeric scFvs that bind to different epitopes (Kriangkum et al.,
2001, Biomol. Eng. 18:31-40). Techniques developed for the
production of single chain antibodies include those described in
U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989,
Nature 334:544, de Graaf et al., 2002, Methods Mol. Biol.
178:379-87.
[0153] Techniques are known for deriving an antibody of a different
subclass or isotype from an antibody of interest, i.e., subclass
switching. Thus, IgG antibodies may be derived from an IgM
antibody, for example, and vice versa. Such techniques allow the
preparation of new antibodies that possess the antigen-binding
properties of a given antibody (the parent antibody), but also
exhibit biological properties associated with an antibody isotype
or subclass different from that of the parent antibody. Recombinant
DNA techniques may be employed. Cloned DNA encoding particular
antibody polypeptides may be employed in such procedures. e.g., DNA
encoding the constant domain of an antibody of the desired isotype
(Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover, if
an IgG4 is desired, it may also be desired to introduce a point
mutation (CPSCP->CPPCP) in the hinge region (Bloom et al., 1997,
Protein Science 6:407) to alleviate a tendency to form intra-H
chain disulfide bonds that can lead to heterogeneity in the IgG4
antibodies.
[0154] In particular embodiments, antigen binding proteins of the
present invention have a binding affinity (K.sub.a) for CXCR3 of at
least 10.sup.6. In other embodiments, the antigen binding proteins
exhibit a K.sub.a of at least 10.sup.7, at least 10.sup.8, at least
10.sup.9, or at least 10.sup.10. In another embodiment, the antigen
binding protein exhibits a K.sub.a substantially the same as that
of an antibody described herein in the Examples.
[0155] In another embodiment, the present disclosure provides an
antigen binding protein that has a low dissociation rate from
CXCR3. In one embodiment, the antigen binding protein has a
K.sub.off of 1.times.10.sup.-4 to .sup.-1 or lower. In another
embodiment, the K.sub.off is 5.times.10.sup.-5 to .sup.-1 or lower.
In another embodiment, the K.sub.off is substantially the same as
an antibody described herein. In another embodiment, the antigen
binding protein binds to CXCR3 with substantially the same
K.sub.off as an antibody described herein.
[0156] In another aspect, the present disclosure provides an
antigen binding protein that inhibits an activity of CXCR3. In one
embodiment, the antigen binding protein has an IC.sub.50 of 1000 nM
or lower. In another embodiment, the IC.sub.50 is 100 nM or lower;
in another embodiment, the IC.sub.50 is 10 nM or lower. In another
embodiment, the IC.sub.50 is substantially the same as that of an
antibody described herein in the Examples. In another embodiment,
the antigen binding protein inhibits an activity of CXCR3 with
substantially the same IC.sub.50 as an antibody described
herein.
[0157] In another aspect, the present disclosure provides an
antigen binding protein that binds to human CXCR3 expressed on the
surface of a cell and, when so bound, inhibits CXCR3 signaling
activity in the cell without causing a significant reduction in the
amount of CXCR3 on the surface of the cell. Any method for
determining or estimating the amount of CXCR3 on the surface and/or
in the interior of the cell can be used. In other embodiments,
binding of the antigen binding protein to the CXCR3-expressing cell
causes less than about 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%,
or 0.1% of the cell-surface CXCR3 to be internalized.
[0158] In another aspect, the present disclosure provides an
antigen binding protein having a half-life of at least one day in
vitro or in vivo (e.g., when administered to a human subject). In
one embodiment, the antigen binding protein has a half-life of at
least three days. In another embodiment, the antigen binding
protein has a half-life of four days or longer. In another
embodiment, the antigen binding protein has a half-life of eight
days or longer. In another embodiment, the antigen binding protein
is derivatized or modified such that it has a longer half-life as
compared to the underivatized or unmodified antigen binding
protein. In another embodiment, the antigen binding protein
contains one or more point mutations to increase serum half life,
such as described in WO00/09560, incorporated by reference
herein.
[0159] The present disclosure further provides multi-specific
antigen binding proteins, for example, bispecific antigen binding
protein, e.g., antigen binding protein that bind to two different
epitopes of CXCR3, or to an epitope of CXCR3 and an epitope of
another molecule, via two different antigen binding sites or
regions. Moreover, bispecific antigen binding protein as disclosed
herein can comprise an CXCR3 binding site from one of the
herein-described antibodies and a second CXCR3 binding region from
another of the herein-described antibodies, including those
described herein by reference to other publications. Alternatively,
a bispecific antigen binding protein may comprise an antigen
binding site from one of the herein described antibodies and a
second antigen binding site from another CXCR3 antibody that is
known in the art, or from an antibody that is prepared by known
methods or the methods described herein.
[0160] Numerous methods of preparing bispecific antibodies are
known in the art. Such methods include the use of hybrid-hybridomas
as described by Milstein et al., 1983, Nature 305:537, and chemical
coupling of antibody fragments (Brennan et al., 1985. Science
229:81; Glennie et al., 1987. J. Immunol. 139:2367; U.S. Pat. No.
6,010,902). Moreover, bispecific antibodies can be produced via
recombinant means, for example by using leucine zipper moieties
(i.e., from the Fos and Jun proteins, which preferentially form
heterodimers; Kostelny et al., 1992, J. Immunol. 148:1547) or other
lock and key interactive domain structures as described in U.S.
Pat. No. 5,582,996. Additional useful techniques include those
described in U.S. Pat. Nos. 5,959,083; and 5,807,706.
[0161] In another aspect, the antigen binding protein comprises a
derivative of an antibody. The derivatized antibody can comprise
any molecule or substance that imparts a desired property to the
antibody, such as increased half-life in a particular use. The
derivatized antibody can comprise, for example, a detectable (or
labeling) moiety (e.g., a radioactive, colorimetric, antigenic or
enzymatic molecule, a detectable bead (such as a magnetic or
electrodense (e.g., gold bead), or a molecule that binds to another
molecule (e.g., biotin or streptavidin), a therapeutic or
diagnostic moiety (e.g., a radioactive, cytotoxic, or
pharmaceutically active moiety), or a molecule that increases the
suitability of the antibody for a particular use (e.g.,
administration to a subject, such as a human subject, or other in
vivo or in vitro uses). Examples of molecules that can be used to
derivatize an antibody include albumin (e.g., human serum albumin)
and polyethylene glycol (PEG). Albumin-linked and PEGylated
derivatives of antibodies can be prepared using techniques well
known in the art. In one embodiment, the antibody is conjugated or
otherwise linked to transthyretin (TTR) or a TTR variant. The TTR
or TTR variant can be chemically modified with, for example, a
chemical selected from the group consisting of dextran,
poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene
glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols and polyvinyl alcohols.
95% Homology
[0162] The present disclosure provides a number of antibodies
structurally characterized by the amino acid sequences of their
variable domain regions. However, the amino acid sequences can
undergo some changes while retaining their high degree of binding
to their specific targets. More specifically, many amino acids in
the variable domain region can be changed with conservative
substitutions and it is predictable that the binding
characteristics of the resulting antibody will not differ from the
binding characteristics of the wild type antibody sequence. There
are many amino acids in an antibody variable domain that do not
directly interact with the antigen or impact antigen binding and
are not critical for determining antibody structure. For example, a
predicted nonessential amino acid residue in any of the disclosed
antibodies is preferably replaced with another amino acid residue
from the same class. Methods of identifying amino acid conservative
substitutions which do not eliminate antigen binding are well-known
in the art (see. e.g., Brummell et al., Biochem. 32: 1180-1187
(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and
Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). Near et
al. Mol. Immunol. 30:369-377, 1993 explains how to impact or not
impact binding through site-directed mutagenesis. Near et al. only
mutated residues that they thought had a high probability of
changing antigen binding. Most had a modest or negative effect on
binding affinity (Near et al. Table 3) and binding to different
forms of digoxin (Near et al. Table 2).
Indications
[0163] In one aspect, the present disclosure provides methods of
treating a subject. The method can, for example, have a generally
salubrious effect on the subject, e.g., it can increase the
subject's expected longevity. Alternatively, the method can, for
example, treat, prevent, cure, relieve, or ameliorate ("treat") a
disease, disorder, condition, or illness ("a condition"). Among the
conditions to be treated are conditions characterized by
inappropriate expression or activity of CXCR3. In some such
conditions, the expression or activity level is too high, and the
treatment comprises administering a CXCR3 antagonist as described
herein. The disorders or conditions are cancer-related. In
particular, those cancers include, but are not limited to, breast,
lung, ovarian and colon carcinoma and various myelomas.
[0164] Specific medical conditions and diseases that are treatable
or preventable with the antigen binding proteins of this disclosure
include various cancers.
Therapeutic Methods and Administration of Antigen Binding
Proteins
[0165] Certain methods provided herein comprise administering a
CXCR3 binding antigen binding protein to a subject, thereby
reducing a CXCR3-induced biological response that plays a role in a
particular condition. In particular embodiments, methods of the
invention involve contacting endogenous CXCR3 with an CXCR3 binding
antigen binding protein, e.g., via administration to a subject or
in an ex vivo procedure.
[0166] The term "treatment" encompasses alleviation or prevention
of at least one symptom or other aspect of a disorder, or reduction
of disease severity, and the like. An antigen binding protein need
not effect a complete cure, or eradicate every symptom or
manifestation of a disease, to constitute a viable therapeutic
agent. As is recognized in the pertinent field, drugs employed as
therapeutic agents may reduce the severity of a given disease
state, but need not abolish every manifestation of the disease to
be regarded as useful therapeutic agents. Similarly, a
prophylactically administered treatment need not be completely
effective in preventing the onset of a condition in order to
constitute a viable prophylactic agent. Simply reducing the impact
of a disease (for example, by reducing the number or severity of
its symptoms, or by increasing the effectiveness of another
treatment, or by producing another beneficial effect), or reducing
the likelihood that the disease will occur or worsen in a subject,
is sufficient. One embodiment of the invention is directed to a
method comprising administering to a patient a CXCR3 antagonist in
an amount and for a time sufficient to induce a sustained
improvement over baseline of an indicator that reflects the
severity of the particular disorder.
[0167] As is understood in the pertinent field, pharmaceutical
compositions comprising the antibodies and fragments thereof of the
disclosure are administered to a subject in a manner appropriate to
the indication. Pharmaceutical compositions may be administered by
any suitable technique, including but not limited to, parenterally,
topically, or by inhalation. If injected, the pharmaceutical
composition can be administered, for example, via intra-articular,
intravenous, intramuscular, intralesional, intraperitoneal or
subcutaneous routes, by bolus injection, or continuous infusion.
Localized administration, e.g. at a site of disease or injury is
contemplated, as are transdermal delivery and sustained release
from implants. Delivery by inhalation includes, for example, nasal
or oral inhalation, use of a nebulizer, inhalation of the
antagonist in aerosol form, and the like. Other alternatives
include eyedrops; oral preparations including pills, syrups,
lozenges or chewing gum; and topical preparations such as lotions,
gels, sprays, and ointments.
[0168] Use of antigen binding proteins in ex vivo procedures also
is contemplated. For example, a patient's blood or other bodily
fluid may be contacted with an antigen binding protein that binds
CXCR3 ex vivo. The antigen binding protein may be bound to a
suitable insoluble matrix or solid support material.
[0169] Advantageously, antigen binding proteins are administered in
the form of a composition comprising one or more additional
components such as a physiologically acceptable carrier, excipient
or diluent. Optionally, the composition additionally comprises one
or more physiologically active agents, for example, a second
inflammation- or immune-inhibiting substance, an anti-angiogenic
substance, an analgesic substance, etc., non-exclusive examples of
which are provided herein. In various particular embodiments, the
composition comprises one, two, three, four, five, or six
physiologically active agents in addition to an CXCR3 binding
antigen binding protein
Combination Therapy
[0170] In another aspect, the present disclosure provides a method
of treating a subject with an CXCR3 inhibiting antigen binding
protein and one or more other treatments. In one embodiment, such a
combination therapy achieves synergy or an additive effect by, for
example, attacking multiple sites or molecular targets in a tumor.
Types of combination therapies that can be used in connection with
the present invention include inhibiting or activating (as
appropriate) multiple nodes in a single disease-related pathway,
multiple pathways in a target cell, and multiple cell types within
a target tissue.
[0171] In another embodiment, a combination therapy method
comprises administering to the subject two, three, four, five, six,
or more of the CXCR3 agonists or antagonists described herein. In
another embodiment, the method comprises administering to the
subject two or more treatments that together inhibit or activate
(directly or indirectly) CXCR3-mediated signal transduction.
Examples of such methods include using combinations of two or more
CXCR3 inhibiting antigen binding proteins, of an CXCR3 inhibiting
antigen binding protein and one or more other therapeutic moiety
having anti-cancer properties (for example, cytotoxic agents,
and/or immunomodulators), or of an CXCR3 inhibiting antigen binding
protein and one or more other treatments (e.g., surgery, or
radiation). Furthermore, one or more anti-CXCR3 antibodies or
antibody derivatives can be used in combination with one or more
molecules or other treatments, wherein the other molecule(s) and/or
treatment(s) do not directly bind to or affect CXCR3, but which
combination is effective for treating or preventing the condition
being treated. In one embodiment, one or more of the molecule(s)
and/or treatment(s) treats or prevents a condition that is caused
by one or more of the other molecule(s) or treatment(s) in the
course of therapy, e.g., nausea, fatigue, alopecia, cachexia,
insomnia, etc. In every case where a combination of molecules
and/or other treatments is used, the individual molecule(s) and/or
treatment(s) can be administered in any order, over any length of
time, which is effective, e.g., simultaneously, consecutively, or
alternately. In one embodiment, the method of treatment comprises
completing a first course of treatment with one molecule or other
treatment before beginning a second course of treatment. The length
of time between the end of the first course of treatment and
beginning of the second course of treatment can be any length of
time that allows the total course of therapy to be effective, e.g.,
seconds, minutes, hours, days, weeks, months, or even years.
[0172] In another embodiment, the method comprises administering
one or more of the CXCR3 antagonists described herein and one or
more other treatments (e.g., a therapeutic or palliative
treatment). Where a method comprises administering more than one
treatment to a subject, it is to be understood that the order,
timing, number, concentration, and volume of the administrations is
limited only by the medical requirements and limitations of the
treatment, i.e., two treatments can be administered to the subject,
e.g., simultaneously, consecutively, alternately, or according to
any other regimen.
Example 1
[0173] This example shows a cellular binding assay to determine the
specific binding of anti-CXCR3 antibodies to CXCR3 expressed on a
stable cell line. CHO-CXCR3 cells were lifted from culture flasks
using non-enzymatic Cell Dissociation Buffer-PBS based (Life
Technologies #13151-014). Cells were resuspended in FACS Buffer (2%
Fetal Bovine Serum in PBS) at 1.times.10.sup.6 cells/ml and 50
.mu.l (0.5.times.10.sup.5 cells) were aliquoted into the wells of a
96-well plate. At the same time, CHO cells expressing an unrelated
protein were treated in the same manner. Aliquots of antibody to
give two different final concentrations, most often 5 and 10
.mu.g/ml, were added to the cells and incubated for 1 hour at
4.degree. C. and then washed twice with FACS Buffer. Cells were
resuspended in 50 .mu.l goat anti-human IgG (.gamma.-chain
specific)-PE conjugated secondary antibody (Southern Biotech
#2040-09) diluted 1:500 in FACS Buffer. Cells were further
incubated for 30 min at 4.degree. C. and then washed 1.times. with
FACS Buffer. The cells were resuspended in a final volume of 30
.mu.l FACS Buffer and analyzed using the Intellicyt Flow Cytometer.
Median fluorescence in the FL-2H channel was determined using
FlowJo software. Binding of each antibody to the CHO-CXCR3 cell
line was compared to binding to a control CHO cell line. The list
of antibodies that showed specific binding to CHO-CXCR3 cells is
shown in Table 1.
TABLE-US-00001 TABLE 1 Antibodies that bind to CHO-CXCR3. Name
Binding to hu-CXCR3 CHO B2 yes C12 yes F7 yes 2A3 yes 3A3 yes 3A11
yes 3C11 yes 4D5 yes 32B12 yes 32D12 yes H1 yes C4 yes 3A7 yes 3A8
yes 3A5 yes
Example 2
[0174] This example shows a cellular binding assay to determine the
EC.sub.50 for anti-CXCR3 antibodies binding to human CXCR3. This
example shows the binding characteristics for these antibodies in
terms of the maximal cell binding and the concentration at which
50% binding saturation (EC.sub.50) is reached. In this example,
CHO-CXCR3 cells were lifted from culture flasks using non-enzymatic
Cell Dissociation Buffer-PBS based (Life Technologies #13151-014).
Cells were resuspended in FACS Buffer (2% Fetal Bovine Serum in
PBS) at 1.times.10.sup.6 cells/ml and 50 .mu.l (0.5.times.10.sup.5
cells) were aliquoted into the wells of a 96-well plate. Plated
cells were spun down and the supernatant discarded. Cells were
resuspended in 30 .mu.l FACS Buffer containing serially diluted
concentrations of antibody in triplicate. Plates were incubated for
1 hr at 4.degree. C. and then washed twice with FACS Buffer. Cells
were resuspended in 50 .mu.l goat anti-human IgG (.gamma.-chain
specific)-PE conjugated secondary antibody (Southern Biotech
#2040-09) diluted 1:500 in FACS Buffer. Cells were further
incubated for 30 min at 4.degree. C. and then washed 1.times. with
FACS Buffer. The cells were resuspended in a final volume of 30
.mu.l FACS Buffer and analyzed using the Intellicyt Flow Cytometer.
Median fluorescence in the FL-2H channel was determined using
FlowJo software and EC.sub.50 value was determined by plotting the
data in GraphPad Prism and analyzing using a variable slope
non-linear regression. The results are shown in Table 2.
TABLE-US-00002 TABLE 2 EC.sub.50 of anti-CXCR3 antibodies to human
CXCR3. Name EC50 (nM), hu-CXCR3 CHO B2 0.7 C12 15 F7 5 2A3 11 3A3
0.5 3A11 1.2 3C11 0.6 4D5 7 32B12 1 32D12 4
Example 3
[0175] This example shows a cellular binding assay to determine the
EC.sub.50 for anti-CXCR3 antibodies binding to murine CXCR3. This
example shows the binding characteristics for these antibodies in
terms of the maximal cell binding and the concentration at which
50% binding saturation (EC.sub.50) is reached. In this example,
murine B16-F10 cells (ATCC, #CRL-6475) were lifted from culture
flasks using non-enzymatic Cell Dissociation Buffer-PBS based (Life
Technologies #13151-014). Cells were harvested, washed and
resuspended in FACS Buffer (2% Fetal Bovine Serum in PBS) at
1.times.10.sup.6 cells/ml and 50 .mu.l (0.5.times.10.sup.5 cells)
were aliquoted into the wells of a 96-well plate. Plated cells were
spun down and the supernatant discarded. Cells were resuspended in
30 .mu.l FACS Buffer serially diluted concentrations of antibody in
triplicate. Plates were incubated for 1 hr at 4.degree. C. and then
washed 2.times. with FACS Buffer. Cells were resuspended in 50
.mu.l goat anti-human IgG (.gamma.-chain specific)-PE conjugated
secondary antibody (Southern Biotech #2040-09) diluted 1:500 in
FACS Buffer. Cells were further incubated for 30 min at 4.degree.
C. and then washed 1.times. with FACS Buffer. The cells were
resuspended in a final volume of 30 .mu.l FACS Buffer and analyzed
using the Intellicyt Flow Cytometer. Median fluorescence in the
FL-2H channel was determined using FlowJo software and the
EC.sub.50 value was determined by plotting the data in GraphPad
Prism and analyzing using a variable slope non-linear regression.
The results are shown in Table 3.
TABLE-US-00003 TABLE 3 EC.sub.50 of anti-CXCR3 antibodies to murine
CXCR3. Name EC50 (nM), mur CXCR3 32B12 25
Example 4
[0176] In this example, CHO-CXCR3 cells were lifted from culture
flasks using non-enzymatic Cell Dissociation Buffer-PBS based (Life
Technologies #13151-014). Cells were resuspended in FACS Buffer (2%
Fetal Bovine Serum in PBS) with sodium azide at 1.times.10.sup.6
cells/ml and 800 .mu.l were aliquoted into a tube for each
antibody. Antibody was added to a final concentration of 5 .mu.g/ml
and incubated at room temperature for 1 h. The samples were placed
on ice and a 105 .mu.l aliquot at various time points was added to
190 .mu.l FACS buffer in a 96-well plate and centrifuged for 3 min.
Supernatant was discarded, the cells were resuspended in 105 .mu.l
of FACS buffer, duplicate aliquots (50 .mu.l) added to another
96-well plate containing 150 .mu.l FACS buffer and incubated at
room temperature. After all of the time points were collected, the
plate was centrifuged for 3 min, supernatant discarded and the
cells resuspended in 50 .mu.l goat anti-human IgG (.gamma.-chain
specific)-PE conjugated secondary antibody (Southern Biotech
#2040-09) diluted 1:500 in FACS Buffer. Cells were incubated for 30
min at 4.degree. C. and then washed 1.times. with FACS Buffer. The
cells were resuspended in a final volume of 30 .mu.l FACS Buffer
and analyzed using the Intellicyt Flow Cytometer. The binding of
clone 32B12 over time is shown in FIG. 1.
Example 5
[0177] This example shows a calcium assay using anti-CXCR3
antibodies. CHO-CXCR3 cells (CHO cells transfected with human CXCR3
gene) were used in calcium assay. Aliquots of 0.1.times.10.sup.5
cells were seeded in 20 .mu.l of growth medium per well of a 384
well plate (#781091, Greiner) and incubated for 20 hours. The
growth medium was F-12K (#21127, Life Technologies) plus 10% FBS. 5
.mu.l of serial diluted antibodies were added into 20 .mu.l cells
and incubated 20 min at room temperature. Then 25 .mu.l of Calcium
4 Assay kit (R8142, Molecular Devices) including 0.5 mM probenecid
(Sigma #P8761-25G) were added to the 25 .mu.l cells pre-incubated
with the antibodies. After 1 h incubation at 37.degree. C. 5%
CO.sub.2 followed by 15 minutes at room temperature, 12.5 .mu.l of
5.times. dose of ligands were added to each well of the plate as
the challenge agonist during detection on the FlexStation3. The
final concentration of each ligand used in the assay was 200 nM of
CXCL9 (#300-26, PeproTech), 164 nM of CXCL10 (#300-12, PeproTech)
and 60 nM of CXCL11 (#300-46, PeproTech). The IC.sub.50 value of
each antibody shown in the table was calculated using Prism5. The
IC.sub.50 of each antibody with each corresponding ligand is shown
in Table 4. Each IC.sub.50 value represents the Mean (n=2). The
control antibody is from R&D (Cat#MAB 160, R&D).
TABLE-US-00004 TABLE 4 IC.sub.50 [nM] of anti-CXCR3 antibodies in a
calcium flux assay using CHO-CXCR3 cells CXCL9 CXCL10 CXCL11
R&D mAb n.d. n.d. 23 32B12 37 7 16 32D12 n.d. n.d. 19 Note:
n.d. = not determined
Example 6
[0178] This example describes chemotaxis assays using activated
human T cells. Primary human T cells activated by anti-CD3 (1
ng/ml) for 2 days were used for prepared. Chemotaxis assays were
set up using a 96-well chemotaxis chamber (ChemoTX; NeuroProbe,
Gaithersburg, Md.) with the 2 compartments separated by a 5-.mu.m
polycarbonate membrane. The assay buffer used for chemotaxis was
RPMI plus 0.5% BSA. Serially-diluted concentrations of antibodies
were pre-incubated with the activated T cells (0.1.times.10.sup.6
cells in 25 .mu.l of the assay buffer) at room temperature for 20
min. The ligands CXCL9 (30 nM), CXCL10 (12.5 nM) and CXCL11 (0.3
nM) were loaded respectively in the wells of plate in the lower
chambers, whereas the cells pre-incubated with antibodies were
loaded on top of membrane. After 2 h incubation at 37.degree. C.,
5% CO.sub.2, migrated cells in the lower chamber were transferred
to an opaque white 96-well plate and incubated with CellTiter GLo
(Cat#G7571, Promega) for 10 min at room temperature. Luminescent
signals generated from the cells in each well were detected by a
fluorescent plate reader (FlexStation3, Molecular Device). The
IC.sub.50 value of each mAb indicated in the table was calculated
by Prism5. The IC.sub.50 of the each mAb against corresponding
ligands is indicated in Table 5. Each IC.sub.50 value represents
Mean (n=2). The control antibody was from R&D (Cat# MAB 160,
R&D).
TABLE-US-00005 TABLE 5 IC.sub.50 [nM] of anti-CXCR3 antibodies in a
chemotaxis assay using activated T cells CXCL9 CXCL10 CXCL11
R&D mAb 1 1.8 0.4 32B12 0.6 0.2 0.3 32D12 2.5 0.03 3.4
TABLE-US-00006 Sequence Listing Heavy chain variable domain region
Light chain variable domain region 32B12
EVQLVQSGGGLVKSGRSLRLSCTVSGFTFGNSAM
SYELTQPPSVSAAPGQKVTISCSGSSSNIGNSYV
SWFRQAPGKGLEWVSSISSSSSYIYYADSVKGRF
SWFQQFPGTAPKLVIYENNKRPSGIPDRFSGSKS
TISRDNAKNSLYLQMNSLRAEDTAVYYCARDLSG
GTSATLGITGLQTGDEADYYCGTWDSSLSGRVFG RGYSYLNYWGQGTLVTVSS SEQ ID NO. 1
GGTKLTVL SEQ ID NO. 2 32D12 EVQLLESGGGLVQPGGSQRLSCAASGFTFSSYPM
LPVLTQPASVSGSPGQPITISCTGTSGNIGSYNY
TWVRQAPGKGLEWVSAISDGGSGTYYADSVKGRF
VSWYQQHPGKAPRLLIYDVSLRPSGISSHFSGSK
TISRDNSKNTLYLQMNSLRAEDTAIYYCARYKMG
SGNTASLTISGLQAEDEAEYFCLSWTSDKTYVFG GSYRGLDDWGQGTLVTVSS SEQ ID NO. 3
SGTKLTVL SEQ ID NO. 4 H1 EVQLLESGGGVVQPGRSLRLSCAASGFTFRYYGM
QAGLTQPPSVSAAPGQKVTISCSGSSSNIGNNYV
HWVRQAPGKGLEWVAAIRNDGSNEYYADSVKGRF
SWYQQLPGTAPKLLIYDNKRRPSGMPDRFSGSKS
TISRDNSKNTLYLEMNSLRAEDTSVYHCARDGFY
GTSATLGITGLQTGDEADYYCGTWDSSLSAVVFG RGSPYYFDSWGQGTLVTVSS SEQ ID NO.
5 GGTKLTVL SEQ ID NO. 6 B7 QVQLVQSGAEVKKPGSSVKVSCKASGGTFKRYSV
DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDG
HWVRQAPGQGLEWVGRIVPFLGLANYAQKFQGRV
NTYLSWFQQRPGQSPRRLIYKVSNRDSGVPDRFS
TITADTSTNTTYMDLRSLRSEDTAVYYCVRSTGY
GSGSGTDFTLKISRVEAEDVGVYFCLQSLQLPIT LDYWGQGTLVTVSS SEQ ID NO. 7
FGQGTKVEIK SEQ ID NO. 8 C12 QVQLVQSGAEVKKPGSSMKVSCKTSGGTFNAYAI
DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDG
TWVRQAPGQGLEWMGRIVPVLGMVSYVQKFQGRL
NTYLNWFQQRPGQSPRRLIYKVSNRESGVPDRFS
TITADKSTSTTYMELSSLTSDDTAVYYCARQTGD
GSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPNT SDHWGPGTLVTVSS SEQ ID NO. 9
FGGXTKLEIK SEQ ID NO. 10 D12 QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYYM
QTVVTQPPSVSGAPGQRVTVSCTGSSSNIGSRYA
HWVRQAPGQGLEWMGIINPSAGSTSYAQKFQGRV
VNWYQQLPGRAPKLLIYGNTNRPSGVPDRFSASK
TMTRDTSTSTVYMELSSLGSEDTAVYYCASRSFG
SGTSASLAISGLQAEDEADYYCQSFDSSLRGYVF NDGVFDIWGQGTMVTVSS SEQ ID NO. 11
GTGTKVTVL SEQ ID NO. 12 E1 EVQLVESGGGVVQPGRSLRLSCAASEFTFSSYTI
SDVVMTQTPSTLSASVGDRVTITCRASQSIKTWL
HWVRQAPGKGLEWVGRIHPKSEGGTTDYAAPVKG
AWYQQRPGKAPKRLIYAASSLQSGVPSRFSGSGS
RFLISRDDSENTMYLQMNSLQTEDTAVYYCATYG
GTEFTLTISSLQPEDFATYYCVQHNSYPITFGQG SGSHWIWGQGTLVTVSS SEQ ID NO. 13
TRLEIK SEQ ID NO. 14 G12 EVQLVESGGGVVQPGKSLRLSCAASGFTFSSYGM
DIVMTQTPLSLPVTLGQPASLSCRSSQSLAHRDG
HWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRF
NTYLSWFQQRPGQSPRRLIYKVSNRDSGVPDRFS
TISRDNSKNTLYLQMNSLRAEDTAVYYCAYGGFS
GSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPT HWGQGTLVTVSS SEQ ID NO. 15
FGGGTKVEIK SEQ ID NO. 16 3A3 QVQLVQSGAEVKKPGSSVKVSCKASGGTFNRYAF
DIVMTQTPLSLPVTLGQPASISCRSSQSLVHNDG
SWVRQAPGQGPELMGRIMPMFDIASYAQKFQGRL
NTYLNWFHQRPGQSPRRLIYKVSNRDFGVPDRFS
TITADKSTSTAYMELSSLRSDDTAVYYCTQVSGD
GSGSGTDFTLRISRVEAEDVGVYYCMQGSHWPLT SDYWGPGTLVTVSS SEQ ID NO. 17
FGGGTKVEIK SEQ ID NO. 18 3A5 QVQLVQSGAEVKKPGSSVKVACKASGGTFSHSAI
DVVMTQTPLSLPVTLGQPASISCRSSQSLVYRDG
SWVRQAPGQGLEWIGRTIPTLEMASYQAKFQGRV
NTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFS
TISADKSTRTGYMELRDLRSEDTAVYYCSTQTPS
GSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPIT YTDHWGQGTLVTVSS SEQ ID NO 19
FGQGTKVEIK SEQ ID NO. 20 3A11 QVQLVQSGGGLVKPGGSLRLSCAASGFTFTSSGM
SYVLTQPPSVSKGLRQTATLTCTGNINNVGNVGA
NWVRQAPGKGLEWVAIYFPNGKTLYPESVKGRFI
TWLQQHQGHPPKLLSDRNNNRPSGISERFSASRS
ITRDNAEKSLFLEMNNLRAEDMGVYYCAIDLNWG
GNAASLTITGLQPEDEADYYCSAWDTSLGAWVFG SGYWGQGTLVTVSS SEQ ID NO 21
GGTKLTVL SEQ ID NO. 22 3B12 EVQLVESGGGVVQPGRSLRLSCAASGFTFSHYGM
QSVLTQPPSVSKDLRQTATLTCTGNSNNVGDKGA
HWVRQAPGKGLEWVAVISYDGSKKSYADSVKGRF
AWLQQHQGHPPKLLSYRNNNRPSGISERLSASRS
TISRDNSKNTLYLQMNSLRADDTAVYYCATSAGG
GNTASLTITGLQPDDEADYYCSAWDSSLSAWVFG GGYWGQGTLVTVSS SEQ ID NO 23
GGKTLTVL SEQ ID NO. 24 3C11 EVQLVESGGGLVQPGGSLRLSCSASGFTFSSYAM
DIQLTQSPSSLSASVGDRVTITCRASQGIGTYLA
HWVRQAPGKGLEYVSAISSNGGSTYYADSVKGRF
WFQQKPGKVPKPLIYAASTLQSGVPSRFSGSGSG
TISRDNSKNTLYLQMSSLRAEDTAVYYCVKSIAA
ADFTLTISSLQPEDVATYYCQKYNSVPQTFGQGT AGTQYYYYYMDVWGKGTTVTVSS KVEIK
SEQ ID NO. 26 SEQ ID NO 25 4C3 QVQLVESGGGLVQPGRSLRLSCIISGFTFGDYTM
AIQLTQSPLALPVPLGQPASISVRASQSLVYSNG
SWFRQAPGKGLEWVGFIRSKAYGGTTQYAASVKG
NTYLNWFQLRPGQSPRRLIYKVSRRDSGVPDRFS
RFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRGD
GSGSGDTDFTLKISRVEAEDVGVYYCMQATQWPI YGDFPWYFDLWGRGTLVTVSS
TFGQGTRLEIK SEQ ID NO. 28 SEQ ID NO 27 B12
QMQLVQSGAEVKKPGSSVKVSCKASGGTFNRYAF
DVVMTQSPLSLPVTGQPASISCRSTESLVHSDGN
SWVRQAPGQGPELMGRIMPMFDIASYAQKGQGRL
TYLNWFHQRPGQSPRRLIYKISNRDSGVPDRFSG
TITADKSTSTAYMELSSLRSDDTAVYYCTQVSGD
SGSGTDFTLKISRVEAEDVGVYYCFQGHTWPPTF SDYWGPGTTVTVSS SEQ ID NO. 29
GQGTKVEIK SEQ ID NO. 30 C4 QVLQVQSGAEVKKPGSSVKVACKASGGTFSHSAI
EIVLTQSPLSLPVTLGQPASISCNSSQSLLHRDG
SWVRQAPGQGLEWIGRTIPTLEMASYAQKFQGRV
HTYLHWFQQRPGQSPRRLIYKVSNRDSGVPDKFS
TISADKSTRTGYMELRDLRSEDTAVYYCSTQTPS
GSGSGTEFTLKISRVEAEDVGVYYCMQGTHWPLA YTDHWGQGTLVTVSS SEQ ID NO. 31
FGGGTKLEIK SEQ ID NO. 32 F7 QVQLVQSGAEVKKPGSSVKVACKASGGTFSHAIS
DIVMTQTPLSLPVTLGQPASISCRSNQSLVHSDG
WVRQAPGQGLEWIGRTIPTLEMASYAQKFQGRVT
NTYLYWFQQRPGQSPRRLIYKVSNRDSGVPDRFS
ISADKSTRTGYMELRDLRSEDTAVYYCSTQTPSY
GSGSGTDFTLKINRVEAEDVGVYYCMQGTHWPNT TDHWGQGTLVTVSS SEQ ID NO. 31
FGQGTKLEIK SEQ ID NO. 33 2A3 QVQLVQSGAEVKKPGSSVKVACKASGGTFSHSAI
DIVMTQTPLSLPVTLGQPASISVRSSQSLVHSDG
SWVRQAPGQGLEWIGRTIPTLEMASYAQKFQGRV
NTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFS
TISADKSTRTGYMELRDLRSEDTAVYYCSTQTPS
GSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPLT YTDHWGQGTLVTVSS SEQ ID NO. 31
FGGGTKVEIK SEQ ID NO. 34 3A7 QVQLVQSGAEVKKPGSSVKVACKASGGTFSHSAI
DVVMTQSPVSLPVTLGQPASISCRSSQSLVHSDG
SWVRQAPGQGLEWIGRTIPTLEMASYAQKFQGRV
NTYFNWFQQRPGQSPRRLIYKVSNRDSGVPDRFS
TISADKSTRTGYMELRDLRSEDTAVYYCSTQTPS
GSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPIT YTDHWGQGTLVTVSS SEQ ID NO. 31
FGQGTRLEIK SEQ ID NO. 35 3A8 QVQLVQSGAEVKKPGSSVKVACKASGGTFSHSAI
DIVMTQTPLSLPVTGQPAAISCRSSQSLVYSDGN
SWVRQAPGQGLEWIGRTIPTLEMASYAQKFQGRV
TYLNWFHQRPGQSPRRLIYKVSNRDSGVPDRFSG
TISADKSTRTGYMELRDLRSEDTAVYYCSTQTPS
SGSGTDFTLKISRVEAEDVGVYYCMQGTHWPLTF YTDHWGQGTLVTVSS SEQ ID NO. 31
GGGTKLEIK SEQ ID NO. 36 4D5 QVLQVQSGAEVKKPGSSVKVACKASGGTFSHSAI
DIVMTQSPLSLPVTGQPASISCRSSHTLVHSDGT
SWVRQAPGQGLEWIGRTIPTLEMASYAQKFQGRV
TYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSG
TISADKSTRTGYMELRDLRSEDTAVYYCSTQTPS
SGSGTDFTLKISRVEAEDVGVYYCMQGTHWPLTF YTDYWGQGTLVTVSS SEQ ID NO. 31
GGGTKVEIK SEQ ID NO. 37
Sequence CWU 1
1
421121PRTHomo Sapiens 1Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu
Val Lys Ser Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser
Gly Phe Thr Phe Gly Asn Ser 20 25 30 Ala Met Ser Trp Phe Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser
Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Leu Ser Gly Arg Gly Tyr Ser Tyr Leu Asn Tyr Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 2110PRTHomo
Sapiens 2Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro
Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
Ile Gly Asn Ser 20 25 30 Tyr Val Ser Trp Phe Gln Gln Phe Pro Gly
Thr Ala Pro Lys Leu Val 35 40 45 Ile Tyr Glu Asn Asn Lys Arg Pro
Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu
Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Gly
Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110
3121PRTHomo Sapiens 3Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Gln Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Asp
Gly Gly Ser Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90
95 Ala Arg Tyr Lys Met Gly Gly Ser Tyr Arg Gly Leu Asp Asp Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 4110PRTHomo
Sapiens 4Leu Pro Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro
Gly Gln 1 5 10 15 Pro Ile Thr Ile Ser Cys Thr Gly Thr Ser Gly Asn
Ile Gly Ser Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro
Gly Lys Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Asp Val Ser Leu Arg
Pro Ser Gly Ile Ser Ser His Phe 50 55 60 Ser Gly Ser Lys Ser Gly
Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp
Glu Ala Glu Tyr Phe Cys Leu Ser Trp Thr Ser Asp 85 90 95 Lys Thr
Tyr Val Phe Gly Ser Gly Thr Lys Leu Thr Val Leu 100 105 110
5122PRTHomo Sapiens 5Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala Ile Arg Asn
Asp Gly Ser Asn Glu Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ser Val Tyr His Cys 85 90
95 Ala Arg Asp Gly Phe Tyr Arg Gly Ser Pro Tyr Tyr Phe Asp Ser Trp
100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
6110PRTHomo Sapiens 6Gln Ala Gly Leu Thr Gln Pro Pro Ser Val Ser
Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln
Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Lys
Arg Arg Pro Ser Gly Met Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys
Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr
Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90
95 Ser Ala Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110 7116PRTHomo Sapiens 7Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Gly Thr Phe Lys Arg Tyr 20 25 30 Ser Val His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Val
Pro Phe Leu Gly Leu Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Thr Tyr 65 70 75 80
Met Asp Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Val Arg Ser Thr Gly Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ser 115 8112PRTHomo Sapiens 8Asp Ile
Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20
25 30 Asp Gly Asn Thr Tyr Leu Ser Trp Phe Gln Gln Arg Pro Gly Gln
Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Phe Cys Leu Gln Ser 85 90 95 Leu Gln Leu Pro Ile Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 9116PRTHomo Sapiens
9Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Met Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Asn Ala
Tyr 20 25 30 Ala Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Arg Ile Val Pro Val Leu Gly Met Val Ser
Tyr Val Gln Lys Phe 50 55 60 Gln Gly Arg Leu Thr Ile Thr Ala Asp
Lys Ser Thr Ser Thr Thr Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Thr Gly
Asp Ser Asp His Trp Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 10112PRTHomo Sapiensmisc_feature(106)..(106)Xaa can be any
naturally occurring amino acid 10Asp Ile Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly Asn Thr
Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg
Arg Leu Ile Tyr Lys Val Ser Asn Arg Glu Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met
Gln Gly 85 90 95 Thr His Trp Pro Asn Thr Phe Gly Gly Xaa Thr Lys
Leu Glu Ile Lys 100 105 110 11120PRTHomo Sapiens 11Gln Met Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35
40 45 Gly Ile Ile Asn Pro Ser Ala Gly Ser Thr Ser Tyr Ala Gln Lys
Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser
Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Gly Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Arg Ser Phe Gly Asn Asp Gly
Val Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser
Ser 115 120 12111PRTHomo Sapiens 12Gln Thr Val Val Thr Gln Pro Pro
Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Val Ser Cys
Thr Gly Ser Ser Ser Asn Ile Gly Ser Arg 20 25 30 Tyr Ala Val Asn
Trp Tyr Gln Gln Leu Pro Gly Arg Ala Pro Lys Leu 35 40 45 Leu Ile
Tyr Gly Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60
Ser Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65
70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Phe Asp
Ser Ser 85 90 95 Leu Arg Gly Tyr Val Phe Gly Thr Gly Thr Lys Val
Thr Val Leu 100 105 110 13119PRTHomo Sapiens 13Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Ser Ser Tyr 20 25 30 Thr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Gly Arg Ile His Pro Lys Ser Glu Gly Gly Thr Thr Asp Tyr Ala Ala
50 55 60 Pro Val Lys Gly Arg Phe Leu Ile Ser Arg Asp Asp Ser Glu
Asn Thr 65 70 75 80 Met Tyr Leu Gln Met Asn Ser Leu Gln Thr Glu Asp
Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Thr Tyr Gly Ser Gly Ser His
Trp Ile Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
14107PRTHomo Sapiens 14Asp Val Val Met Thr Gln Thr Pro Ser Thr Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Lys Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Arg
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Val Gln His Asn Ser Tyr Pro Ile 85 90
95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 15114PRTHomo
Sapiens 15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Tyr
Gly Gly Phe Ser His Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110
Ser Ser 16112PRTHomo Sapiens 16Asp Ile Val Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Leu Ser Cys
Arg Ser Ser Gln Ser Leu Ala His Arg 20 25 30 Asp Gly Asn Thr Tyr
Leu Ser Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg
Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
Gly 85 90 95 Thr His Trp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105 110 17116PRTHomo Sapiens 17Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Arg Tyr 20 25 30 Ala
Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Pro Glu Leu Met 35 40
45 Gly Arg Ile Met Pro Met Phe Asp Ile Ala Ser Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Leu Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Gln Val Ser Gly Asp Ser Asp Tyr Trp
Gly Pro Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
18112PRTHomo Sapiens 18Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Val His Asn 20 25 30 Asp Gly Asn Thr Tyr Leu Asn
Trp Phe His Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile
Tyr Lys Val Ser Asn Arg Asp Phe Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90
95 Ser His Trp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 19117PRTHomo Sapiens 19Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ala Cys
Lys Ala Ser Gly Gly Thr Phe Ser His Ser 20 25 30 Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg
Thr Ile Pro Thr Leu Glu Met Ala Ser Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Thr Arg Thr Gly Tyr 65
70 75 80 Met Glu Leu Arg Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ser Thr Gln Thr Pro Ser Tyr Thr Asp His Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 20112PRTHomo
Sapiens 20Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val Tyr Arg 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln
Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val
Ser Asn Arg Asp Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Gly 85 90 95 Thr His Trp Pro Ile Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 110 21116PRTHomo Sapiens 21Gln Val
Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Ser 20
25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Tyr Ile Phe Pro Asn Gly Lys Thr Leu Tyr Pro Glu
Ser Val Lys 50 55 60 Gly Arg Phe Ile Ile Thr Arg Asp Asn Ala Glu
Lys Ser Leu Phe Leu 65 70 75 80 Glu Met Asn Asn Leu Arg Ala Glu Asp
Met Gly Val Tyr Tyr Cys Ala 85 90 95 Ile Asp Leu Asn Trp Gly Ser
Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
22110PRTHomo Sapiens 22Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser
Lys Gly Leu Arg Gln 1 5 10 15 Thr Ala Thr Leu Thr Cys Thr Gly Asn
Ile Asn Asn Val Gly Asn Val 20 25 30 Gly Ala Thr Trp Leu Gln Gln
His Gln Gly His Pro Pro Lys Leu Leu 35 40 45 Ser Asp Arg Asn Asn
Asn Arg Pro Ser Gly Ile Ser Glu Arg Phe Ser 50 55 60 Ala Ser Arg
Ser Gly Asn Ala Ala Ser Leu Thr Ile Thr Gly Leu Gln 65 70 75 80 Pro
Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ala Trp Asp Thr Ser Leu 85 90
95 Gly Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
110 23116PRTHomo Sapiens 23Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Lys Lys Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Thr Ser Ala Gly Gly Gly Gly Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ser 115 24110PRTHomo Sapiens 24Gln Ser
Val Leu Thr Gln Pro Pro Ser Val Ser Lys Asp Leu Arg Gln 1 5 10 15
Thr Ala Thr Leu Thr Cys Thr Gly Asn Ser Asn Asn Val Gly Asp Lys 20
25 30 Gly Ala Ala Trp Leu Gln Gln His Gln Gly His Pro Pro Lys Leu
Leu 35 40 45 Ser Tyr Arg Asn Asn Asn Arg Pro Ser Gly Ile Ser Glu
Arg Leu Ser 50 55 60 Ala Ser Arg Ser Gly Asn Thr Ala Ser Leu Thr
Ile Thr Gly Leu Gln 65 70 75 80 Pro Asp Asp Glu Ala Asp Tyr Tyr Cys
Ser Ala Trp Asp Ser Ser Leu 85 90 95 Ser Ala Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu 100 105 110 25125PRTHomo Sapiens 25Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Tyr Val 35 40 45 Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Lys Ser Ile Ala Ala
Ala Gly Thr Gln Tyr Tyr Tyr Tyr Tyr Met 100 105 110 Asp Val Trp Gly
Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 125 26107PRTHomo
Sapiens 26Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Gly Thr Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys
Val Pro Lys Pro Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala
Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Val Pro Gln 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 27123PRTHomo Sapiens
27Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1
5 10 15 Ser Leu Arg Leu Ser Cys Ile Ile Ser Gly Phe Thr Phe Gly Asp
Tyr 20 25 30 Thr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Gly Phe Ile Arg Ser Lys Ala Tyr Gly Gly Thr
Thr Gln Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser
Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Gly
Asp Tyr Gly Asp Phe Pro Trp Tyr Phe Asp Leu 100 105 110 Trp Gly Arg
Gly Thr Leu Val Thr Val Ser Ser 115 120 28112PRTHomo Sapiens 28Ala
Ile Gln Leu Thr Gln Ser Pro Leu Ala Leu Pro Val Pro Leu Gly 1 5 10
15 Gln Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Ser Leu Val Tyr Ser
20 25 30 Asn Gly Asn Thr Tyr Leu Asn Trp Phe Gln Leu Arg Pro Gly
Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Arg Arg Asp
Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Asp Thr
Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Thr Gln Trp Pro Ile Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 29116PRTHomo
Sapiens 29Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr
Phe Asn Arg Tyr 20 25 30 Ala Phe Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Pro Glu Leu Met 35 40 45 Gly Arg Ile Met Pro Met Phe Asp
Ile Ala Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Leu Thr Ile
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Gln
Val Ser Gly Asp Ser Asp Tyr Trp Gly Pro Gly Thr Thr Val 100 105 110
Thr Val Ser Ser 115 30112PRTHomo Sapiens 30Asp Val Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser
Ile Ser Cys Arg Ser Thr Glu Ser Leu Val His Ser 20 25 30 Asp Gly
Asn Thr Tyr Leu Asn Trp Phe His Gln Arg Pro Gly Gln Ser 35 40 45
Pro Arg Arg Leu Ile Tyr Lys Ile Ser Asn Arg Asp Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Phe Gln Gly 85 90 95 Thr His Trp Pro Pro Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 110 31117PRTHomo Sapiens 31Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser
Val Lys Val Ala Cys Lys Ala Ser Gly Gly Thr Phe Ser His Ser 20 25
30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45 Gly Arg Thr Ile Pro Thr Leu Glu Met Ala Ser Tyr Ala Gln
Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Thr
Arg Thr Gly Tyr 65 70 75 80 Met Glu Leu Arg Asp Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Thr Gln Thr Pro Ser Tyr Thr
Asp His Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
32112PRTHomo Sapiens 32Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Asn Ser
Ser Gln Ser Leu Leu His Arg 20 25 30 Asp Gly His Thr Tyr Leu His
Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile
Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Lys Phe
Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90
95 Thr His Trp Pro Leu Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110 33117PRTHomo Sapiens 33Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ala Cys
Lys Ala Ser Gly Gly Thr Phe Ser His Ser 20 25 30 Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg
Thr Ile Pro Thr Leu Glu Met Ala Ser Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Ile Ser Ala Asp Lys Ser Thr Arg Thr Gly Tyr 65
70 75 80 Met Glu Leu Arg Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ser Thr Gln Thr Pro Ser Tyr Thr Asp His Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 34112PRTHomo
Sapiens 34Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Asn Gln Ser
Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Tyr Trp Phe Gln
Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val
Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Asn Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His
Trp Pro Asn Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
35117PRTHomo Sapiens 35Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ala Cys Lys Ala Ser
Gly Gly Thr Phe Ser His Ser 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Thr Ile Pro
Thr Leu Glu Met Ala Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Ser Ala Asp Lys Ser Thr Arg Thr Gly Tyr 65 70 75 80 Met
Glu Leu Arg Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ser Thr Gln Thr Pro Ser Tyr Thr Asp His Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 36112PRTHomo Sapiens 36Asp Ile
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20
25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln
Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 37117PRTHomo
Sapiens 37Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ala Cys Lys Ala Ser Gly Gly Thr
Phe Ser His Ser 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Thr Ile Pro Thr Leu Glu
Met Ala Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile
Ser Ala Asp Lys Ser Thr Arg Thr Gly Tyr 65 70 75 80 Met Glu Leu Arg
Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Thr
Gln Thr Pro Ser Tyr Thr Asp His Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 38112PRTHomo Sapiens 38Asp Val Val Met Thr
Gln Ser Pro Val Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp
Gly Asn Thr Tyr Phe Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40
45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Met Gln Gly 85 90 95 Thr His Trp Pro Ile Thr Phe Gly Gln Gly
Thr Arg Leu Glu Ile Lys 100 105 110 39117PRTHomo Sapiens 39Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15
Ser Val Lys Val Ala Cys Lys Ala Ser Gly Gly Thr Phe Ser His Ser 20
25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Arg Thr Ile Pro Thr Leu Glu Met Ala Ser Tyr Ala
Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ser Ala Asp Lys Ser
Thr Arg Thr Gly Tyr 65 70 75 80 Met Glu Leu Arg Asp Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Thr Gln Thr Pro Ser Tyr
Thr Asp His Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
115 40112PRTHomo Sapiens
40Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1
5 10 15 Gln Pro Ala Ala Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr
Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe His Gln Arg Pro
Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg
Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Leu
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110
41117PRTHomo Sapiens 41Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ala Cys Lys Ala Ser
Gly Gly Thr Phe Ser His Ser 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Thr Ile Pro
Thr Leu Glu Met Ala Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Ser Ala Asp Lys Ser Thr Arg Thr Gly Tyr 65 70 75 80 Met
Glu Leu Arg Asp Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ser Thr Gln Thr Pro Ser Tyr Thr Asp His Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 42112PRTHomo Sapiens 42Asp Ile
Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser His Thr Leu Val His Ser 20
25 30 Asp Gly Thr Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln
Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Leu Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110
* * * * *
References