U.S. patent application number 14/593950 was filed with the patent office on 2015-07-23 for novel anti- alpha5 beta1 antibodies and uses thereof.
The applicant listed for this patent is GENENTECH, INC.. Invention is credited to Wei-Ching LIANG, Gregory D. PLOWMAN, Yan WU, Weilan YE.
Application Number | 20150203581 14/593950 |
Document ID | / |
Family ID | 42184446 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150203581 |
Kind Code |
A1 |
LIANG; Wei-Ching ; et
al. |
July 23, 2015 |
NOVEL ANTI- ALPHA5 BETA1 ANTIBODIES AND USES THEREOF
Abstract
The present invention provides new anti-.alpha.5.beta.1
antibodies, compositions and kits comprising the antibodies, and
methods of making and using the antibodies.
Inventors: |
LIANG; Wei-Ching; (South San
Francisco, CA) ; PLOWMAN; Gregory D.; (South San
Francisco, CA) ; WU; Yan; (South San Francisco,
CA) ; YE; Weilan; (South San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Family ID: |
42184446 |
Appl. No.: |
14/593950 |
Filed: |
January 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13335632 |
Dec 22, 2011 |
8962275 |
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14593950 |
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12729999 |
Mar 23, 2010 |
8124740 |
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13335632 |
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61163241 |
Mar 25, 2009 |
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Current U.S.
Class: |
424/133.1 ;
424/172.1; 435/188; 435/334; 435/69.6; 435/7.21; 436/501;
530/387.3; 530/389.1; 530/391.3; 530/391.7; 536/23.53 |
Current CPC
Class: |
A61P 13/12 20180101;
A61K 2039/505 20130101; Y02A 50/412 20180101; C07K 2317/31
20130101; A61P 37/06 20180101; Y02A 50/386 20180101; A61P 17/06
20180101; A61P 37/00 20180101; A61P 37/02 20180101; A61P 27/00
20180101; A61K 47/6835 20170801; C07K 2317/92 20130101; C07K
16/2842 20130101; Y02A 50/30 20180101; C07K 16/22 20130101; C07K
2317/565 20130101; A61K 2039/507 20130101; A61P 35/00 20180101;
A61P 1/00 20180101; C07K 2317/56 20130101; Y02A 50/41 20180101;
A61P 19/02 20180101; A61P 9/00 20180101; C07K 2317/24 20130101;
A61P 27/02 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/22 20060101 C07K016/22; A61K 47/48 20060101
A61K047/48 |
Claims
1. An anti-.beta.5.beta.1 antibody comprising TABLE-US-00003 (SEQ
ID NO: 15) a VL domain comprising a CDR-L1 comprising TL-S/
T-S/P/T-Q/N-H-F/S-T/I-Y-K/T-I-G/D/S; (SEQ ID NO: 16) a CDR-L2
comprising L/I-N/T-S-D/H/S-G/S-S/L/T-H/ Y-N/K/Q/I-K/T-G/A-D/S/V;
(SEQ ID NO: 17) a CDR-L3 comprising G/A-S/A/Y-S/Y-Y-S/A/Y-S/Y/T-
GY-V/I; and (SEQ ID NO: 18) a VH domain comprising a CDR-H1
comprising GFTFS- N/A-RW-I/V-Y; (SEQ ID NO: 19) a CDR-H2 comprising
GIKTKP-N/A/T-I/R-YAT-E/Q- YADSVKG; and (SEQ ID NO: 20) a CDR-H3
comprising L/V-TG-M/K-R/K-YFDY.
2. The antibody of claim 1, wherein the VL domain comprises a
CDR-L1, CDR-L2, and CDR-L3, each comprising a sequence set forth in
FIG. 3 and the VH domain comprises a CDR-H1, CDR-H2, and CDR-H3,
each comprising a sequence set forth in FIG. 3.
3. The anti-.alpha..sub.5.beta..sub.1 antibody of claim 1, wherein
the VL domain comprises any one of SEQ ID NOS:3-8 and the VH domain
comprises any one of SEQ ID NOS: 11-14.
4. The antibody of claim 1, wherein the VL domain comprises SEQ ID
NO:8 and the VH domain comprises SEQ ID NO:14.
5. The antibody of claim 1, which is a human, humanized, chimeric,
bispecifc or multispecific antibody.
6. The antibody of claim 1, which is an antibody fragment that
binds .alpha.5.beta.1.
7. The antibody of claim 1, which is a full length IgG1
antibody.
8. The antibody according to claim 7, wherein the antibody
comprises an Fc portion comprising a N297A substitution.
9. An isolated nucleic acid molecule encoding the antibody of claim
1.
10. A host cell comprising the nucleic acid of claim 9.
11. A method of producing an anti-.beta.5.beta.1 antibody
comprising culturing the host cell of claim 10 so that the antibody
is produced.
12. An immunoconjugate comprising the antibody of any one of claim
1 and a cytotoxic agent.
13. A pharmaceutical composition comprising the antibody of claim 1
and a pharmaceutically acceptable carrier.
14. The pharmaceutical composition of claim 13, further comprising
a VEGF antagonist.
15. The pharmaceutical composition of claim 14, wherein the VEGF
antagonist is an anti-VEGF antibody.
16. The pharmaceutical composition of claim 15, wherein the
anti-VEGF antibody is bevacizumab.
17. The antibody of claim 1 further comprising a detectable
label.
18. The antibody of claim 17, wherein the detectable label is a
member selected from the group consisting of: a radioisotope, a
fluorescent dye, and an enzyme.
19. The antibody of claim 1 for use as a medicament.
20. The antibody of claim 1 any one for use in treating a disease
or disorder involving abnormal angiogenesis and/or vascular
permeability or leakage.
21. The antibody of claim 20, wherein the disease is a member
selected from the group consisting of: cancer, ocular disease,
autoimmune disease.
22. The antibody of claim 1 any one for use in inhibiting
angiogenesis.
23. A method of detecting .alpha.5.beta.1 protein in a sample
suspected of containing the .alpha.5.beta.1 protein, the method
comprising (a) contacting the antibody of claim 17 with the sample;
and (b) detecting formation of a complex between the
anti-.beta.5.beta.1 antibody and the .alpha.5.beta.1 protein.
24. The method of claim 23, wherein the sample is from a patient
diagnosed with a disease involving abnormal angiogenesis, abnormal
vascular permeability, and/or vascular leakage.
25. A method for treating an individual having a disease or
disorder involving abnormal angiogenesis and/or vascular
permeability or leakage, the method comprising administering to the
individual an effective amount of an antibody of claim 1.
26. The method of claim 25, wherein the disease or disorder is a
member selected from the group consisting of: cancer, an ocular
disease, and an autoimmune disease.
27. The method of claim 26, wherein the cancer is a selected from
the group consisting of colorectal cancer, lung cancer, breast
cancer, renal cell cancer, and glioblastoma.
28. The method of claim 25, wherein the disease or disorder is a
member selected from the group consisting of retinopathy,
age-related macular degeneration, corneal neovascularization,
corneal graft neovascularization, retinal neovascularization, and
neovascular glaucoma.
29. The method of claim 25, further comprising administering an
effective amount of a VEGF antagonist to the individual.
30. The method of claim 29, wherein the VEGF antagonist is an
anti-VEGF antibody.
31. The method of claim 30, wherein the anti-VEGF antibody is
bevacizumab.
32. The method of claim 29, wherein the VEGF antagonist is
administered prior to the anti-.beta.5.beta.1 antibody.
33. The method of claim 29, wherein the VEGF antagonist and the
anti-.alpha.5.beta.1 antibody are administered simultaneously.
34. The method of claim 29, where the subject is first treated with
the VEGF antagonist until the subject is unresponsive to VEGF
antagonist treatment and then the subject is treated with the
anti-.beta.5.beta.1 antibody.
35. The method of claim 29, further comprising administering a
therapeutic agent selected from the group consisting of an
anti-neoplastic agent, a chemotherapeutic agent, a growth
inhibitory agent and a cytotoxic agent.
36. A kit for detecting .alpha.5.beta.1 in a subject who has been
treated with a VEGF antagonist, said kit comprising (a) an
anti-.beta.5.beta.1 antibody of claim 1; and (b) instructions for
use.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/729,999, filed Mar. 23, 2010, which claims the benefit of
U.S. Provisional Patent Application No. 61/163,241, filed Mar. 25,
2009, the disclosures of which is incorporated by reference in
their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to novel
.alpha..sub.5.beta..sub.1 antibodies, compositions and kits
comprising the antibodies and methods for using the antibodies.
BACKGROUND OF THE INVENTION
[0003] .alpha.5.beta.1 integrin is a cell membrane glycoprotein
that mediates cell-ECM interactions through its major ligand,
fibronectin. .beta.5.beta.1 integrin plays a role in cell
migration, differentiation and survivial. Levels of .alpha.5.beta.1
integrin are elevated in tumor vascular endothelium (e.g., gastric,
colorectal hepatocellular, uterocervial, and breast carcinomas) and
other angiogenic vessels. .alpha.5.beta.1 integrin modulates mural
cell association with endothelial cells and the assembly of the
endothelial extracellular matrix during angiogenesis. As such,
.alpha.5.beta.1 integrin is a useful target for inhibition of
angiogenesis and sensitization of cells to the effects of a VEGF
antagonist.
[0004] Thus, there is a need in the art for compositions and
methods for targeting .beta.5.beta.1 integrin. The present
invention meets this and other needs.
SUMMARY OF THE INVENTION
[0005] The present invention provides novel
anti-.alpha..sub.5.beta..sub.1 antibodies derived from the
monoclonal antibody 18C12, kits and compositions comprising the
novel anti-.alpha..sub.5.beta..sub.1 antibodies, and methods of
making and/or using them.
[0006] One embodiment of the invention provides an
anti-.alpha..sub.5.beta..sub.1 antibody comprising a VL domain
comprising a CDR-L1 comprising
TL-S/T-S/P/T-Q/N-H-F/S-T/I-Y-K/T-I-G/D/S (SEQ ID NO:15); a CDR-L2
comprising L/I-N/T-S-D/H/S-G/S-S/L/T-H/Y-N/K/Q/I-K/T-G/A-D/S/V (SEQ
ID NO:16); a CDR-L3 comprising G/A-S/A/Y-S/Y-Y-S/A/Y-S/Y/T-GY-V/I
(SEQ ID NO:17); and a VH domain comprising a CDR-H1 comprising
GFTFS-N/A-RW-1/V-Y (SEQ ID NO:18); a CDR-H2 comprising
GIKTKP-N/A/T-I/R-YAT-E/Q-YADSVKG (SEQ ID NO:19); and a CDR-H3
comprising L/V-TG-M/K-R/K-YFDY (SEQ ID NO:20). In some embodiments,
the anti-.alpha..sub.5.beta..sub.1 antibody comprises a VL domain
comprising a CDR-L1, CDR-L2, and CDR-L3 each comprising a sequence
set forth in FIG. 3 and a VH domain comprising a CDR-H1, CDR-H2,
and CDR-H3 each comprising a sequence set forth in FIG. 3, i.e., a
VL domain comprising a CDR-L1 comprising a sequence set forth in
SEQ ID NO: 21, 22, 23, or 24, a CDR-L2 comprising a sequence set
forth in SEQ ID NO: 25, 26, 27, or 28, and a CDR-L3 comprising a
sequence set forth in SEQ ID NO: 29, 30, 31, 32 or 33; and a VH
domain comprising a CDR-H1 comprising a sequence set forth in SEQ
ID NO: 34 or 35, a CDR-H2 comprising a sequence set forth in SEQ ID
NO: 36 or 37, and a CDR-H3 comprising a sequence set forth in SEQ
ID NO: 38, 39, or 40. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody comprises a VL domain
comprising any one of SEQ ID NOS:3-8 and a VH domain comprising any
one of SEQ ID NOS:11-14. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody comprises a VL domain
comprising SEQ ID NO:4 and a VH domain comprising SEQ ID NO:11. In
some embodiments, the anti-.alpha..sub.5.beta..sub.1 antibody
comprises a VL domain comprising SEQ ID NO:5 and a VH domain
comprising SEQ ID NO:12. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody comprises a VL domain
comprising SEQ ID NO:6 and a VH domain comprising SEQ ID NO:13. In
some embodiments, the anti-.alpha..sub.5.beta..sub.1 antibody
comprises a VL domain comprising SEQ ID NO:7 and a VH domain
comprising SEQ ID NO:13. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody comprises a VL domain
comprising SEQ ID NO:8 and a VH domain comprising SEQ ID NO:14. In
some embodiments, the anti-.alpha..sub.5.beta..sub.1 antibody is
human, humanized or chimeric. In one embodiment, the
anti-.alpha..sub.5.beta..sub.1 antibody is a monoclonal antibody.
In some embodiments, the anti-.alpha..sub.5.beta..sub.1 antibody
competes with the 18C12 antibody for binding to
.alpha..sub.5.beta..sub.1 integrin. In some embodiments, the
monoclonal antibody is a chimeric antibody. In some embodiments,
the anti-.alpha..sub.5.beta..sub.1 antibody is an antibody fragment
that binds .alpha..sub.5.beta..sub.1. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody is selected from: a Fab,
Fab', a F(ab)'.sub.2, single-chain Fv (scFv), an Fv fragment; a
diabody and a linear antibody. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody is a full length IgG1 or a
full length IgG4. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody is a bispecific antibody or
a multispecific antibody. In some embodiments, the bispecific
antibody binds VEGF and .alpha..sub.5.beta..sub.1 and is a VEGF
antagonist. In some embodiments, the anti-.alpha..sub.5.beta..sub.1
antibody has an altered effector function. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody is altered to decrease or
prevent antibody dependent cellular cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC) activity (e.g., by altering
the nucleic acid sequence encoding the Fc portion of the antibody).
In some embodiments, the Fc portion of the antibody comprises a
N297A substitution. In some embodiments, the
anti-.alpha..sub.5.beta..sub.1 antibody has been modified to
increase or decrease its half-life in humans (e.g., by altering the
nucleic acid sequence encoding the Fc portion of the antibody). In
some embodiment, the .alpha..sub.5.beta..sub.1 antibody is part of
an immunoonjugate conjugated to another entity (e.g., a therapeutic
agent or a detectable label). In some embodiments, the therapeutic
entity is a cytotoxic agent (e.g., radioactive isotope, a toxin, a
growth-inhibitory agent, or a chemotherapeutic agent). In some
embodiments, the detectable label is a fluorescent dye, a
radioisotope, or an enzyme.
[0007] Further embodiments of the invention provide nucleic acid
molecules encoding any of the anti-.alpha..sub.5.beta..sub.1
antibodies described herein, expression vectors comprising the
nucleic acids, and host cells comprising the nucleic acids. Yet
further embodiments of the invention provide methods of producing
any of the anti-.alpha..sub.5.beta..sub.1 antibodies described
herein, the methods comprising culturing the host cells so that the
antibody is produced. In some embodiments, the methods further
comprise recovering the antibody from the host cell.
[0008] Additional embodiments of the invention provide
pharmaceutical compositions comprising a .alpha..sub.5.beta..sub.1
antibody of this invention and a pharmaceutically acceptable
carrier. In some embodiments, the pharmaceutical compositions
further comprise at least one, two, three, four, or more additional
agent(s) including, e.g., a VEGF antagonist. In some embodiments,
the additional agent(s) are selected from a cytotoxic agent, a
chemotherapeutic agent, a growth-inhibitory agent, or an
anti-angiogenic agent. In some embodiments, the VEGF antagonist is
an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody
is bevacizumab. The invention also provides articles of manufacture
and kits comprising instructions for detecting
.alpha..sub.5.beta..sub.1 (e.g., in a subject who has been treated
with a VEGF antagonist).
[0009] Another embodiment of the invention provides methods of
treating subjects suffering from a disease or disorder involving
abnormal angiogenesis, vascular permeability, or vascular leakage.
The methods comprising administering a therapeutically effective
amount of the anti-.alpha..sub.5.beta..sub.1 antibodies described
herein to the subject, thereby treating the disease or disorder
(e.g., by partially or completely inhibiting abnormal angiogenesis,
vascular permeability, or vascular leakage). In some embodiments, a
VEGF antagonist is also administered to the subject. In some
embodiments, the VEGF antagonist and the
anti-.alpha..sub.5.beta..sub.1 antibody are administered
concurrently. In some embodiments, the VEGF antagonist and the
anti-.alpha..sub.5.beta..sub.1 antibody are administered
sequentially. In some embodiments, the disease or disorder is
responsive to VEGF antagonist therapies. In some embodiments, the
disease or disorder is selected from: a cancer, an immune disease
or an ocular disease. According to one embodiment, the disease or
disorder is selected from: a solid tumor, a metastatic tumor, a
soft tissue tumor, a disease having ocular neovascularisation, an
inflammatory disease having abnormal angiogenesis, a disease
arising after transplantation into the subject and a disease having
abnormal proliferation of fibrovascular tissue. According to
another embodiment, the cancer is selected from: breast cancer
(including metastatic breast cancer), cervical cancer, colorectal
cancer (including metastatic colorectal cancer), lung cancer
(including non-small cell lung cancer), non-Hodgkins lymphoma
(NHL), chronic lymphocytic leukemia, renal cell cancer, prostate
cancer including hormone refractory prostate cancer, liver cancer,
head and neck cancer, melanoma, ovarian cancer, mesothelioma, soft
tissue cancer, gastrointestinal stromal tumor, glioblastoma
multiforme and multiple myeloma. According to another embodiment,
the disease is selected from: retinopathy, age-related macular
degeneration (e.g., wet AMD), diabetic macular edema, retinal vein
occlusion (RVO), and dry AMD/geographic atrophy (for prevention of
progression to wet AMD) rubeosis; psoriasis, an inflammatory renal
disease, haemolytic uremic syndrome, diabetic nephropathy (e.g.,
proliferative diabetic retinopathy), arthritis (e.g., psoriatic
arthritis, osteoarthritis, rheumatoid arthritis), inflammatory
bowel disease, chronic inflammation, chronic retinal detachment,
chronic uveitis, chronic vitritis, corneal graft rejection, corneal
ncovascularization, corneal graft ncovascularization, Crohn's
disease, myopia, ocular neovascular disease, Pagets disease,
pemphigoid, polyarteritis, post-laser radial keratotomy, retinal
neovascularization, Sogrens syndrome, ulcerative colitis, graft
rejection, lung inflammation, nephrotic syndrome, edema, ascites
associated with malignancies, stroke, angiofibroma and neovascular
glaucoma. In one embodiment, the methods further comprise
administering an additional therapeutic agent (e.g., an
anti-neoplastic agent, a chemotherapeutic agent, a growth
inhibitory agent, or a cytotoxic agent) to the subject.
[0010] Yet another embodiment of the invention provides methods of
treating cancer in a subject, the methods comprising: administering
therapeutically effective amount of a VEGF antagonist and an
anti-.alpha..sub.5.beta..sub.1 antibody to the subject. In some
embodiments, the VEGF antagonist and the
anti-.alpha..sub.5.beta..sub.1 antibody are administered
concurrently. In some embodiments, the VEGF antagonist and the
anti-.alpha..sub.5.beta..sub.1 antibody are administered
sequentially. In some embodiments, the cancer is responsive to VEGF
antagonist therapies.
[0011] A further embodiment of the invention provides methods of
treating age related macular degeneration (AMD) (including, e.g.,
wet age-related macular degeneration) in a subject suffering from
AMD, the methods comprising: administering a therapeutically
effective amount of a VEGF antagonist and an
anti-.alpha..sub.5.beta..sub.1 antibody to the subject. In some
embodiments, the VEGF antagonist and the
anti-.alpha..sub.5.beta..sub.1 antibody are administered
concurrently. In some embodiments, the VEGF antagonist and the
anti-.alpha..sub.5.beta..sub.1 antibody are administered
sequentially. In yet another embodiment, a method of treating an
autoimmune disease in a subject comprising the step of
administering a therapeutically effective amount of a VEGF
antagonist and an .alpha..sub.5.beta..sub.1 antagonist concurrently
or sequentially is provided.
[0012] In some embodiments, the VEGF antagonist is initially
administered to the subject to be treated and the
anti-.alpha..sub.5.beta..sub.1 antibody is subsequently
administered to the subject. In some embodiments, the VEGF
antagonist and the .alpha..sub.5.beta..sub.1 antagonist are
administered to the subject simultaneously. In some embodiments,
the anti-.alpha..sub.5.beta..sub.1 antibody VEGF antagonist is
initially administered to the subject to be treated and the VEGF
antagonist is subsequently administered to the subject. In some
embodiments, the subject is treated with the VEGF antagonist until
the subject is unresponsive to VEGF antagonist treatment and then
the subject is treated with the anti-.alpha..sub.5.beta..sub.1
antibody. In one particular embodiment, the subject is treated with
the VEGF antagonist when the cancer is non-invasive or early stage
and treated with the anti-.alpha..sub.5.beta..sub.1 antibody when
the cancer is invasive. In another embodiment, subject being
treated with the anti-.alpha..sub.5.beta..sub.1 antibody has
elevated .alpha..sub.5.beta..sub.1 levels in a diseased tissue
compared to tissue from a subject not suffering from the disease or
compared to non-diseased tissue. In this instance, the method can
further include the step of detecting .alpha..sub.5.beta..sub.1 in
the subject, e.g., in a diseased tissue after treatment with a VEGF
antagonist. According to one embodiment, the invasive cancer is a
metastasized cancer. According to another embodiment, the early
stage cancer is a cancer treated by adjuvant therapy (e.g.,
chemotherapy or surgical removal).
[0013] According to one embodiment of this invention, the subject
to be treated with an anti-.alpha..sub.5.beta..sub.1 antibody is
suffering from a relapse after VEGF antagonist treatment or has
become refractory to VEGF antagonist treatment. According to
another embodiment, the subject to be treated with an
anti-.alpha..sub.5.beta..sub.1 antibody and a VEGF antagonist is
suffering from a metastatic cancer or has previously been treated
with adjuvant therapy. In one embodiment, the candidate patient is
relapsed, refractory or resistant to a chemotherapeutic agent such
as irinotecan. Examples of such diseases, include but are not
limited to, metastatic colorectal cancer, relapsed metastatic
colorectal cancer, metastatic breast cancer, relapsed metastatic
breast cancer, metastatic HER2.sup.+ breast cancer, adjuvant breast
cancer, adjuvant HER2.sup.+ breast cancer, metastatic pancreatic
cancer, adjuvant colon cancer, adjuvant non-small cell lung cancer,
adjuvant rectal cancer, adjuvant non small cell lung cancer,
metastatic non small cell lung cancer, metastatic ovarian cancer,
metastatic renal cell cancer and adjuvant renal cell cancer.
[0014] According to one embodiment, the subject suffering from a
disease described herein is administered a maintenance therapy
after treatment for the disease with a VEGF antagonist, wherein the
maintenance therapy is an .alpha..sub.5.beta..sub.1 antagonist
alone or sequentially or concurrently with a VEGF antagonist.
[0015] In some embodiments, the VEGF antagonist is selected from:
an antibody, an immunoadhesin, a peptibody, a small molecule and a
nucleic acid that hybridizes to a nucleic acid molecule encoding
VEGF under stringent conditions (e.g., ribozyme, siRNA and
aptamer). In some embodiments, the VEGF antagonist is an antibody
(e.g., a monoclonal antibody). According to one embodiment, the
anti-VEGF antibody is capable of being competitively inhibited from
binding to human VEGF by the Avastin.RTM. antibody. According to
another embodiment, the anti-VEGF antibody is human, humanized or
chimeric. According one specific embodiment, the anti-VEGF antibody
is the Avastin.RTM. antibody. According to another embodiment, the
anti-VEGF antibody is selected from the group consisting of a Fab,
Fab', a F(ab)'.sub.2, single-chain Fv (scFv), an Fv fragment; a
diabody and a linear antibody. According to another embodiment, the
VEGF antagonist is a bispecific antibody that binds both VEGF and
.alpha..sub.5.beta..sub.1 and is also a .alpha..sub.5.beta..sub.1
antagonist.
[0016] A further embodiment of the invention provides methods of
detecting .alpha..sub.5.beta..sub.1 protein in a sample suspected
of containing the .alpha..sub.5.beta..sub.1 protein. The methods
comprising (1) contacting the antibodies described herein with the
sample; and detecting formation of a complex between the
anti-.alpha..sub.5.beta..sub.1 antibody and the
.alpha..sub.5.beta..sub.1 protein. In some embodiments, the sample
is from a patient diagnosed with a disease characterized by
abnormal angiogenesis, abnormal vascular permeability, and/or
vascular leakage.
[0017] These and other embodiments of the invention are further
described in the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A and 1B depict alignment of sequences of light-chain
variable domains for the following: Human lambda III; Hamster
18C12; chimeric 18C12.v.1.1; h18C12.v3; h18C12.v6;
h18C12.v6.1.Lam3; h18C12.v6.2Lam3; and h18C12.v6.1.5.
[0019] FIGS. 2A and 2B depict alignment of sequences of the
heavy-chain variable domain for the following: Hamster 18C12;
h18C12.v3; h18C12.v6; h18C12.v6.1.Lam3; h18C12.v6.2Lam3; and
h18C12.v6.1.5.
[0020] FIG. 3 depicts CDR sequences of h18C12.v3 and h18C12.v3
affinity matured variants h18C12.v6; h18C12.v7; h18C12.v9;
h18C12.v15; h18C12.v16; h18C12.v28; h18C12.v30; h18C12.v51;
h18C12.v54; h18C12.v70; and h18C12.v78.
[0021] FIG. 4 depicts the results of a phage competition ELISA
demonstrating binding of h18C12.v3 parental clone, and 18C12
affinity matured variants h18C12.v6; h18C12.v7; h18C12.v9;
h18C12.v15; h18C12.v16; h18C12.v28; h18C12.v30; h18C12.v51;
h18C12.v54; h18C12.v70; and h18C12.v78 to human
.alpha..sub.5.beta..sub.1 integrin.
[0022] FIG. 5 depicts the results of a BIACORE.RTM. analysis of
binding of h18C12.v3 parental clone and h18C12.v3 affinity matured
variants h18C12.v6; h18C12.v15; h18C12.v54; and h18C12.v70 to human
.beta.5.beta.1 integrin.
[0023] FIG. 6 depicts the results of a BIACORE.RTM. analysis of
binding of chimeric 18C12 and h18C12.v6.1.Lam3 to human
.alpha..sub.5.beta..sub.1 integrin.
[0024] FIG. 7 depicts the results of a BIACORE.RTM. analysis of
binding of h18C12.v6.1 clones h18C12.v6.1.1, h18C12.v6.1.2,
h18C12.v6.1.3, h18C12.v6.1.4, and h18C12.v6.1.5 and sets forth the
CDR-L2 sequence at positions 50a, 50b, 50c, and 50d for each of the
h18C12.v6.1 clones
[0025] FIG. 8 depicts the results of a fibronectin binding assay
comparing the ability of chimeric 18C12 and h18C12.v6.1 to
interfere with the binding of U937 cells to fibronectin.
[0026] FIG. 9 depicts the results of a fibronectin binding assay
comparing the ability of hamster 18C12 and h18C12.v6.1.5 to
interfere with the binding of U937 cells to fibronectin.
[0027] FIG. 10 depicts the results of a fibronectin binding assay
comparing the ability of hamster 18C12 and h18C12.v6.1.5 to
interfere with the binding of .alpha..sub.5.beta..sub.1 to
fibronectin.
[0028] FIG. 11 depicts the results of a HUVEC migration assay
comparing the ability of chimeric 18C12 and h18C12.v6.1 to
interfere with migration of HUVEC cells on fibronectin.
[0029] FIG. 12 depicts the results of a tumor xenograft assay
measuring the ability of h18C12.v6.1.5+/- anti-VEGF to enhance
survival.
[0030] FIG. 13 depicts the results of a tumor xenograft assay
measuring the ability of h18C12.v6.1.5+/- anti-VEGF to reduce tumor
burden.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0031] The present invention is based on the identification of
novel antibodies that bind .alpha..sub.5.beta..sub.1 integrin. The
.alpha..sub.5.beta..sub.1 antibodies are derived from the
monoclonal antibody 18C12 and can be used in a variety of
therapeutic and diagnostic methods. For example, the
.alpha..sub.5.beta..sub.1 antibodies can be used alone or in
combination with other agents for treating abnormal angiogenesis,
neoplasia, ocular diseases and autoimmune diseases. The antibodies
can also be used for detecting .alpha..sub.5.beta..sub.1 protein in
patients or patient samples by administering the antibodies to
.alpha..sub.5.beta..sub.1 protein in patients and detecting the
anti-.alpha..sub.5.beta..sub.1 antibody bound to the
.alpha..sub.5.beta..sub.1 protein in a sample from the patient
(e.g., in vivo or ex vivo) or by contacting the antibodies with
samples from patients and detecting qualitatively or quantitatively
the anti-.alpha..sub.5.beta..sub.1 antibody bound to the
.alpha..sub.5.beta..sub.1 protein.
II. Definitions
[0032] "Alpha5beta1" or ".alpha.5.beta.1" or ".alpha.5b1" or
".alpha..sub.5.beta..sub.1" is an integrin comprising two different
proteins (i.e., subunits Alpha5 and beta1). .alpha.5.beta.1 has
been shown to bind to fibronectin, L1-CAM and fibrinogen.
.beta.5.beta.1 integrin is also known as Very Late Activation-5,
VLA-5, alpha5beta1, CD49e/CD29, fibronectin receptor, FNR and
GPIc-IIa. According to a embodiment, the .alpha.5.beta.1 is a human
.alpha.5.beta.1.
[0033] "Alpha5" is used herein interchangeably with CD49c,
.alpha.5, integrin alpha5 subunit, VLA-5 alpha subunit, IC subunit
of GPIc-IIa and FNR alpha chain refers to one subunit of the
.alpha..sub.5.beta..sub.1 integrin. Alpha5 has four isoforms
generated by alternative splicing (A-D) and which vary within their
cytoplasmic domains. Amino acid sequences for human isforms of
alpha5 can be found at, e.g., Genbank accession numbers: X07979,
U33879, U33882 and U33880, respectively.
[0034] "Beta1" also called CD29, beta1, Platelet GPIIa; VLA-beta
chain; beta-1 integrin chain, CD29; FNRB; MDF2; VLAB; GPIIA; MSK12
and VLA5B. Amino acid sequences for human Beta1 can be found, e.g.,
at Genbank Accession No. X06256.
[0035] The term "VEGF" as used herein refers to the 165-amino acid
human vascular endothelial cell growth factor and related 121-,
189-, and 206-amino acid human vascular endothelial cell growth
factors, as described by Leung et al. Science, 246:1306 (1989), and
Houck et al. Mol. Endocrin., 5:1806 (1991), together with the
naturally occurring allelic and processed forms thereof. The term
"VEGF" also refers to VEGFs from non-human species such as mouse,
rat or primate. Sometimes the VEGF from a specific species are
indicated by terms such as hVEGF for human VEGF, mVEGF for murine
VEGF, and etc. The term "VEGF" is also used to refer to truncated
forms of the polypeptide comprising amino acids 8 to 109 or 1 to
109 of the 165-amino acid human vascular endothelial cell growth
factor. Reference to any such forms of VEGF may be identified in
the present application, e.g., by "VEGF (8-109)," "VEGF (1-109)" or
"VEGF.sub.165." The amino acid positions for a "truncated" native
VEGF are numbered as indicated in the native VEGF sequence. For
example, amino acid position 17 (methionine) in truncated native
VEGF is also position 17 (methionine) in native VEGF. The truncated
native VEGF has binding affinity for the KDR and Flt-1 receptors
comparable to native VEGF. According to a embodiment, the VEGF is a
human VEGF.
[0036] A "VEGF antagonist" refers to a molecule capable of
neutralizing, blocking, inhibiting, abrogating, reducing or
interfering with VEGF activities including its binding to VEGF or
one or more VEGF receptors or the nucleic acid encoding them.
Preferrably, the VEGF antagonist binds VEGF or a VEGF receptor.
VEGF antagonists include anti-VEGF antibodies and antigen-binding
fragments thereof, polypeptides that bind VEGF and VEGF receptors
and block ligand-receptor interaction (e.g., immunoadhesins,
peptibodies), anti-VEGF receptor antibodies and VEGF receptor
antagonists such as small molecule inhibitors of the VEGFR tyrosine
kinases, aptamers that bind VEGF and nucleic acids that hybridize
under stringent conditions to nucleic acid sequences that encode
VEGF or VEGF receptor (e.g., RNAi). According to one embodiment,
the VEGF antagonist binds to VEGF and inhibits VEGF-induced
endothelial cell proliferation in vitro. According to one
embodiment, the VEGF antagonist binds to VEGF or a VEGF receptor
with greater affinity than a non-VEGF or non-VEGF receptor.
According to one embodiment, the VEG antagonist binds to VEGF or a
VEGF receptor with a Kd of between 1 .mu.M and 1 pM. According to
another embodiment, the VEGF antagonist binds to VEGF or a VEGF
receptor between 500 nM and 1 pM.
[0037] According a embodiment, the VEGF antagonist is selected from
the group consisting of a polypeptidc such as an antibody, a
peptibody, an immunoadhesin, a small molecule or an aptamer. In a
embodiment, the antibody is an anti-VEGF antibody such as the
AVASTIN.COPYRGT. antibody or an anti-VEGF receptor antibody such as
an anti-VEGFR2 or an anti-VEGFR3 antibody. Other examples of VEGF
antagonists include: VEGF-Trap, Mucagen, PTK787, SU11248,
AG-013736, Bay 439006 (sorafenib), ZD-6474, CP632, CP-547632,
AZD-2171, CDP-171, SU-14813, CHIR-258, AEE-788, SB786034,
BAY579352, CDP-791, EG-3306, GW-786034, RWJ-417975/CT6758 and
KRN-633.
[0038] An "anti-VEGF antibody" is an antibody that binds to VEGF
with sufficient affinity and specificity. Preferably, the anti-VEGF
antibody of the invention can be used as a therapeutic agent in
targeting and interfering with diseases or conditions wherein the
VEGF activity is involved. An anti-VEGF antibody will usually not
bind to other VEGF homologues such as VEGF-B or VEGF-C, nor other
growth factors such as P1GF, PDGF or bFGF. A anti-VEGF antibody is
a monoclonal antibody that binds to the same epitope as the
monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB
10709. More preferably the anti-VEGF antibody is a recombinant
humanized anti-VEGF monoclonal antibody generated according to
Presta et al. (1997) Cancer Res. 57:4593-4599, including but not
limited to the antibody known as bevacizumab (BV; Avastin.RTM.).
According to another embodiment, anti-VEGF antibodies that can be
used include, but are not limited to the antibodies disclosed in WO
2005/012359. According to one embodiment, the anti-VEGF antibody
comprises the variable heavy and variable light region of any one
of the antibodies disclosed in FIGS. 24, 25, 26, 27 and 29 of WO
2005/012359 (e.g., G6, G6-23, G6-31, G6-23.1, G6-23.2, B20, B20-4
and B20.4.1). In another embodiment, the anti-VEGF antibody known
as ranibizumab is the VEGF antagonist administered for ocular
disease such as diabetic retinopathy and wet AMD.
[0039] The anti-VEGF antibody "Bevacizumab (BV)", also known as
"rhuMAb VEGF" or "Avastin.RTM.", is a recombinant humanized
anti-VEGF monoclonal antibody generated according to Presta et al.
(1997) Cancer Res. 57:4593-4599. It comprises mutated human IgG1
framework regions and antigen-binding complementarity-determining
regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that
blocks binding of human VEGF to its receptors. Approximately 93% of
the amino acid sequence of Bevacizumab, including most of the
framework regions, is derived from human IgG1, and about 7% of the
sequence is derived from the murine antibody A4.6.1. Bevacizumab
has a molecular mass of about 149,000 daltons and is glycosylated.
Other anti-VEGF antibodies include the antibodies described in U.S.
Pat. No. 6,884,879 and WO 2005/044853.
[0040] The anti-VEGF antibody Ranibizumab or the LUCENTIS.RTM.
antibody or rhuFab V2 is a humanized, affinity-matured anti-human
VEGF Fab fragment. Ranibizumab is produced by standard recombinant
technology methods in E. coli expression vector and bacterial
fermentation. Ranibizumab is not glycosylated and has a molecular
mass of .about.48,000 daltons. See WO98/45331 and U.S.
2003/0190317.
[0041] Molecules, such as antibodies, characterized by binding to
overlapping or the similar areas on a target can be identified by
competitive inhibition/binding assays.
[0042] In one embodiment, HUVEC or other cells expressing
.alpha..sub.5.beta..sub.1 are used in a competitive inhibition
assay and FACS is used to evaluate binding localities of two
anti-.alpha..sub.5.beta..sub.1 antibodies relative to each other.
For example, HUVEC cells can be washed in conical tube and spun 5
min @ 1000 rpm. The pellet is typically washed two times. Then, the
cells can be resuspended, counted and kept on ice until use. 100
.mu.l of a first anti-.alpha..sub.5.beta..sub.1 antibody (e.g.,
start at a 1 .mu.g/ml concentration or lower concentration) can be
added to the well. Next, 100 .mu.l (e.g., 20.times.10.sup.5 cells)
of cells can be added into per well and incubated on ice for 30
min. Next, 100 .mu.l of a biotinylated
anti-.alpha..sub.5.beta..sub.1 antibody (5 .mu.g/ml stock) can be
added to each well and incubated on ice for 30 min. The cells are
then washed and pelleted for 5 min. @ 1000 rpm. The supernatant is
aspirated. R-Phycoerythrin conjugated streptavidin (Jackson
016-110-084) is added to the well (100 .mu.l @ 1:1000). Next, the
plate can be wrapped in foil and incubated on ice 30 min. Following
the incubation, the pellet can be washed and pelleted 5 min. @ 1000
rpm. The pellet can be resuspended and transferred to microtiter
tubes for FACS analysis.
[0043] An "angiogenic factor or agent" is a growth factor or its
receptor which is involved in stimulating the development of blood
vessels, e.g., promote angiogenesis, endothelial cell growth,
stabiliy of blood vessels, and/or vasculogenesis, etc. For example,
angiogenic factors, include, but are not limited to, e.g., VEGF and
members of the VEGF family and their receptors (VEGF-B, VEGF-C,
VEGF-D, VEGFR1, VEGFR2 and VEGFR3), P1GF, PDGF family, fibroblast
growth factor family (FGFs), TIE ligands (Angiopoietins, ANGPT1,
ANGPT2), TIE1, TIE2, ephrins, Bv8, Delta-like ligand 4 (DLL4),
Del-1, fibroblast growth factors: acidic (aFGF) and basic (bFGF),
FGF4, FGF9, BMP9, BMP10, Follistatin, Granulocyte
colony-stimulating factor (G-CSF), GM-CSF, Hepatocyte growth factor
(HGF)/scatter factor (SF), Interleukin-8 (IL-8), CXCL12, Leptin,
Midkine, neuropilins, NRP1, NRP2, Placental growth factor,
Platelet-derived endothelial cell growth factor (PD-ECGF),
Platelet-derived growth factor, especially PDGF-BB, PDGFR-alpha, or
PDGFR-beta, Pleiotrophin (PTN), Progranulin, Proliferin,
Transforming growth factor-alpha (TGF-alpha), Transforming growth
factor-beta (TGF-beta), Tumor necrosis factor-alpha (TNF-alpha),
Alk1, CXCR4, Notch1, Notch4, Sema3A, Sema3C, Sema3F, Robo4, etc. It
would further include factors that promote angiogenesis, such as
ESM1 and Perlecan. It would also include factors that accelerate
wound healing, such as growth hormone, insulin-like growth factor-I
(IGF-I), VIGF, epidermal growth factor (EGF), EGF-like domain,
multiple 7 (EGFL7), CTGF and members of its family, and TGF-alpha
and TGF-beta. See, e.g., Klagsbrun and D'Amore (1991) Annu. Rev.
Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179;
Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364;
Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 1 listing
known angiogenic factors); and, Sato (2003) Int. J. Clin. Oncol.
8:200-206.
[0044] An "anti-angiogenic agent" or "angiogenic inhibitor" refers
to a small molecular weight substance, a polynucleotide (including,
e.g., an inhibitory RNA (RNAi or siRNA)), a polypeptide, an
isolated protein, a recombinant protein, an antibody, or conjugates
or fusion proteins thereof, that inhibits angiogenesis,
vasculogenesis, or undesirable vascular permeability, either
directly or indirectly. It should be understood that the
anti-angiogenic agent includes those agents that bind and block the
angiogenic activity of the angiogenic factor or its receptor. For
example, an anti-angiogenic agent is an antibody or other
antagonist to an angiogenic agent as defined above, e.g.,
antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptor
or Flt-1 receptor), anti-PDGFR inhibitors, small molecules that
block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668,
SUTENT.RTM./SU11248 (sunitinib malate), AMG706, or those described
in, e.g., international patent application WO 2004/113304).
Anti-angiogenic agents include, but are not limited to, the
following agents: VEGF inhibitors such as a VEGF-specific
antagonist, EGF inhibitor, EGFR inhibitors, Erbitux.RTM.
(cetuximab, ImClone Systems, Inc., Branchburg, N.J.), Vectibix.RTM.
(panitumumab, Amgen, Thousand Oaks, Calif.), TIE2 inhibitors, IGF1R
inhibitors, COX-II (cyclooxygenase II) inhibitors, MMP-2
(matrix-metalloproteinase 2) inhibitors, and MMP-9
(matrix-metalloproteinase 9) inhibitors, CP-547,632 (Pfizer Inc.,
NY, USA), Axitinib (Pfizer Inc.; AG-013736), ZD-6474 (AstraZeneca),
AEE788 (Novartis), AZD-2171), VEGF Trap (Regeneron/Aventis),
Vatalanib (also known as PTK-787, ZK-222584: Novartis &
Schering A G), Macugen (pegaptanib octasodium, NX-1838, EYE-001,
Pfizer Inc./Gilead/Eyetech), IM862 (Cytran Inc. of Kirkland, Wash.,
USA); and angiozyme, a synthetic ribozyme from Ribozyme (Boulder,
Colo.) and Chiron (Emeryville, Calif.) and combinations thereof.
Other angiogenesis inhibitors include thrombospondin1,
thrombospondin2, collagen IV and collagen XVIII. VEGF inhibitors
are disclosed in U.S. Pat. Nos. 6,534,524 and 6,235,764, both of
which arc incorporated in their entirety for all purposes.
Anti-angiogenic agents also include native angiogenesis inhibitors,
e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and
D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar
(2003) Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic
therapy in malignant melanoma); Ferrara & Alitalo (1999) Nature
Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene
22:6549-6556 (e.g., Table 2 listing known antiangiogenic factors);
and, Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1
listing anti-angiogenic agents used in clinical trials).
[0045] The term "anti-angiogenic therapy" refers to a therapy
useful for inhibiting angiogenesis which comprises the
administration of an anti-angiogenic agent.
[0046] The "Kd" or "Kd value" for an antibody an anti-VEGF antibody
according to this invention is in one embodiment measured by a
radiolabeled VEGF binding assay (RIA) performed with the Fab
version of the antibody and a VEGF molecule as described by the
following assay that measures solution binding affinity of Fabs for
VEGF or by equilibrating Fab with a minimal concentration of
(.sup.125I)-labeled molecule VEGF(109) in the presence of a
titration series of unlabeled VEGF, then capturing bound VEGF with
an anti-Fab antibody-coated plate (Chen, et al., (1999) J. Mol Biol
293:865-881). To establish conditions for the assay, microtiter
plates (Dynex) are coated overnight with 5 ug/ml of a capturing
anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6),
and subsequently blocked with 2% (w/v) bovine serum albumin in PBS
for two to five hours at room temperature (approximately 23.degree.
C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM
[.sup.125I]VEGF(109) are mixed with serial dilutions of a Fab of
interest, (e.g., Fab-12 (Presta et al., (1997) Cancer Res.
57:4593-4599)). The Fab of interest is then incubated overnight;
however, the incubation may continue for 65 hours to insure that
equilibrium is reached. Thereafter, the mixtures are transferred to
the capture plate for incubation at room temperature for one hour.
The solution is then removed and the plate washed eight times with
0.1% Tween-20 in PBS. When the plates had dried, 150 ul/well of
scintillant (MicroScint-20; Packard) is added, and the plates are
counted on a Topcount gamma counter (Packard) for ten minutes.
Concentrations of each Fab that give less than or equal to 20% of
maximal binding are chosen for use in competitive binding assays.
According to another embodiment the Kd or Kd value is measured by
using surface plasmon resonance assays using a BIAcore.TM.-2000 or
a BIAcore.TM.-3000 (BIAcore, Inc., Piscataway, N.J.) at 25.degree.
C. with immobilized target molecule hVEGF (8-109) CM5 chips at
.about.10 response units (RU). Briefly, carboxymethylated dextran
biosensor chips (CM5, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Human VEGF is diluted with 10 mM sodium acetate, pH
4.8, into 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow
rate of 5 .mu.l/minute to achieve approximately 10 response units
(RU) of coupled protein. Following the injection of human VEGF, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 .mu.l/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIA core Evaluation
Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
was calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen,
Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds
10.sup.6 M.sup.-1 S.sup.-1 bythe surface plasmon resonance assay
above, then the on-rate is can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-VEGF antibody
(Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of human VEGF short form (8-109) or mouse VEGF as
measured in a spectrometer, such as a stop-flow equipped
spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco
spectrophotometer (ThermoSpectronic) with a stirred cuvette.
Similar binding assays can be performed for determining the Kd of
an anti-.alpha.5.beta.1 Fab or antibody using .alpha.5.beta.1 as
the target.
[0047] As used herein, a subject to be treated is a mammal (e.g.,
human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat,
dog, cat, etc.). The subject may be a clinical patient, a clinical
trial volunteer, an experimental animal, etc. The subject may be
suspected of having or at risk for having a cancer, an immune
disease, or any other disease having abnormal angiogenesis, be
diagnosed with a cancer, immune disease, or any other disease
having abnormal angiogenesis. Many diagnostic methods for cancer,
immune disease or any other disease exhibiting abnormal
angiogenesis and the clinical delineation of those diseases are
known in the art. According to one embodiment, the subject to be
treated according to this invention is a human.
[0048] "Abnormal angiogenesis" occurs when new blood vessels grow
either excessively or otherwise inappropriately (e.g., the
location, timing, degree, or onset of the angiogenesis being
undesired from a medical standpoint) in a diseased state or such
that it causes a diseased state. In some cases, excessive,
uncontrolled, or otherwise inappropriate angiogenesis occurs when
there is new blood vessel growth that contributes to the worsening
of the diseased state or cause of a diseased state, such as in
cancer, especially vascularized solid tumors and metastatic tumors
(including colon, lung cancer (especially small-cell lung cancer),
or prostate cancer), diseases caused by ocular neovascularisation,
especially diabetic blindness, retinopathies, primarily diabetic
retinopathy or age-related macular degeneration, choroidal
neovascularization (CNV), diabetic macular edema, pathological
myopia, von Hippel-Lindau disease, histoplasmosis of the eye,
Central Retinal Vein Occlusion (CRVO), corneal neovascularization,
retinal neovascularization and rubeosis; psoriasis, psoriatic
arthritis, haemangioblastoma such as haemangioma; inflammatory
renal diseases, such as glomerulonephritis, especially
mesangioproliferative glomerulonephritis, haemolytic uremic
syndrome, diabetic nephropathy or hypertensive nephrosclerosis;
various inflammatory diseases, such as arthritis, especially
rheumatoid arthritis, inflammatory bowel disease, psoriasis,
sarcoidosis, arterial arteriosclerosis and diseases occurring after
transplants, endometriosis or chronic asthma and more than 70 other
conditions. The new blood vessels can feed the diseased tissues,
destroy normal tissues, and in the case of cancer, the new vessels
can allow tumor cells to escape into the circulation and lodge in
other organs (tumor metastases). The present invention contemplates
treating those patients that are at risk of developing the
above-mentioned illnesses.
[0049] "Abnormal vascular permeability" occurs when the flow of
fluids, molecules (e.g., ions and nutrients) and cells (e.g.,
lymphocytes) between the vascular and extravascular compartments is
excessive or otherwise inappropriate (e.g., the location, timing,
degree, or onset of the vascular permeability being undesired from
a medical standpoint) in a diseased state or such that it causes a
diseased state. Abnormal vascular permeability may lead to
excessive or otherwise inappropriate "leakage" of ions, water,
nutrients, or cells through the vasculature. In some cases,
excessive, uncontrolled, or otherwise inappropriate vascular
permeability or vascular leakage exacerbates or induces disease
states including, e.g., edema associated with tumors including,
e.g., brain tumors; ascites associated with malignancies; Meigs'
syndrome; lung inflammation; nephrotic syndrome; pericardial
effusion; pleural effusion; permeability associated with
cardiovascular diseases such as the condition following myocardial
infarctions and strokes and the like. The present invention
contemplates treating those patients that have developed or are at
risk of developing the diseases and disorders associated with
abnormal vascular permeability or leakage.
[0050] Other patients that are candidates for receiving the
antibodies or polypeptides of this invention have, or are at risk
for developing, abnormal proliferation of fibrovascular tissue,
acne rosacea, acquired immune deficiency syndrome, artery
occlusion, atopic keratitis, bacterial ulcers, Bechets disease,
blood borne tumors, carotid obstructive disease, choroidal
neovascularization, chronic inflammation, chronic retinal
detachment, chronic uveitis, chronic vitritis, contact lens
overwear, corneal graft rejection, corneal neovascularization,
corneal graft neovascularization, Crohn's disease, Eales disease,
epidemic keratoconjunctivitis, fungal ulcers, Herpes simplex
infections, Herpes zoster infections, hyperviscosity syndromes,
Kaposi's sarcoma, leukemia, lipid degeneration, Lyme's disease,
marginal keratolysis, Mooren ulcer, Mycobacteria infections other
than leprosy, myopia, ocular neovascular disease, optic pits,
Osler-Weber syndrome (Osler-Weber-Rendu, osteoarthritis, Pagets
disease, pars planitis, pemphigoid, phylectenulosis, polyarteritis,
post-laser complications, protozoan infections, pseudoxanthoma
elasticum, pterygium keratitis sicca, radial keratotomy, retinal
neovascularization, retinopathy of prematurity, retrolental
fibroplasias, sarcoid, scleritis, sickle cell anemia, Sogrens
syndrome, solid tumors, Stargarts disease, Steven's Johnson
disease, superior limbic keratitis, syphilis, systemic lupus,
Terrien's marginal degeneration, toxoplasmosis, trauma, tumors of
Ewing sarcoma, tumors of neuroblastoma, tumors of osteosarcoma,
tumors of retinoblastoma, tumors of rhabdomyosarcoma, ulcerative
colitis, vein occlusion, Vitamin A deficiency and Wegeners
sarcoidosis, undesired angiogenesis associated with diabetes,
parasitic diseases, abnormal wound healing, hypertrophy following
surgery, injury or trauma, inhibition of hair growth, inhibition of
ovulation and corpus luteum formation, inhibition of implantation
and inhibition of embryo development in the uterus.
[0051] Anti-angiogenesis therapies are useful in the general
treatment of graft rejection, lung inflammation, nephrotic
syndrome, preeclampsia, pericardial effusion, such as that
associated with pericarditis, and pleural effusion, diseases and
disorders characterized by undesirable vascular permeability or
leakage, e.g., edema associated with tumors including, e.g., brain
tumors, ascites associated with malignancies, Meigs' syndrome, lung
inflammation, nephrotic syndrome, pericardial effusion, pleural
effusion, permeability associated with cardiovascular diseases such
as the condition following myocardial infarctions and strokes and
the like.
[0052] Other angiogenesis-dependent diseases according to this
invention include angiofibroma (abnormal blood of vessels which are
prone to bleeding), neovascular glaucoma (growth of blood vessels
in the eye), arteriovenous malformations (abnormal communication
between arteries and veins), nonunion fractures (fractures that
will not heal), atherosclerotic plaques (hardening of the
arteries), pyogenic granuloma (common skin lesion composed of blood
vessels), scleroderma (a form of connective tissue disease),
hemangioma (tumor composed of blood vessels), trachoma (leading
cause of blindness in the third world), hemophilic joints, vascular
adhesions and hypertrophic scars (abnormal scar formation).
[0053] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to administering a
compound or a pharmaceutical composition for prophylactic and/or
therapeutic purposes. To "treat disease" or use for "therapeutic
treatment" refers to administering treatment to a subject already
suffering from a disease to improve the subject's condition.
Desirable effects of treatment include, but are not limited to,
preventing recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease. Preferably, the subject is diagnosed
as suffering from a disease having abnormal angiogenesis, based on
identification of any of the characteristic symptoms described
below or the use of the diagnostic methods described herein. To
"prevent disease" refers to prophylactic treatment of a subject who
is not yet ill, but who is susceptible to, or otherwise at risk of,
developing a particular disease. Preferably a subject is determined
to be at risk of developing a disease having abnormal angiogenesis,
using the diagnostic methods described herein.
[0054] By "treating or ameliorating" is meant ameliorating a
condition or symptoms of the condition before or after its onset.
As compared with an equivalent untreated control, such amelioration
or degree of treatment is at least 5%, 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, or 100% as measured by any standard
technique.
[0055] The terms "recurrence," "relapse" or "relapsed" refers to
the return of a cancer or disease after clinical assessment of the
disappearance of disease. A diagnosis of distant metastasis or
local recurrence can be considered a relapse.
[0056] "Refractory" refers to the resistance or non-responsiveness
of a disease or condition to a treatment (e.g., the number of
neoplastic plasma cells increases even though treatment if given).
In certain embodiments, the term "refractory" refers a resistance
or non-responsiveness to any previous treatment including, but not
limited to, VEGF antagonist, anti-angiogenic agents and
chemotherapy treatments. In certain embodiments, the term
"refractory" refers an intrinsically non-responsiveness of a
disease or condition to any previous treatment comprising a VEGF
antagonist, anti-angiogenic agents and/or chemotherapy treatments.
In certain embodiments, the VEGF antagonist is an anti-VEGF
antibody.
[0057] "Relapsed" refers to the regression of the patient's illness
back to its former diseased state, especially the return of
symptoms following an apparent recovery or partial recovery. In
certain embodiments, relapsed state refers to the process of
returning to or the return to illness before the previous treatment
including, but not limited to, VEGF antagonist, anti-angiogenic
agents and/or chemotherapy treatments. In certain embodiments,
relapsed state refers to the process of returning to or the return
to illness after an initial strong response to a cancer therapy
comprising a VEGF antagonist, anti-angiogenic agents and/or
chemotherapy treatments. In certain embodiments, the VEGF
antagonist is an anti-VEGF antibody.
[0058] The term "adjuvant therapy" refers to treatment given after
the primary therapy, usually surgery. Adjuvant therapy for cancer
or disease may include immune therapy, chemotherapy, radiation
therapy or hormone therapy.
[0059] The term "maintenance therapy" refers to scheduled
retreatment that is given to help maintain a previous treatment's
effects. Maintenance therapy is often given to help keep cancer in
remission or prolong a response to a specific therapy regardless of
disease progression.
[0060] The term "invasive cancer" refers to cancer that has spread
beyond the layer of tissue in which it started into the normal
surrounding tissues. Invasive cancers may or may not be
metastatic.
[0061] The term "non-invasive cancer" refers to a very early cancer
or a cancer that has not spread beyond the tissue of origin.
[0062] The term "progression-free survival" in oncology refers to
the length of time during and after treatment that a cancer does
not grow. Progression-free survival includes the amount of time
patients have experienced a complete response or a partial
response, as well as the amount of time patients have experienced
stable disease.
[0063] The term "progressive disease" in oncology can refer to a
tumor growth of more than 20 percent since treatment began--either
due to an increase in mass or a spread in the tumor.
[0064] A "disorder" is any condition that would benefit from
treatment with the antibody. For example, mammals who suffer from
or need prophylaxis against abnormal angiogenesis (excessive,
inappropriate or uncontrolled angiogenesis) or abnormal vascular
permeability or leakage. This includes chronic and acute disorders
or diseases including those pathological conditions which
predispose the mammal to the disorder in question. Non-limiting
examples of disorders to be treated herein include malignant and
benign tumors; non-leukemias and lymphoid malignancies; neuronal,
glial, astrocytal, hypothalamic and other glandular, macrophagal,
epithelial, stromal and blastocoelic disorders; and inflammatory,
angiogenic and immunologic disorders.
[0065] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell
cancer, glioblastoma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
colorectal cancer, endometrial carcinoma, salivary gland carcinoma,
kidney cancer, renal cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, head and neck cancer, rectal
cancer, colorectal cancer, lung cancer including small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung and
squamous carcinoma of the lung, squamous cell cancer (e.g.
epithelial squamous cell cancer), prostate cancer, cancer of the
peritoneum, hepatocellular cancer, gastric or stomach cancer
including gastrointestinal cancer, pancreatic cancer, glioblastoma,
retinoblastoma, astrocytoma, thecomas, arrhenoblastomas, hepatoma,
hematologic malignancies including non-Hodgkins lymphoma (NHL),
multiple myeloma and acute hematologic malignancies, endometrial or
uterine carcinoma, endometriosis, fibrosarcomas, choriocarcinoma,
salivary gland carcinoma, vulval cancer, thyroid cancer, esophageal
carcinomas, hepatic carcinoma, anal carcinoma, penile carcinoma,
nasopharyngeal carcinoma, laryngeal carcinomas, Kaposi's sarcoma,
melanoma, skin carcinomas, Schwannoma, oligodendroglioma,
neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma,
leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas,
Wilm's tumor, as well as B-cell lymphoma (including low
grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, and Meigs' syndrome.
[0066] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0067] The term "anti-neoplastic composition" or "anti-neoplastic
agent" refers to a composition useful in treating cancer comprising
at least one active therapeutic agent, e.g., "anti-cancer agent."
Examples of therapeutic agents (anti-cancer agents) include, but
are limited to, e.g., chemotherapeutic agents, growth inhibitory
agents, cytotoxic agents, agents used in radiation therapy,
anti-angiogenesis agents, apoptotic agents, anti-tubulin agents,
and other-agents to treat cancer, such as anti-HER-2 antibodies,
anti-CD20 antibodies, an epidermal growth factor receptor (EGFR)
antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR inhibitor
(e.g., erlotinib (Tarceva.TM.), platelet derived growth factor
inhibitors (e.g., Gleevec.TM. (Imatinib Mesylate)), a COX-2
inhibitor (e.g., celecoxib), interferons, cytokines, antagonists
(e.g., neutralizing antibodies) that bind to one or more of the
following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BAFF, BR3,
APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2, and other bioactive
and organic chemical agents, etc. Combinations thereof are also
contemplated in this invention.
[0068] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth or proliferation of a
cell in vitro and/or in vivo. Thus, the growth inhibitory agent may
be one which significantly reduces the percentage of cells in S
phase. Examples of growth inhibitory agents include agents that
block cell cycle progression (at a place other than S phase), such
as agents that induce G1 arrest and M-phase arrest. Classical
M-phase blockers include the vincas (vincristine and vinblastine),
TAXOL.RTM., and topo II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest G1
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in The Molecular Basis of Cancer,
Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(WB Saunders: Philadelphia, 1995), especially p. 13.
[0069] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., I.sup.131, I.sup.125, Y.sup.90 and
Re.sup.186), growth inhibitory agents, chemotherapeutic agents, and
toxins such as enzymatically active toxins of bacterial, fungal,
plant or animal origin, or fragments thereof.
[0070] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include is a chemical compound useful in the treatment of cancer.
Examples of chemotherapeutic agents include alkylating agents such
as thiotepa and CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates
such as busulfan, improsulfan and piposulfan; aziridines such as
benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
carminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulene Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate;
irinotecan (Camptosar, CPT-11) (including the treatment regimen of
irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS
2000; difluorometlhylornithine (DMFO); retinoids such as retinoic
acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin,
including the oxaliplatin treatment regimen (FOLFOX); inhibitors of
PKC-alpha, Raf, H-Ras and EGFR (e.g., erlotinib (Tarceva.TM.)) that
reduce cell proliferation and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0071] Chemotherapeutic agents also include anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those which inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Raf and H-Ras;
ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME.RTM.
ribozyme) and a HER2 expression inhibitor; vaccines such as gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; PROLEUKIN.RTM.
rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; Vinorelbine and Esperamicins (see U.S. Pat. No. 4,675,187),
and pharmaceutically acceptable salts, acids or derivatives of any
of the above.
[0072] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
(e.g., small molecule) that is less cytotoxic to diseased cells
compared to the parent drug and is capable of being enzymatically
activated or converted into the more active parent form. See, e.g.,
Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society
Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and
Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug
Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp.
247-267, Humana Press (1985). The prodrugs of this invention
include, but are not limited to, phosphate-containing prodrugs,
thiophosphate-containing prodrugs, sulfate-containing prodrugs,
peptide-containing prodrugs, D-amino acid-modified prodrugs,
glycosylated prodrugs, .beta.-lactam-containing prodrugs,
optionally substituted phenoxyacetamide-containing prodrugs or
optionally substituted phenylacetamide-containing prodrugs,
5-fluorocytosine and other 5-fluorouridine prodrugs which can be
converted into the more active cytotoxic free drug. Examples of
cytotoxic drugs that can be derivatized into a prodrug form for use
in this invention include, but are not limited to, those
chemotherapeutic agents described above.
[0073] "Isolated," when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a cell or cell culture from which
it was expressed. Contaminant components of its natural environment
are materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and can include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
embodiments, the polypeptide will be purified (1) to a degree
sufficient to obtain at least 15 residues of N-terminal or internal
amino acid sequence by use of a spinning cup sequenator, or (2) to
homogeneity by SDS-PAGE under non-reducing or reducing conditions
using Coomassie blue or, preferably, silver stain. Isolated
polypeptide includes polypeptide in situ within recombinant cells,
since at least one component of the polypeptide natural environment
will not be present. Ordinarily, however, isolated polypeptide will
be prepared by at least one purification step.
[0074] An "isolated" polypeptide-encoding nucleic acid or other
polypeptide-encoding nucleic acid is a nucleic acid molecule that
is identified and separated from at least one contaminant nucleic
acid molecule with which it is ordinarily associated in the natural
source of the polypeptide-encoding nucleic acid. An isolated
polypeptide-encoding nucleic acid molecule is other than in the
form or setting in which it is found in nature. Isolated
polypeptide-encoding nucleic acid molecules therefore are
distinguished from the specific polypeptide-encoding nucleic acid
molecule as it exists in natural cells. However, an isolated
polypeptide-encoding nucleic acid molecule includes
polypeptide-encoding nucleic acid molecules contained in cells that
ordinarily express the polypeptide where, for example, the nucleic
acid molecule is in a chromosomal location different from that of
natural cells.
[0075] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0076] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0077] "Stringent conditions" or "high stringency conditions", as
defined herein, can be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50 C; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42 C; or (3) overnight hybridization in a
solution that employs 50% formamide, 5.times.SSC (0.75 M NaCl,
0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1%
sodium pyrophosphate, 5.times.Denhardt's solution, sonicated salmon
sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at
42.degree. C., with a 10 minute wash at 42.degree. C. in
0.2.times.SSC (sodium chloride/sodium citrate) followed by a 10
minute high-stringency wash consisting of 0.1.times.SSC containing
EDTA at 55.degree. C.
[0078] The amino acid sequences described herein are contiguous
amino acid sequences unless otherwise specified.
[0079] As used herein, the term "immunoadhesin" designates
antibody-like molecules that combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity that is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand--such as a VEGFR or a fibronectin ligand. The immunoglobulin
constant domain sequence in the immunoadhesin can be obtained from
any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes,
IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM. Peptibodies,
which often comprise a sequence derived from phage display
selection of sequences that specifically bind a target fused to an
Fc portion of an immunoglobulin, can be considered immunadhesins
herein.
[0080] The term "antibody" is used in the broadest sense and
specifically covers, for example, single monoclonal antibodies
(including agonist, antagonist, and neutralizing antibodies),
antibody compositions with polyepitopic specificity, polyclonal
antibodies, single chain anti-antibodies, and fragments of
antibodies (see below) as long as they specifically bind a native
polypeptide and/or exhibit a biological activity or immunological
activity of this invention. According to one embodiment, the
antibody binds to an oligomeric form of a target protein, e.g., a
trimeric form. According to another embodiment, the antibody
specifically binds to a protein, which binding can be inhibited by
a monoclonal antibody of this invention (e.g., a deposited antibody
of this invention, etc.). The phrase "functional fragment or
analog" of an antibody is a compound having a qualitative
biological activity in common with an antibody to which it is being
referred. For example, a functional fragment or analog of an
antibody of this invention can be one which can specifically bind
to VEGF or .alpha.5.beta.1. In one embodiment, the antibody can
prevent or substantially reduce the ability of a VEGF to induce
cell proliferation.
[0081] An "isolated antibody" is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and can include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In embodiments, the
antibody will be purified (1) to greater than 95% by weight of
antibody as determined by the Lowry method, and most preferably
more than 99% by weight, (2) to a degree sufficient to obtain at
least 15 residues of N-terminal or internal amino acid sequence by
use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE
under reducing or nonreducing conditions using Coomassie blue or,
preferably, silver stain. Isolated antibody includes the antibody
in situ within recombinant cells since at least one component of
the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one
purification step.
[0082] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains (an IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called J chain, and therefore contain 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to a H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (C.sub.H1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
domains. The pairing of a V.sub.H and V.sub.L together forms a
single antigen-binding site. For the structure and properties of
the different classes of antibodies, see, e.g., BASIC AND CLINICAL
IMMUNOLOGY, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71 and Chapter 6.
[0083] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (C.sub.H), immunoglobulins can be assigned to different
classes or isotypes. There are five classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, having heavy chains designated
.alpha., .delta., .gamma., .epsilon., and .mu., respectively. The
.gamma. and .alpha. classes are further divided into subclasses on
the basis of relatively minor differences in C.sub.H sequence and
function, e.g., humans express the following subclasses: IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
[0084] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and define
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
110-amino acid span of the variable domains. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a beta-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the beta-sheet
structure. The hypervariable regions in each chain are held
together in close proximity by the FRs and, with the hypervariable
regions from the other chain, contribute to the formation of the
antigen-binding site of antibodies (see Kabat et al., SEQUENCES OF
PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). The constant
domains are not involved directly in binding an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody dependent cellular
cytotoxicity (ADCC).
[0085] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 31-35B (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H (in one embodiment, H1 is around about 31-35);
Kabat et al., supra and/or those residues from a "hypervariable
loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the
V.sub.L, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the V.sub.H;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
[0086] Throughout the present specification and claims, the Kabat
numbering system is generally used when referring to a residue in
the variable domain (approximately, residues 1-107 of the light
chain and residues 1-113 of the heavy chain) (e.g, Kabat et al.,
supra (1991)). The "EU numbering system" or "EU index" is generally
used when referring to a residue in an immunoglobulin heavy chain
constant region (e.g., the EU index reported in Kabat et al., supra
(1991) expressly incorporated herein by reference). Unless stated
otherwise herein, references to residues numbers in the variable
domain of antibodies means residue numbering by the Kabat numbering
system. Unless stated otherwise herein, references to residue
numbers in the constant domain of antibodies means residue
numbering by the EU numbering system.
[0087] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that can be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they can be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies useful in the present invention can be
prepared by the hybridoma methodology first described by Kohler et
al., Nature, 256:495 (1975), or can be made using recombinant DNA
methods in bacterial, eukaryotic animal or plant cells (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" can also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991), Marks et
al., J. Mol. Biol., 222:581-597 (1991) or using the methods set
forth in the Examples below.
[0088] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit a biological activity of this
invention (see U.S. Pat. No. 4,816,567; and Morrison et al., PNAS
USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein
include "primatized" antibodies comprising variable domain
antigen-binding sequences derived from a non-human primate (e.g.
Old World Monkey, Ape etc), and human constant region
sequences.
[0089] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al., Bio/Technology 10:779-783 (1992) describes affinity maturation
by VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by: Barbas et al., PNAS USA
91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton
et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J.
Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.
226:889-896 (1992).
[0090] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
binds. For example blocking or antagonist anti-.alpha.5.beta.1
antibodies partially or completely inhibit the angiogenesis by
binding .alpha.5.beta.1.
[0091] An "agonist" antibody is one which enhances or increases
biological activity of the antigen it binds. For example agonist
antibodies anti-.beta.5.beta.1 antibodies enhance angiogenesis by
binding .alpha.5.beta.1.
[0092] An "intact" antibody is one which comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H1, C.sub.H2 and C.sub.H3. The constant
domains can be native sequence constant domains (e.g. human native
sequence constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0093] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.
8(10): 1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0094] The expression "linear antibodies" generally refers to the
antibodies described in Zapata et al., Protein Eng.,
8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair
of tandem Fd segments (VH-CH1-VH-CH1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0095] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment which roughly corresponds to two
disulfide linked Fab fragments having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having additional few
residues at the carboxy terminus of the C.sub.H1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s) of
the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0096] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0097] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one "amino acid modification" as herein defined. Preferably,
the variant Fc region has at least one amino acid substitution
compared to a native sequence Fc region or to the Fc region of a
parent polypeptide, e.g. from about one to about ten amino acid
substitutions, and preferably from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. In one embodiment, the variant Fc
region herein will possess at least about 80% homology, at least
about 85% homology, at least about 90% homology, at least about 95%
homology or at least about 99% homology with a native sequence Fc
region. According to another embodiment, the variant Fc region
herein will possess at least about 80% homology, at least about 85%
homology, at least about 90% homology, at least about 95% homology
or at least about 99% homology with an Fc region of a parent
polypeptide.
[0098] The term "Fc region-comprising polypeptide" refers to a
polypeptide, such as an antibody or immunoadhesin (see definitions
below), which comprises an Fc region. The C-terminal lysine
(residue 447 according to the EU numbering system) of the Fc region
may be removed, for example, during purification of the polypeptide
or by recombinantly engineering the nucleic acid encoding the
polypeptide. Accordingly, a composition comprising polypeptides,
including antibodies, having an Fc region according to this
invention can comprise polypeptides populations with all K447
residues removed, polypeptide populations with no K447 residues
removed or polypeptide populations having a mixture of polypeptides
with and without the K447 residue.
[0099] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although at a lower affinity than
the entire binding site.
[0100] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Borrebaeck 1995, infra.
[0101] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Bispecific
diabodies arc heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., PNAS USA, 90:6444-6448 (1993).
[0102] "Humanized" antibodies are forms of non-human (e.g., rodent)
antibodies that are chimeric antibodies that contain minimal
sequence derived from the non-human antibody. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody)
in which residues from a hypervariable region of the recipient are
replaced by residues from a hypervariable region of a non-human
species (donor antibody) such as mouse, rat, rabbit or non-human
primate having the desired antibody specificity, affinity, and
capability. In some instances, framework region (FR) residues of
the human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, humanized antibodies can comprise residues
that are not found in the recipient antibody or in the donor
antibody. These modifications are made to further refine antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the hypervariable
loops correspond to those of a non-human immunoglobulin and all or
substantially all of the FRs are those of a human immunoglobulin
sequence. The humanized antibody optionally also will comprise at
least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin. For further details, see
Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596
(1992).
[0103] A "species-dependent antibody" is an antibody which has a
stronger binding affinity for an antigen from a first mammalian
species than it has for a homologue of that antigen from a second
mammalian species. Normally, the species-dependent antibody "bind
specifically" to a human antigen (i.e., has a binding affinity (Kd)
value of no more than about 1.times.10.sup.-7 M, no more than about
1.times.10.sup.-8 or no more than about 1.times.10.sup.-9 M) but
has a binding affinity for a homologue of the antigen from a second
non-human mammalian species which is at least about 50 fold, or at
least about 500 fold, or at least about 1000 fold, weaker than its
binding affinity for the human antigen. The species-dependent
antibody can be of any of the various types of antibodies as
defined above, but preferably is a humanized or human antibody.
[0104] In such embodiments, the extent of binding of the
polypeptide, antibody, antagonist or composition to a "non-target"
protein will be less than about 10% of the binding of the
polypeptide, antibody, antagonist or composition to its particular
target protein as determined by fluorescence activated cell sorting
(FACS) analysis or radioimmunoprecipitation (RIA). With regard to
the binding of a polypeptide, antibody, antagonist or composition
to a target molecule, the term "specific binding" or "specifically
binds to" or is "specific for" a particular polypeptide or an
epitope on a particular polypeptide target means binding that is
measurably different from a non-specific interaction. Specific
binding can be measured, for example, by determining binding of a
molecule compared to binding of a control molecule, which generally
is a molecule of similar structure that does not have binding
activity. For example, specific binding can be determined by
competition with a control molecule that is similar to the target,
for example, an excess of non-labeled target. In this case,
specific binding is indicated if the binding of the labeled target
to a probe is competitively inhibited by excess unlabeled target.
The term "specific binding" or "specifically binds to" or is
"specific for" a particular polypeptide or an epitope on a
particular polypeptide target as used herein can be exhibited, for
example, by a molecule having a Kd for the target of at least about
10.sup.-4 M, at least about 10.sup.-5 M, at least about 10.sup.-6
M, at least about 10.sup.-7 M, at least about 10.sup.-8 M, at least
about 10.sup.-9 M, alternatively at least about 10.sup.-10 M, at
least about 10.sup.-11 M, at least about 10.sup.-12 M, or greater.
In one embodiment, the term "specific binding" refers to binding
where a molecule binds to a particular polypeptide or epitope on a
particular polypeptide without substantially binding to any other
polypeptide or polypeptide epitope.
[0105] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors; and B cell activation. A "native sequence Fc region"
comprises an amino acid sequence identical to the amino acid
sequence of an Fc region found in nature. Examples of Fc sequences
are described in, for example, but not limited to, Kabat et al.,
supra (1991)).
[0106] "Percent (%) amino acid sequence identity" or "homology"
with respect to the polypeptide and antibody sequences identified
herein is defined as the percentage of amino acid residues in a
candidate sequence that are identical with the amino acid residues
in the polypeptide being compared, after aligning the sequences
considering any conservative substitutions as part of the sequence
identity. Alignment for purposes of determining percent amino acid
sequence identity can be achieved in various ways that arc within
the skill in the art, for instance, using publicly available
computer software such as BLAST, BLAST-2, ALIGN or Megalign
(DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are generated using the
sequence comparison computer program ALIGN-2. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc. and the
source code has been filed with user documentation in the U.S.
Copyright Office, Washington D.C., 20559, where it is registered
under U.S. Copyright Registration No. TXU510087. The ALIGN-2
program is publicly available through Genentech, Inc., South San
Francisco, Calif. The ALIGN-2 program should be compiled for use on
a UNIX operating system, preferably digital UNIX V4.0D. All
sequence comparison parameters are set by the ALIGN-2 program and
do not vary.
[0107] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. In one
embodiment, an FcR of this invention is one that binds an IgG
antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RITA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). The term includes
allotypes, such as Fc.gamma.RIIIA allotypes: Fc.gamma.RIIIA-Phe158,
Fc.gamma.RIIIA-Val158, Fc.gamma.RIIA-R131 and/or
Fc.gamma.RIIA-H131. FcRs are reviewed in Ravetch and Kinet, Annu.
Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34
(1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).
Other FcRs, including those to be identified in the future, are
encompassed by the term "FcR" herein. The term also includes the
neonatal receptor, FcRn, which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587
(1976) and Kim et al., J. Immunol. 24:249 (1994)).
[0108] The term "FcRn" refers to the neonatal Fc receptor (FcRn).
FcRn is structurally similar to major histocompatibility complex
(MHC) and consists of an .alpha.-chain noncovalently bound to
.beta.2-microglobulin. The multiple functions of the neonatal Fc
receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev.
Immunol. 18, 739-766. FcRn plays a role in the passive delivery of
immunoglobulin IgGs from mother to young and the regulation of
serum IgG levels. FcRn can act as a salvage receptor, binding and
transporting pinocytosed IgGs in intact form both within and across
cells, and rescuing them from a default degradative pathway.
[0109] WO 00/42072 (Presta) and Shields et al. J. Biol. Chem. 9(2):
6591-6604 (2001) describe antibody variants with improved or
diminished binding to FcRs. The contents of those publications are
specifically incorporated herein by reference.
[0110] The "CH1 domain" of a human IgG Fc region (also referred to
as "C1" of "H1" domain) usually extends from about amino acid 118
to about amino acid 215 (EU numbering system).
[0111] "Hinge region" is generally defined as stretching from
Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206
(1985)). Hinge regions of other IgG isotypes may be aligned with
the IgG1 sequence by placing the first and last cysteine residues
forming inter-heavy chain S-S bonds in the same positions.
[0112] The "lower hinge region" of an Fc region is normally defined
as the stretch of residues immediately C-terminal to the hinge
region, i.e. residues 233 to 239 of the Fc region. In previous
reports, FcR binding was generally attributed to amino acid
residues in the lower hinge region of an IgG Fc region.
[0113] The "CH2 domain" of a human IgG Fc region (also referred to
as "C2" of "H2" domain) usually extends from about amino acid 231
to about amino acid 340. The CH2 domain is unique in that it is not
closely paired with another domain. Rather, two N-linked branched
carbohydrate chains are interposed between the two CH2 domains of
an intact native IgG molecule. It has been speculated that the
carbohydrate may provide a substitute for the domain-domain pairing
and help stabilize the CH2 domain. Burton, Molec. Immunol.
22:161-206 (1985).
[0114] The "CH3 domain" (also referred to as "C2" or "H3" domain)
comprises the stretch of residues C-terminal to a CH2 domain in an
Fc region (i.e. from about amino acid residue 341 to the C-terminal
end of an antibody sequence, typically at amino acid residue 446 or
447 of an IgG).
[0115] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
C1q binding; complement dependent cytotoxicity; Fc receptor
binding; antibody-dependent cell-mediated cytotoxicity (ADCC);
phagocytosis; down regulation of cell surface receptors (e.g. B
cell receptor; BCR), etc. Such effector functions generally require
the Fc region to be combined with a binding domain (e.g. an
antibody variable domain) and can be assessed using various assays
as herein disclosed, for example.
[0116] "C1q" is a polypeptide that includes a binding site for the
Fc region of an immunoglobulin. C1q together with two serine
proteases, C1r and C1s, forms the complex C1, the first component
of the complement dependent cytotoxicity (CDC) pathway. Human C1q
can be purchased commercially from, e.g. Quidel, San Diego,
Calif.
[0117] The term "binding domain" refers to the region of a
polypeptide that binds to another molecule. In the case of an FcR,
the binding domain can comprise a portion of a polypeptide chain
thereof (e.g. the alpha chain thereof) which is responsible for
binding an Fc region. One useful binding domain is the
extracellular domain of an FcR alpha chain.
[0118] An antibody or peptibody with a variant IgG Fc with
"altered" FcR binding affinity or ADCC activity is one which has
either enhanced or diminished FcR binding activity (e.g, Fc.gamma.R
or FcRn) and/or ADCC activity compared to a parent polypeptide or
to a polypeptide comprising a native sequence Fc region. The
variant Fc which "exhibits increased binding" to an FcR binds at
least one FcR with higher affinity (e.g., lower apparent Kd or 1050
value) than the parent polypeptide or a native sequence IgG Fc.
According to some embodiments, the improvement in binding compared
to a parent polypeptide is about 3 fold, preferably about 5, 10,
25, 50, 60, 100, 150, 200, 250, 300, 350, 400, 450, or 500 fold, or
about 25% to 1000% improvement in binding. The polypeptide variant
which "exhibits decreased binding" to an FcR, binds at least one
FcR with lower affinity (e.g, higher apparent Kd or higher IC50
value) than a parent polypeptide. The decrease in binding compared
to a parent polypeptide may be about 5%, 10%, 20%, 30%, 40%, 50%,
60%, or more decrease in binding.
[0119] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound to Fc
receptors (FcRs) present on certain cytotoxic cells (e.g. Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 or in the
Examples below may be performed. Useful effector cells for such
assays include peripheral blood mononuclear cells (PBMC) and
Natural Killer (NK) cells. Alternatively, or additionally, ADCC
activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. PNAS
(USA) 95:652-656 (1998).
[0120] The polypeptide comprising a variant Fc region which
"exhibits increased ADCC" or mediates antibody-dependent
cell-mediated cytotoxicity (ADCC) in the presence of human effector
cells more effectively than a polypeptide having wild type IgG Fc
or a parent polypeptide is one which in vitro or in vivo is
substantially more effective at mediating ADCC, when the amounts of
polypeptide with variant Fc region and the polypeptide with wild
type Fc region (or the parent polypeptide) in the assay are
essentially the same. Generally, such variants will be identified
using the in vitro ADCC assay as herein disclosed, but other assays
or methods for determining ADCC activity, e.g. in an animal model
etc, are contemplated. In one embodiment, the preferred variant is
from about 5 fold to about 100 fold, e.g. from about 25 to about 50
fold, more effective at mediating ADCC than the wild type Fc (or
parent polypeptide).
[0121] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0122] Polypeptide variants with altered Fc region amino acid
sequences and increased or decreased C1q binding capability are
described in U.S. Pat. No. 6,194,551 and WO 99/51642. The contents
of those patent publications are specifically incorporated herein
by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184
(2000).
[0123] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. According to one
embodiment, the cells express at least Fc.gamma.RIII and perform
ADCC effector function. Examples of human leukocytes which mediate
ADCC include peripheral blood mononuclear cells (PBMC), natural
killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;
with PBMCs and NK cells being preferred. The effector cells may be
isolated from a native source thereof, e.g. from blood or PBMCs as
described herein.
[0124] Methods of measuring binding to FcRn are known (see, e.g.,
Ghetie 1997, Hinton 2004). Binding to human FcRn in vivo and scrum
half life of human FcRn high affinity binding polypeptides can be
assayed, e.g, in transgenic mice or transfected human cell lines
expressing human FcRn, or in primates administered with the Fc
variant polypeptides. In one embodiment, specifically the
anti-.beta.5.beta.1 antibodies of the invention having a variant
IgG Fc exhibits increased binding affinity for human FcRn over a
polypeptide having wild-type IgG Fc, by at least 2 fold, at least 5
fold, at least 10 fold, at least 50 fold, at least 60 fold, at
least 70 fold, at least 80 fold, at least 100 fold, at least 125
fold, at least 150 fold. In a specific embodiment, the binding
affinity for human FcRn is increased about 170 fold.
[0125] For binding affinity to FcRn, in one embodiment, the EC50 or
apparent Kd (at pH 6.0) of the polypeptide is less than 1 .mu.M,
more preferably less than or equal to 100 nM, more preferably less
than or equal to 10 nM. In one embodiment, for increased binding
affinity to Fc.gamma.RIII (F158; i.e. low-affinity isotype) the
EC50 or apparent Kd less is than or equal to 10 nM, and for
Fc.gamma.RIII (V158; high-affinity isotype) the EC50 or apparent Kd
is less than or equal to 3 nM. According to another embodiment, a
reduction in binding of an antibody to a Fc receptor relative to a
control antibody (e.g., the Herceptin.RTM. antibody) may be
considered significant relative to the control antibody if the
ratio of the values of the absorbances at the midpoints of the test
antibody and control antibody binding curves (e.g, A.sub.450
nm(antibody)/A.sub.450 nm(control Ab)) is less than or equal to
40%. According to another embodiment, an increase in binding of an
antibody to a Fc receptor relative to a control antibody (e.g., the
Herceptin.RTM. antibody) may be considered significant relative to
the control antibody if the ratio of the values of the absorbances
at the midpoints of the test antibody and control antibody binding
curves (e.g, A.sub.450 nm(antibody)/A.sub.450 nm(control Ab)) is
greater than or equal to 125%.
[0126] A "parent polypeptide" or "parent antibody" is a polypeptide
or antibody comprising an amino acid sequence from which the
variant polypeptide or antibody arose and against which the variant
polypeptide or antibody is being compared. Typically the parent
polypeptide or parent antibody lacks one or more of the Fc region
modifications disclosed herein and differs in effector function
compared to a polypeptide variant as herein disclosed. The parent
polypeptide may comprise a native sequence Fc region or an Fc
region with pre-existing amino acid sequence modifications (such as
additions, deletions and/or substitutions).
[0127] Antibodies of this invention can be derived from phage
display. As used herein, "library" refers to a plurality of
antibody or antibody fragment sequences, or the nucleic acids that
encode these sequences, the sequences being different in the
combination of variant amino acids that are introduced into these
sequences according to the methods of the invention.
[0128] "Phage display" is a technique by which variant polypeptides
are displayed as fusion proteins to at least a portion of coat
protein on the surface of phage, e.g., filamentous phage,
particles. A utility of phage display lies in the fact that large
libraries of randomized protein variants can be rapidly and
efficiently sorted for those sequences that bind to a target
antigen with high affinity. Display of peptide and protein
libraries on phage has been used for screening millions of
polypeptides for ones with specific binding properties. Polyvalent
phage display methods have been used for displaying small random
peptides and small proteins through fusions to either gene III or
gene VIII of filamentous phage. Wells and Lowman, Curr. Opin.
Struct. Biol., 3:355-362 (1992), and references cited therein. In a
monovalent phage display, a protein or peptide library is fused to
a gene III or a portion thereof, and expressed at low levels in the
presence of wild type gene III protein so that phage particles
display one copy or none of the fusion proteins. Avidity effects
are reduced relative to polyvalent phage so that sorting is on the
basis of intrinsic ligand affinity, and phagemid vectors are used,
which simplify DNA manipulations. Lowman and Wells, Methods: A
companion to Methods in Enzymology, 3:205-0216 (1991).
[0129] A "phagemid" is a plasmid vector having a bacterial origin
of replication, e.g., Co1E1, and a copy of an intergenic region of
a bacteriophage. The phagemid may be used on any known
bacteriophage, including filamentous bacteriophage and lambdoid
bacteriophage. The plasmid will also generally contain a selectable
marker for antibiotic resistance. Segments of DNA cloned into these
vectors can be propagated as plasmids. When cells harboring these
vectors are provided with all genes necessary for the production of
phage particles, the mode of replication of the plasmid changes to
rolling circle replication to generate copies of one strand of the
plasmid DNA and package phage particles. The phagemid may form
infectious or non-infectious phage particles. This term includes
phagemids which contain a phage coat protein gene or fragment
thereof linked to a heterologous polypeptide gene as a gene fusion
such that the heterologous polypeptide is displayed on the surface
of the phage particle.
[0130] The term "phage vector" means a double stranded replicative
form of a bacteriophage containing a heterologous gene and capable
of replication. The phage vector has a phage origin of replication
allowing phage replication and phage particle formation. The phage
is preferably a filamentous bacteriophage, such as an M13, fl, fd,
Pf3 phage or a derivative thereof, or a lambdoid phage, such as
lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative
thereof.
[0131] Covalent modifications of polypeptides such as peptibodies,
immunoadesins, antibodies and short peptides are included within
the scope of this invention. One type of covalent modification
includes reacting targeted amino acid residues of a polypeptide
with an organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of the
polypeptide. Derivatization with bifunctional agents is useful, for
instance, for crosslinking the polypeptide to a water-insoluble
support matrix or surface for use in the method for purifying
antibodies, and vice-versa. Commonly used crosslinking agents
include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxy-succinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0132] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0133] Other modifications include the conjugation of toxins to the
antagonists such as maytansine and maytansinoids, calicheamicin and
other cytotoxic agents.
[0134] Another type of covalent modification of the polypeptide
comprises linking the polypeptide to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, or polyoxyalkylenes, in the manner 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.
[0135] The polypeptide of the present invention can also be
modified if advantageous in a way to form a chimeric molecule
comprising the polypeptide fused to another, heterologous
polypeptide or amino acid sequence (e.g., immunoadhesins or
peptibodies).
[0136] In one embodiment, such a chimeric molecule comprises a
fusion of the polypeptide with a protein transduction domain which
targets the polypeptide for delivery to various tissues and more
particularly across the brain blood barrier, using, for example,
the protein transduction domain of human immunodeficiency virus TAT
protein (Schwarze et al., 1999, Science 285: 1569-72).
[0137] In another embodiment, such a chimeric molecule comprises a
fusion of the polypeptide with a tag polypeptide which provides an
epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino- or carboxyl-terminus
of the polypeptide. The presence of such epitope-tagged forms of
the polypeptide can be detected using an antibody against the tag
polypeptide. Also, provision of the epitope tag enables the
polypeptide to be readily purified by affinity purification using
an anti-tag antibody or another type of affinity matrix that binds
to the epitope tag. Various tag polypeptides and their respective
antibodies are known in the art. Examples include poly-histidine
(poly-His) or poly-histidine-glycine (poly-His-gly) tags; the flu
HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell.
Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10,
G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and
Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)];
the KT3 epitope peptide [Martin et al., Science, 255:192-194
(1992)]; an .alpha.-tubulin epitope peptide [Skinner et al., J.
Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein
peptide tag [Lutz-Freyermuth et al., PNAS USA, 87:6393-6397
(1990)].
[0138] In an alternative embodiment, the chimeric molecule can
comprise a fusion of the polypeptide with an immunoglobulin or a
particular region of an immunoglobulin. For a bivalent form of the
chimeric molecule (e.g., an "immunoadhesin"), such a fusion could
be to the Fc region of an IgG molecule. Ig fusions of this
invention include polypeptides that comprise approximately or only
residues 94-243, residues 33-53 or residues 33-52 of human in place
of at least one variable region within an Ig molecule. In a
particularly embodiment, the immunoglobulin fusion includes the
hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an
IgG1 molecule. For the production of immunoglobulin fusions see
also, U.S. Pat. No. 5,428,130.
[0139] The invention provides methods and compositions for
inhibiting or preventing relapse tumor growth or relapse cancer
cell growth. In various embodiments, a cancer is relapse tumor
growth or relapse cancer cell growth where the number of cancer
cells has not been significantly reduced, or has increased, or
tumor size has not been significantly reduced, or has increased, or
fails any further reduction in size or in number of cancer cells.
The determination of whether the cancer cells are relapse tumor
growth or relapse cancer cell growth can be made either in vivo or
in vitro by any method known in the art for assaying the
effectiveness of treatment on cancer cells. A tumor resistant to
anti-VEGF treatment is an example of a relapse tumor growth.
[0140] An "effective amount" of a polypeptide, antibody, antagonist
or composition as disclosed herein is an amount sufficient to carry
out a specifically stated purpose. An "effective amount" can be
determined empirically and by known methods relating to the stated
purpose.
[0141] The term "therapeutically effective amount" refers to an
amount of an antibody, polypeptide or antagonist of this invention
effective to "treat" a disease or disorder in a mammal (aka
patient). In the case of cancer, the therapeutically effective
amount of the drug can reduce the number of cancer cells; reduce
the tumor size or weight; inhibit (i.e., slow to some extent and
preferably stop) cancer cell infiltration into peripheral organs;
inhibit (i.e., slow to some extent and preferably stop) tumor
metastasis; inhibit, to some extent, tumor growth; and/or relieve
to some extent one or more of the symptoms associated with the
cancer. To the extent the drug can prevent growth and/or kill
existing cancer cells, it can be cytostatic and/or cytotoxic. In
one embodiment, the therapeutically effective amount is a growth
inhibitory amount. In another embodiment, the therapeutically
effective amount is an amount that extends the survival of a
patient. In another embodiment, the therapeutically effective
amount is an amount that improves progression free survival of a
patient.
[0142] In the case of wound healing, the term "effective amount" or
"therapeutically effective amount" refers to an amount of a drug
effective to accelerate or improve wound healing in a subject. A
therapeutic dose is a dose which exhibits a therapeutic effect on
the patient and a sub-therapeutic dose is a dose which does not
exhibit a therapeutic effect on the patient treated.
[0143] A "chronic wound" refers a wound that does not heal. See,
e.g., Lazarus et al., Definitions and guidelines for assessment of
wounds and evaluation of healing, Arch. Dermatol. 130:489-93
(1994). Chronic wounds include, but are not limited to, e.g.,
arterial ulcers, diabetic ulcers, pressure ulcers, venous ulcers,
etc. An acute wound can develop into a chronic wound. Acute wounds
include, but are not limited to, wounds caused by, e.g., thermal
injury, trauma, surgery, excision of extensive skin cancer, deep
fungal and bacterial infections, vasculitis, scleroderma,
pemphigus, toxic epidermal necrolysis, etc. See, e.g., Buford,
Wound Healing and Pressure Sores, HealingWell.com, published on:
Oct. 24, 2001. A "normal wound" refers a wound that undergoes
normal wound healing repair.
[0144] A "growth inhibitory amount" of a polypeptide, antibody,
antagonist or composition of this invention is an amount capable of
inhibiting the growth of a cell, especially tumor, e.g., cancer
cell, either in vitro or in vivo. A "growth inhibitory amount" of a
polypeptide, antibody, antagonist or composition of this invention
for purposes of inhibiting neoplastic cell growth can be determined
empirically and by known methods or by examples provided
herein.
[0145] A "cytotoxic amount" of a polypeptide, antibody, antagonist
or composition of this invention is an amount capable of causing
the destruction of a cell, especially tumor, e.g., cancer cell,
either in vitro or in vivo. A "cytotoxic amount" of a polypeptide,
antibody, antagonist or composition of this invention for purposes
of inhibiting neoplastic cell growth can be determined empirically
and by methods known in the art.
[0146] An "autoimmune disease" herein is a disease or disorder
arising from and directed against an individual's own tissues or a
co-segregate or manifestation thereof or resulting condition
therefrom. Examples of autoimmune diseases or disorders include,
but are not limited to arthritis (rheumatoid arthritis such as
acute arthritis, chronic rheumatoid arthritis, gouty arthritis,
acute gouty arthritis, chronic inflammatory arthritis, degenerative
arthritis, infectious arthritis, Lyme arthritis, proliferative
arthritis, psoriatic arthritis, vertebral arthritis, and
juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis
chronica progrediente, arthritis deformans, polyarthritis chronica
primaria, reactive arthritis, and ankylosing spondylitis),
inflammatory hyperproliferative skin diseases, psoriasis such as
plaque psoriasis, gutatte psoriasis, pustular psoriasis, and
psoriasis of the nails, dermatitis including contact dermatitis,
chronic contact dermatitis, allergic dermatitis, allergic contact
dermatitis, dermatitis herpetiformis, and atopic dermatitis,
x-linked hyper IgM syndrome, urticaria such as chronic idiopathic
urticaria, including chronic autoimmune urticaria,
polymyositis/dermatomyositis, juvenile dermatomyositis, toxic
epidermal necrolysis, scleroderma (including systemic scleroderma),
sclerosis such as systemic sclerosis, multiple sclerosis (MS) such
as spino-optical MS, primary progressive MS, and relapsing
remitting MS, progressive systemic sclerosis, atherosclerosis,
arteriosclerosis, sclerosis disseminata, and ataxic sclerosis,
inflammatory bowel disease (IBD) (for example, Crohn's disease,
colitis such as ulcerative colitis, colitis ulcerosa, microscopic
colitis, collagenous colitis, colitis polyposa, necrotizing
enterocolitis, and transmural colitis, and autoimmune inflammatory
bowel disease), pyoderma gangrenosum, erythema nodosum, primary
sclerosing cholangitis, episcleritis), respiratory distress
syndrome, including adult or acute respiratory distress syndrome
(ARDS), meningitis, inflammation of all or part of the uvea,
iritis, choroiditis, an autoimmune hematological disorder,
rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases
such as anaphylaxis and allergic and atopic rhinitis, encephalitis
such as Rasmussen's encephalitis and limbic and/or brainstem
encephalitis, uveitis, such as anterior uveitis, acute anterior
uveitis, granulomatous uveitis, nongranulomatous uveitis,
phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,
glomerulonephritis (GN) with and without nephrotic syndrome such as
chronic or acute glomerulonephritis such as primary GN,
immune-mediated GN, membranous GN (membranous nephropathy),
idiopathic membranous GN, membranous proliferative GN (MPGN),
including Type I and Type II, and rapidly progressive GN, allergic
conditions, allergic reaction, eczema including allergic or atopic
eczema, asthma such as asthma bronchiale, bronchial asthma, and
auto-immune asthma, conditions involving infiltration of T cells
and chronic inflammatory responses, chronic pulmonary inflammatory
disease, autoimmune myocarditis, leukocyte adhesion deficiency,
systemic lupus erythematosus (SLE) or systemic lupus erythematodes
such as cutaneous SLE, subacute cutaneous lupus erythematosus,
neonatal lupus syndrome (NLE), lupus erythematosus disseminatus,
lupus (including nephritis, cerebritis, pediatric, non-renal,
discoid, alopecia), juvenile onset (Type I) diabetes mellitus,
including pediatric insulin-dependent diabetes mellitus (IDDM),
adult onset diabetes mellitus (Type II diabetes), autoimmune
diabetes, idiopathic diabetes insipidus, immune responses
associated with acute and delayed hypersensitivity mediated by
cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including lymphomatoid granulomatosis, Wegener's
granulomatosis, agranulocytosis, vasculitides, including vasculitis
(including large vessel vasculitis (including polymyalgia
rheumatica and giant cell (Takayasu's) arteritis), medium vessel
vasculitis (including Kawasaki's disease and polyarteritis nodosa),
microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous,
or hypersensitivity vasculitis, systemic necrotizing vasculitis,
and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or
syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
hemolytic anemia or immune hemolytic anemia including autoimmune
hemolytic anemia (AIHA), pernicious anemia (anemia perniciosa),
Addison's disease, pure red cell anemia or aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome such as
those secondary to septicemia, trauma or hemorrhage,
antigen-antibody complex-mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Bechet's or Behcet's disease, Castleman's
syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's
syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid
bullous and skin pemphigoid, pemphigus (including pemphigus
vulgaris, pemphigus foliaccus, pemphigus mucus-membrane pemphigoid,
and pemphigus erythematosus), autoimmune polyendocrinopathies,
Reiter's disease or syndrome, immune complex nephritis,
antibody-mediated nephritis, chronic neuropathy such as IgM
polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as
developed by myocardial infarction patients, for example),
including thrombotic thrombocytopenic purpura (TTP) and autoimmune
or immune-mediated thrombocytopenia such as idiopathic
thrombocytopenic purpura (ITP) including chronic or acute ITP,
autoimmune disease of the testis and ovary including autoimmune
orchitis and oophoritis, primary hypothyroidism,
hypoparathyroidism, autoimmune endocrine diseases including
thyroiditis such as autoimmune thyroiditis, Hashimoto's disease,
chronic thyroiditis (Hashimoto's thyroiditis), or subacute
thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism,
Grave's disease, polyglandular syndromes such as autoimmune
polyglandular syndromes (or polyglandular endocrinopathy
syndromes), paraneoplastic syndromes, including neurologic
paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome
or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome,
encephalomyelitis such as allergic encephalomyelitis or
encephalomyelitis allergica and experimental allergic
encephalomyelitis (EAE), myasthenia gravis, cerebellar
degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus
syndrome (OMS), and sensory neuropathy, Sheehan's syndrome,
autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant
cell hepatitis, chronic active hepatitis or autoimmune chronic
active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis
obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome,
Berger's disease (IgA nephropathy), idiopathic IgA nephropathy,
linear IgA dermatosis, primary biliary cirrhosis,
pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,
Coeliac disease, celiac sprue (gluten enteropathy), refractory
sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral
sclerosis (ALS; Lou Gehrig's disease), coronary artery disease,
autoimmune inner ear disease (AIED), or autoimmune hearing loss,
opsoclonus myoclonus syndrome (OMS), polychondritis such as
refractory or relapsed polychondritis, pulmonary alveolar
proteinosis, amyloidosis, scleritis, a non-cancerous lymphocytosis,
a primary lymphocytosis, which includes monoclonal B cell
lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal
gammopathy of undetermined significance, MGUS), peripheral
neuropathy, paraneoplastic syndrome, channelopathies such as
epilepsy, migraine, arrhythmia, muscular disorders, deafness,
blindness, periodic paralysis, and channelopathies of the CNS,
autism, inflammatory myopathy, focal segmental glomerulosclerosis
(FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis,
autoimmune hepatological disorder, fibromyalgia, multiple endocrine
failure, Schmidt's syndrome, adrenalitis, gastric atrophy,
presenile dementia, demyelinating diseases such as autoimmune
demyelinating diseases, diabetic nephropathy, Dressler's syndrome,
alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon,
esophageal dysmotility, sclerodactyly, and telangiectasia), male
and female autoimmune infertility, mixed connective tissue disease,
Chagas' disease, rheumatic fever, recurrent abortion, farmer's
lung, erythema multiforme, post-cardiotomy syndrome, Cushing's
syndrome, bird-fancier's lung, allergic granulomatous angiitis,
benign lymphocytic angiitis, Alport's syndrome, alveolitis such as
allergic alveolitis and fibrosing alveolitis, interstitial lung
disease, transfusion reaction, leprosy, malaria, leishmaniasis,
kypanosomiasis, schistosomiasis, ascariasis, aspergillosis,
Sampter's syndrome, Caplan's syndrome, dengue, endocarditis,
endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis,
interstitial lung fibrosis, idiopathic pulmonary fibrosis, cystic
fibrosis, endophthalmitis, erythema elevatum et diutinum,
erythroblastosis fetalis, eosinophilic faciitis, Shulman's
syndrome, Felty's syndrome, flariasis, cyclitis such as chronic
cyclitis, heterochronic cyclitis, iridocyclitis, or Fuch's
cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus
(HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's
disease, parvovirus infection, rubella virus infection,
post-vaccination syndromes, congenital rubella infection,
Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune
gonadal failure, Sydenham's chorea, post-streptococcal nephritis,
thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis,
chorioiditis, giant cell polymyalgia, endocrine ophthamopathy,
chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca,
epidemic keratoconjunctivitis, idiopathic nephritic syndrome,
minimal change nephropathy, benign familial and
ischemia-reperfusion injury, retinal autoimmunity, joint
inflammation, bronchitis, chronic obstructive airway disease,
silicosis, aphthae, aphthous stomatitis, arteriosclerotic
disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease,
cryoglobulinemia, Dupuytren's contracture, endophthalmia
phacoanaphylactica, enteritis allergica, erythema nodosum leprosum,
idiopathic facial paralysis, chronic fatigue syndrome, febris
rheumatica, Hamman-Rich's disease, sensoneural hearing loss,
haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,
leucopenia, mononucleosis infectiosa, traverse myelitis, primary
idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis
granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma
gangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,
infertility due to antispermatozoan antobodies, non-malignant
thymoma, vitiligo, SCID and Epstein-Barr virus-associated diseases,
acquired immune deficiency syndrome (AIDS), parasitic diseases such
as Lesihmania, toxic-shock syndrome, food poisoning, conditions
involving infiltration of T cells, leukocyte-adhesion deficiency,
immune responses associated with acute and delayed hypersensitivity
mediated by cytokines and T-lymphocytes, diseases involving
leukocyte diapedesis, multiple organ injury syndrome,
antigen-antibody complex-mediated diseases, antiglomerular basement
membrane disease, allergic neuritis, autoimmune
polyendocrinopathies, oophoritis, primary myxedema, autoimmune
atrophic gastritis, sympathetic ophthalmia, rheumatic diseases,
mixed connective tissue disease, nephrotic syndrome, insulitis,
polyendocrine failure, peripheral neuropathy, autoimmune
polyglandular syndrome type I, adult-onset idiopathic
hypoparathyroidism (AOIH), alopecia totalis, dilated
cardiomyopathy, epidermolisis bullosa acquisita (EBA),
hemochromatosis, myocarditis, nephrotic syndrome, primary
sclerosing cholangitis, purulent or nonpurulent sinusitis, acute or
chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid
sinusitis, an eosinophil-related disorder such as eosinophilia,
pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,
Loffler's syndrome, chronic eosinophilic pneumonia, tropical
pulmonary eosinophilia, bronchopneumonic aspergillosis,
aspergilloma, or granulomas containing eosinophils, anaphylaxis,
seronegative spondyloarthritides, polyendocrine autoimmune disease,
sclerosing cholangitis, sclera, episclera, chronic mucocutaneous
candidiasis, Bruton's syndrome, transient hypogammaglobufinemi a of
infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia,
autoimmune disorders associated with collagen disease, rheumatism,
neurological disease, ischemic re-perfusion disorder, reduction in
blood pressure response, vascular dysfunction, antgiectasis, tissue
injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia,
and disease accompanying vascularization, allergic hypersensitivity
disorders, glomerulonephritides, reperfusion injury, reperfusion
injury of myocardial or other tissues, dermatoses with acute
inflammatory components, acute purulent meningitis or other central
nervous system inflammatory disorders, granulocyte
transfusion-associated syndromes, cytokine-induced toxicity, acute
serious inflammation, chronic intractable inflammation, pyelitis,
pneumonocirrhosis, diabetic retinopathy, diabetic large-artery
disorder, endarterial hyperplasia, peptic ulcer, valvulitis, and
endometriosis.
[0147] The term "detecting" is intended to include determining the
presence or absence of a substance or quantifying the amount of a
substance. The term thus refers to the use of the materials,
compositions, and methods of the present invention for qualitative
and quantitative determinations. In general, the particular
technique used for detection is not critical for practice of the
invention.
[0148] For example, "detecting" according to the invention may
include: observing the presence or absence of .alpha.5 gene
product, a .beta.1 gene product (e.g., mRNA molecules), or an
.alpha.5 or .alpha.5.beta.1 polypeptide; a change in the levels of
an .alpha.5 or .alpha.5.beta.1 polypeptide or amount bound to a
target; a change in biological function/activity of an .alpha.5 or
.beta.5.beta.1 polypeptide. In some embodiments, "detecting" may
include detecting wild type .alpha.5 or .beta.5.beta.1 levels
(e.g., mRNA or polypeptide levels). Detecting may include
quantifying a change (e.g., an increase or decrease) of any value
of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more when
compared to a control. Detecting may include quantifying a change
of any value of at least about 2-fold, 3-fold, 4-fold, 5-fold,
6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more e.g., 20-fold,
30-fold, 4-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,
100-fold or more.
[0149] "Label" when used herein refers to a detectable compound or
composition which is conjugated directly or indirectly to the
antibody. The label may itself be detectable by itself (e.g.,
radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical alteration of a substrate
compound or composition which is detectable.
III. Anti-.alpha.5.beta.1 Antibodies
[0150] Antibodies that can bind human .alpha.5.beta.1 and
competitively inhibit the binding of the anti-.alpha.5.beta.1,
18C12 antibody to human .alpha.5.beta.1 are provided herein.
Accordingly, one embodiment of the invention provides antibodies
comprising a variable light (VL) sequence set forth in any one of
SEQ ID NOS: 2, 3, 4, 5, 6, 7, or 8 and a variable heavy (VH)
sequence set forth in any one of SEQ ID NOS: 9, 10, 11, 12, 13, or
14. Human or chimeric (including, e.g., humanized) forms of the
antibodies of the anti-.beta.5.beta.1 antibodies described herein
are also contemplated.
[0151] According to one embodiment, the antibody binds a human
.alpha.5.beta.1 with a Kd between 500 nM and 1 pM. In certain
embodiments, an antibody provided herein has a dissociation
constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM
(e.g. 10.sup.-8M or less, e.g. from 10.sup.-8M to 10.sup.13M, e.g.,
from 10.sup.-9M to 10.sup.13 M). According to another embodiment,
the antibody does not bind .alpha.V.beta.3 or .alpha.V.beta.5 or
.alpha.V.beta.1. According to another embodiment, the antibody
comprises an Fc sequence of a human IgG, e.g., human IgG1 or human
IgG4. In another embodiment, an Fc sequence has been altered or
otherwise changed so that it that lacks antibody dependent cellular
cytotoxicity (ADCC) effector function, often related to their
binding to Fc receptors (FcRs). There are many examples of changes
or mutations to Fc sequences that can alter effector function. For
example, WO 00/42072 and Shields et al. J. Biol. Chem. 9(2):
6591-6604 (2001) describe antibody variants with improved or
diminished binding to FcRs. The contents of those publications are
specifically incorporated herein by reference. The antibody can be
in the form of a Fab, Fab', a F(ab)'.sub.2, single-chain Fv (scFv),
an Fv fragment; a diabody and a linear antibody. Also, the antibody
can be a multi-specific antibody that binds to .alpha.5.beta.1 and
is an .alpha.5.beta.1 antagonist, but also binds one or more other
targets and inhibits their function (e.g., VEGF) or a
multi-specific antibody that binds to two or more different sites
on .alpha.5.beta.1. The antibody can be conjugated to a therapeutic
agent (e.g., cytotoxic agent, a radioisotope and a chemotherapeutic
agent) or a label for detecting .alpha.5.beta.1 in patient samples
or in vivo by imaging (e.g., radioisotope, fluorescent dye and
enzyme).
[0152] Nucleic acid molecules encoding the anti-.alpha.5.beta.1
antibodies, expression vectors comprising nucleic acid molecules
encoding one or both variable domains, and cells comprising the
nucleic acid molecules are also contemplated. These antibodies can
be used in the therapies described herein and to detect
.alpha.5.beta.1 protein in patient samples (e.g., using FACS,
immunohistochemistry (IHC), ELISA assays) or in patients.
[0153] A. Monoclonal Antibodies
[0154] Monoclonal antibodies can be prepared, e.g., using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975) or can be made by recombinant DNA methods (U.S. Pat.
No. 4,816,567) or can be produced by the methods described herein
in the Examples below. In a hybridoma method, a hamster, mouse, or
other appropriate host animal is typically immunized with an
immunizing agent (e.g., an .alpha.5, .beta.1, or .alpha.5.beta.1
polypeptide alone or in combination with a suitable carrier) to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the immunizing agent.
Alternatively, the lymphocytes can be immunized in vitro.
[0155] The immunizing agent will typically include a polypeptide or
a fusion protein of the protein of interest or a composition
comprising the protein. Generally, either peripheral blood
lymphocytes ("PBLs") are used if cells of human origin are desired,
or spleen cells or lymph node cells are used if non-human mammalian
sources are desired. The lymphocytes are then fused with an
immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell. Goding, MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE (New York: Academic Press,
1986), pp. 59-103. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine, and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells can be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0156] Preferred immortalized cell lines are those that fuse
efficiently, support stable high-level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human mycloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies. Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., MONOCLONAL ANTIBODY
PRODUCTION TECHNIQUES AND APPLICATIONS (Marcel Dekker, Inc.: New
York, 1987) pp. 51-63.
[0157] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the polypeptide. The binding specificity of
monoclonal antibodies produced by the hybridoma cells can be
determined by immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RTA) or enzyme-linked immunoabsorbent
assay (ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980).
[0158] After the desired hybridoma cells are identified, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods. Goding, supra. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells can be
grown in vivo as ascites in a mammal.
[0159] The monoclonal antibodies secreted by the subclones can be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0160] The monoclonal antibodies can also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA can be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also can be modified, for example, by
substituting the coding sequence for human heavy- and light-chain
constant domains in place of the homologous murine sequences (U.S.
Pat. No. 4,816,567; Morrison et al., supra) or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0161] The antibodies can be monovalent antibodies. Methods for
preparing monovalent antibodies are known in the art. For example,
one method involves recombinant expression of immunoglobulin light
chain and modified heavy chain. The heavy chain is truncated
generally at any point in the Fc region so as to prevent
heavy-chain crosslinking. Alternatively, the relevant cysteine
residues are substituted with another amino acid residue or are
deleted so as to prevent crosslinking.
[0162] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly Fab fragments, can be accomplished using, but not
limited to, techniques known in the art.
[0163] B. Human Antibodies
[0164] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0165] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HUMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELOCIMOUSE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0166] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., PNAS.
USA, 103:3557-3562 (2006). Additional methods include those
described, for example, in U.S. Pat. No. 7,189,826 (describing
production of monoclonal human IgM antibodies from hybridoma cell
lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing
human-human hybridomas). Human hybridoma technology (Trioma
technology) is also described in Vollmers and Brandlein, Histology
and Histopathology, 20(3):927-937 (2005) and Vollmers and
Brandlein, Methods and Findings in Experimental and Clinical
Pharmacology, 27(3):185-91 (2005).
[0167] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0168] C. Library-Derived Antibodies
[0169] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, PNAS USA 101(34): 12467-12472 (2004); and Lee et
al. Immunol. Methods 284(1-2): 119-132(2004).
[0170] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0171] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0172] D. Chimeric and Humanized Antibodies
[0173] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., PNAS USA,
81:6851-6855 (1984)). In one example, a chimeric antibody comprises
a non-human variable region (e.g., a variable region derived from a
mouse, rat, hamster, rabbit, or non-human primate, such as a
monkey) and a human constant region. In a further example, a
chimeric antibody is a "class switched" antibody in which the class
or subclass has been changed from that of the parent antibody.
Chimeric antibodies include antigen-binding fragments thereof.
[0174] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) arc derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0175] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., PNAS USA
86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791,
6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005)
(describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498
(1991) (describing "resurfacing"); Dall'Acqua et al., Methods
36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al.,
Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,
83:252-260 (2000) (describing the "guided selection" approach to FR
shuffling).
[0176] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. PNAS USA, 89:4285
(1992); and Presta et al. J. Immunol., 151:2623 (1993)); human
mature (somatically mutated) framework regions or human germline
framework regions (see, e.g., Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008)); and framework regions derived from screening
FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0177] E. Multi-Specific Antibodies
[0178] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for .beta.5.beta.1
and the other is for any other antigen (e.g., VEGF). In certain
embodiments, bispecific antibodies may bind to two different
epitopes of .alpha.5.beta.1. Bispecific antibodies may also be used
to localize cytotoxic agents to cells which express
.alpha.5.beta.1. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments.
[0179] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., PNAS
USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers
(see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); and
preparing trispecific antibodies as described, e.g., in Tutt et al.
J. Immunol. 147: 60 (1991).
[0180] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576A1). The antibody or fragment
herein also includes a "Dual Acting FAb" or "DAF" comprising an
antigen binding site that binds to .beta.5.beta.1 as well as
another, different antigen (see, US 2008/0069820, for example).
[0181] F. Antibody Variants
[0182] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0183] 1. Substitution, Insertion, and Deletion Variants
[0184] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 1 under the heading of
"conservative substitutions." More substantial changes are provided
in Table 1 under the heading of "exemplary substitutions," and as
further described below in reference to amino acid side chain
classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or improved ADCC or CDC.
TABLE-US-00001 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile
Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine
Amino acids may be grouped according to common side-chain
properties:
[0185] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0186] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0187] (3) acidic: Asp, Glu;
[0188] (4) basic: His, Lys, Arg;
[0189] (5) residues that influence chain orientation: Gly, Pro;
[0190] (6) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another class.
[0191] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0192] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs
(a-CDRs), with the resulting variant VH or VL being tested for
binding affinity. Affinity maturation by constructing and
reselecting from secondary libraries has been described, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien
et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0193] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may be outside of HVR "hotspots" or SDRs. In certain
embodiments of the variant VH and VL sequences provided above, each
HVR either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0194] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0195] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0196] 2. Glycosylation Variants
[0197] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0198] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0199] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e. g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US Patent Publication Nos. US 2003/0157108 (Presta, L.); US
2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications
related to "defucosylated" or "fucose-deficient" antibody variants
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines capable of producing defucosylated antibodies include
Lec13 CHO cells deficient in protein fucosylation (Ripka et al.
Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,
especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0200] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). 3. Fc Region
Variants
[0201] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant, so as to enhance,
e.g., the effectiveness of the antibody in treating a disease or
disorder involving abnormal angiogenesis and/or vascular
permeability or leakage. The Fc region variant may comprise a human
Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc
region) comprising an amino acid modification (e.g. a substitution)
at one or more amino acid positions.
[0202] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting
examples of in vitro assays to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.
Hellstrom, I. et al. PNAS USA 83:7059-7063 (1986)) and Hellstrom, I
et al., PNAS USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M.
et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively,
non-radioactive assays methods may be employed (see, for example,
ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful
effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in a animal model such as that disclosed in
Clynes et al. PNAS USA 95:652-656 (1998). C1q binding assays may
also be carried out to confirm that the antibody is unable to bind
C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding
ELISA in WO 2006/029879 and WO 2005/100402. To assess complement
activation, a CDC assay may be performed (see, for example,
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg,
M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M.
J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo
clearance/half life determinations can also be performed using
methods known in the art (see, e.g., Petkova, S. B. et al., Int'l.
Immunol. 18(12):1759-1769 (2006)).
[0203] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0204] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0205] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0206] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0207] Mutations or alterations in the Fc region sequences can be
made to improve FcR binding (e.g., FcgammaR, FcRn). According to
one embodiment, an antibody of this invention has at least one
altered effector function selected from the group consisting of
ADCC, CDC, and improved FcRn binding compared to a native IgG or a
parent antibody. Examples of several useful specific mutations are
described in, e.g., Shields, R L et al. (2001) JBC 276(6)6591-6604;
Presta, L. G., (2002) Biochemical Society Transactions
30(4):487-490; and WO 00/42072. Antibodies with increased half
lives and improved binding to the neonatal Fc receptor (FcRn),
which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.
Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton
et al.). Those antibodies comprise an Fc region with one or more
substitutions therein which improve binding of the Fc region to
FcRn. Such Fc variants include those with substitutions at one or
more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,
311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or
434, e.g., substitution of Fc region residue 434 (U.S. Pat. No.
7,371,826).
[0208] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0209] 4. Cysteine Engineered Antibody Variants
[0210] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody arc substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
5400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0211] In some embodiments, cysteine residue(s) can be introduced
into the Fc region, thereby allowing interchain disulfide bond
formation in this region. The homodimeric antibody thus generated
can have improved internalization capability and/or increased
complement-mediated cell killing and antibody-dependent cellular
cytotoxicity (ADCC). See, Caron et al., J. Exp. Med., 176:
1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992).
Homodimeric antibodies with enhanced anti-tumor activity can also
be prepared using heterobifunctional cross-linkers as described in
Wolff et al., Cancer Research, 53: 2560-2565 (1993). Alternatively,
an antibody can be engineered that has dual Fc regions and can
thereby have enhanced complement lysis and ADCC capabilities. See,
Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
[0212] 5. Antibody Derivatives
[0213] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0214] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
[0215] G. Immunoconjugates
[0216] The invention also provides immunoconjugates comprising an
anti-.alpha.5.beta.1 antibody herein conjugated to one or more
cytotoxic agents, such as chemotherapeutic agents or drugs, growth
inhibitory agents, toxins (e.g., protein toxins, enzymatically
active toxins of bacterial, fungal, plant, or animal origin, or
fragments thereof), or radioactive isotopes.
[0217] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC 1065.
[0218] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0219] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the radioconjugate is used for detection, it
may comprise a radioactive atom for scintigraphic studies, for
example tc99m or 1123, or a spin label for nuclear magnetic
resonance (NMR) imaging (also known as magnetic resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0220] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-malcimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0221] The immunoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
[0222] H. Immunoliposomes
[0223] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al., PNAS
USA, 82: 3688 (1985); Hwang et al., PNAS USA, 77: 4030 (1980); and
U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced
circulation time are disclosed in U.S. Pat. No. 5,013,556.
[0224] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. An anti-neoplastic agent, a growth inhibitory agent, or a
chemotherapeutic agent (such as Doxorubicin) is optionally also
contained within the liposome. See, Gabizon et al., J. National
Cancer Inst., 81(19): 1484 (1989).
IV. Therapeutic Methods Using Anti-.beta.5.beta.1 Antibodies
[0225] Any of the anti-.alpha.5.beta.1 antibodies provided herein
may be used in therapeutic methods. It is understood that any of
the formulations or therapeutic methods described herein may be use
an immunoconjugate of the invention in place of or in addition to
an anti-.alpha.5.beta.1 antibody.
[0226] In one aspect, an anti-.beta.5.beta.1 antibody for use as a
medicament is provided. In further aspects, an anti-.beta.5.beta.1
antibody for use in treating diseases and disorders involving
abnormal angiogenesis and/or vascular permeability or leakage,
including, for example, cancer, ocular diseases, and immune
disorders (e.g., autoimmune disorders) is provided. In certain
embodiments, an anti-.beta.5.beta.1 antibody for use in a method of
treatment is provided. In certain embodiments, the invention
provides an anti-.beta.5.beta.1 antibody for use in a method of
treating an individual having a disease or disorder involving
abnormal angiogenesis and/or vascular permeability or leakage
comprising administering to the individual an effective amount of
the anti-.beta.5.beta.1 antibody. In one such embodiment, the
method further comprises administering to the individual an
effective amount of at least one, two, three, four, or more
additional therapeutic agent(s), including, e.g., an antiangiogenic
agent, an anti-neoplastic agent, a growth inhibitory agent, or a
chemotherapeutic agent as described herein. In further embodiments,
the invention provides an anti-.alpha.5.beta.1 antibody for use in
inhibiting abnormal angiogenesis and/or vascular permeability or
leakage. In certain embodiments, the invention provides an
anti-.alpha.5.beta.1 antibody for use in a method of inhibiting
abnormal angiogenesis and/or vascular permeability or leakage in an
individual comprising administering to the individual an effective
of the anti-.alpha.5.beta.1 antibody to inhibit abnormal
angiogenesis and/or vascular permeability or leakage. An
"individual" according to any of the above embodiments is
preferably a human.
[0227] In a further aspect, the invention provides for the use of
an anti-.alpha.5.beta.1 antibody in the manufacture or preparation
of a medicament. In one embodiment, the medicament is for treatment
of a disease or disorder involving abnormal angiogenesis and/or
vascular permeability or leakage In a further embodiment, the
medicament is for use in a method of treating a disease or disorder
involving abnormal angiogenesis and/or vascular permeability or
leakage comprising administering to an individual having a disease
or disorder involving abnormal angiogenesis and/or vascular
permeability or leakage an effective amount of the medicament. In
one such embodiment, the method further comprises administering to
the individual an effective amount of at least one, two, three,
four, or more additional therapeutic agent(s) including, e.g., an
antiangiogenic agent, an anti-neoplastic agent, a growth inhibitory
agent, or a chemotherapeutic agent as described herein. In a
further embodiment, the medicament is for inhibiting abnormal
angiogenesis and/or vascular permeability or leakage. In a further
embodiment, the medicament is for use in a method of inhibiting
abnormal angiogenesis and/or vascular permeability or leakage in an
individual comprising administering to the individual an amount
effective of the medicament to inhibit abnormal angiogenesis and/or
vascular permeability or leakage. An "individual" according to any
of the above embodiments may be a human.
[0228] In a further aspect, the invention provides a method for
treating a disease or disorder involving abnormal angiogenesis
and/or vascular permeability or leakage. In one embodiment, the
method comprises administering to an individual having such a
disease or disorder involving abnormal angiogenesis and/or vascular
permeability or leakage an effective amount of an
anti-.alpha.5.beta.1 antibody. In one such embodiment, the method
further comprises administering to the individual an effective
amount of at least one additional therapeutic agent, as described
below. An "individual" according to any of the above embodiments
may be a human.
[0229] In a further aspect, the invention provides a method for
inhibiting abnormal angiogenesis and/or vascular permeability or
leakage in an individual. In one embodiment, the method comprises
administering to the individual an effective amount of an
anti-.alpha.5.beta.1 antibody to inhibit abnormal angiogenesis
and/or vascular permeability or leakage. In one embodiment, an
"individual" is a human.
[0230] In a further aspect, the invention provides pharmaceutical
formulations comprising any of the anti-.beta.5.beta.1 antibodies
provided herein, e.g., for use in any of the above therapeutic
methods. In one embodiment, a pharmaceutical formulation comprises
any of the anti-.beta.5.beta.1 antibodies provided herein and a
pharmaceutically acceptable carrier. In another embodiment, a
pharmaceutical formulation comprises any of the anti-.beta.5.beta.1
antibodies provided herein and at least one additional therapeutic
agent, e.g., as described herein.
[0231] Combination therapies described herein encompass combined
administration (where two, three, four, or more therapeutic agents
are included in the same or separate formulations), and separate
administration, in which case, administration of the antibody of
the invention can occur prior to, simultaneously, and/or following,
administration of the additional therapeutic agent and/or adjuvant.
Antibodies of the invention can also be used in combination with
radiation therapy.
[0232] In certain embodiments, an additional therapeutic agent is a
VEGF antagonist (e.g., an anti-VEGF antibody such as, for example,
bevacizumab). In some embodiments, the anti-.beta.5.beta.1
antibodies are administered in combination with a VEGF antagonist.
The anti-.beta.5.beta.1 antibody and additional agent (e.g., a VEGF
antagonist) can be administered concurrently or sequentially.
Alternatively, the subject can be treated with the VEGF antagonist
and subsequently administered the .beta.5.beta.1 antagonist, e.g.,
treating with the VEGF antagonist until the subject is unresponsive
to VEGF antagonist treatment and then treating the subject is
treated with an .beta.5.beta.1 antagonist. According to one
embodiment, the subject is treated with the VEGF antagonist when
the cancer is non-invasive and then treated with the
.alpha.5.beta.1 antagonist when the cancer is invasive. Some
patients who experience elevated .beta.5.beta.1 levels naturally or
in response to VEGF antagonist therapy, compared to non-diseased
patients or control, can be especially responsive to this
combination treatment. Combinations further comprising a
therapeutic agent (e.g., an anti-neoplastic agent, a
chemotherapeutic agent, a growth inhibitory agent and a cytotoxic
agent) are contemplated. For example, patients who are to be
treated with chemotherapy (e.g., irinotecan) and .alpha.5.beta.1
antagonists, or who have been treated with chemotherapy and
.alpha.5.beta.1 antagonists, can benefit from VEGF antagonist
therapy. Alternatively, patients who have been treated with
chemotherapy and VEGF antagonists can benefit from .beta.5.beta.1
antagonist therapy. In one embodiment, the anti-VEGF antibody is
bevacizumab. In another embodiment, the anti-.alpha.5.beta.1
antibody is an anti-.beta.5.beta.1 antibody described herein.
[0233] An antibody of the invention (and any additional therapeutic
agent) can be administered by any suitable means, including
parenteral, intrapulmonary, and intranasal, and, if desired for
local treatment, intralesional administration. Parenteral infusions
include intramuscular, intravenous, intraarterial, intraperitoneal,
or subcutaneous administration. Dosing can be by any suitable
route, e.g. by injections, such as intravenous or subcutaneous
injections, depending in part on whether the administration is
brief or chronic. Various dosing schedules including but not
limited to single or multiple administrations over various
time-points, bolus administration, and pulse infusion are
contemplated herein.
[0234] Antibodies of the invention would be formulated, dosed, and
administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular
disorder being treated, the particular mammal being treated, the
clinical condition of the individual patient, the cause of the
disorder, the site of delivery of the agent, the method of
administration, the scheduling of administration, and other factors
known to medical practitioners. The antibody need not be, but is
optionally formulated with one or more agents currently used to
prevent or treat the disorder in question. The effective amount of
such other agents depends on the amount of antibody present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as described herein, or about from 1 to
99% of the dosages described herein, or in any dosage and by any
route that is empirically/clinically determined to be
appropriate.
[0235] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of antibody can be an initial candidate dosage for administration
to the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the antibody would be in the range from about 0.05 mg/kg
to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0
mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be
administered to the patient. Such doses may be administered
intermittently, e.g. every week or every three weeks (e.g. such
that the patient receives from about two to about twenty, or e.g.
about six doses of the antibody). An initial higher loading dose,
followed by one or more lower doses may be administered. However,
other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[0236] Depending on the indication to be treated and factors
relevant to the dosing that a physician of skill in the field would
be familiar with, the antibodies of the invention will be
administered at a dosage that is efficacious for the treatment of
that indication while minimizing toxicity and side effects.
[0237] Cancer treatments can be evaluated by, e.g., but not limited
to, tumor regression, tumor weight or size shrinkage, time to
progression, duration of survival, progression free survival,
overall response rate, duration of response, quality of life,
protein expression and/or activity. Because the anti-angiogenic
agents described herein target the tumor vasculature and not
necessarily the neoplastic cells themselves, they represent a
unique class of anticancer drugs, and therefore can require unique
measures and definitions of clinical responses to drugs. For
example, tumor shrinkage of greater than 50% in a 2-dimensional
analysis is the standard cut-off for declaring a response. However,
the .alpha.5.beta.1 antagonists and VEGF antagonists of the
invention may cause inhibition of metastatic spread without
shrinkage of the primary tumor, or may simply exert a tumouristatic
effect. Accordingly, approaches to determining efficacy of the
therapy can be employed, including for example, measurement of
plasma or urinary markers of angiogenesis and measurement of
response through radiological imaging.
[0238] Evaluation of treatments for age-related macular
degeneration (AMD) includes, but it is not limited to, a decrease
in the rate of further vision loss or the prevention of further
vision loss. For AMD therapy, efficacy in vivo can, for example, be
measured by one or more of the following: assessing the mean change
in the best corrected visual acuity (BCVA) from baseline to a
desired time, assessing the proportion of subjects who lose fewer
than 15 letters in visual acuity at a desired time compared with
baseline, assessing the proportion of subjects who gain greater
than or equal to 15 letters in visual acuity at a desired time
compared with baseline, assessing the proportion of subjects with a
visual-acuity Snellen equivalent of 20/2000 or worse at desired
time, assessing the NEI Visual Functioning Questionnaire, assessing
the size of CNV and amount of leakage of CNV at a desired time, as
assessed by fluorescein angiography, and the like.
V. Pharmaceutical Formulations
[0239] The anti-.alpha.5.beta.1 antibodies can be formulated with
suitable carriers or excipients so that they are suitable for
administration. Suitable formulations of the antibodies are
obtained by mixing an antibody having the desired degree of purity
with optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
olyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG). Exemplary antibody formulations are
described in WO98/56418, expressly incorporated herein by
reference. Exemplary pharmaceutically acceptable carriers herein
further include interstitial drug dispersion agents such as soluble
neutral-active hyaluronidase glycoproteins (sHASEGP), for example,
human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20
(HYLENEX.RTM., Baxter International, Inc.). Certain exemplary
sHASEGPs and methods of use, including rHuPH20, are described in US
Patent Publication Nos. 2005/0260186 and 2006/0104968. In one
aspect, a sHASEGP is combined with one or more additional
glycosaminoglycanases such as chondroitinases. Lyophilized
formulations adapted for subcutaneous administration are described
in WO97/04801. Such lyophilized formulations may be reconstituted
with a suitable diluent to a high protein concentration and the
reconstituted formulation may be administered subcutaneously to the
mammal to be treated herein.
[0240] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
further provide an anti-neoplastic agent, a growth inhibitory
agent, a cytotoxic agent, and/or a chemotherapeutic agent. Such
molecules are suitably present in combination in amounts that are
effective for the purpose intended. The effective amount of such
other agents depends on the amount of antibody present in the
formulation, the type of disease or disorder or treatment, and
other factors discussed above. These are generally used in the same
dosages and with administration routes as described herein or about
from 1 to 99% of the heretofore employed dosages.
[0241] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0242] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antagonist,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPOT.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0243] Lipofectins or liposomes can be used to deliver the
polypeptides and antibodies or compositions of this invention into
cells. Where antibody fragments arc used, the smallest inhibitory
fragment that specifically binds to the binding domain of the
target protein is preferred. For example, based upon the
variable-region sequences of an antibody, peptide molecules can be
designed that retain the ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA technology. See, e.g., Marasco et al.,
PNAS USA, 90: 7889-7893 (1993).
[0244] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethyl cellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's PHARMACEUTICAL SCIENCES,
supra.
[0245] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they can denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S-S bond formation through
thio-disulfide interchange, stabilization can be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0246] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
VI. Methods of Diagnosis and Imaging Using Anti-.beta.5.beta.1
Antibodies
[0247] Labeled anti-.alpha.5.beta.1 antibodies, and derivatives and
analogs thereof, which specifically bind to an .beta.5.beta.1
polypeptide can be used for diagnostic purposes to detect,
diagnose, or monitor diseases and/or disorders associated with the
expression, aberrant expression and/or activity of .alpha.5.beta.1.
For example, the anti-.alpha.5.beta.1 antibodies of the invention
can be used in in situ, in vivo, ex vivo, and in vitro diagnostic
assays or imaging assays.
[0248] Methods for detecting expression of an .beta.5.beta.1
polypeptide, comprising (a) assaying the expression of the
polypeptide in cells (e.g., tissue) or body fluid of an individual
using one or more antibodies of this invention and (b) comparing
the level of gene expression with a standard gene expression level,
whereby an increase or decrease in the assayed gene expression
level compared to the standard expression level is indicative of
aberrant expression.
[0249] Additional embodiments of the invention include methods of
diagnosing a disease or disorder associated with expression or
aberrant expression of .alpha.5.beta.1 in an animal (e.g., a mammal
such as a human). The methods comprise detecting .beta.5.beta.1
molecules in the mammal. In one embodiment, after administering a
VEGF antagonist, diagnosis comprises: (a) administering an
effective amount of a labeled anti-.beta.5.beta.1 antibody to a
mammal (b) waiting for a time interval following the administering
for permitting the labeled .alpha.5.beta.1 antibody to
preferentially concentrate at sites in the subject where the
.beta.5.beta.1 molecule is expressed (and for unbound labeled
molecule to be cleared to background level); (c) determining
background level; and (d) detecting the labeled molecule in the
subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with expression or aberrant
expression of .alpha.5.beta.1. Background level can be determined
by various methods including, comparing the amount of labeled
molecule detected to a standard value previously determined for a
particular system.
[0250] Anti-.alpha.5.beta.1 antibodies of the invention can be used
to assay protein levels in a biological sample using classical
immunohistological methods known to those of skill in the art
(e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985);
Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other
antibody-based methods useful for detecting protein gene expression
include immunoassays, such as the enzyme linked immunosorbent assay
(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay
labels are known in the art and include enzyme labels, such as,
glucose oxidase; radioisotopes, such as iodine (.sup.131I,
.sup.125I, .sup.123I, .sup.121I), carbon (.sup.14C), sulfur
(.sup.35S), tritium (.sup.3H), indium (.sup.115mIn, .sup.113mIn,
.sup.112In, .sup.111In), and technetium (.sup.99Tc, .sup.99mTc),
thallium (.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium
(.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.153SM, .sup.177Lu, .sup.159Gd, .sup.149Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru; luminol;
and fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0251] Techniques known in the art may be applied to labeled
antibodies of the invention. Such techniques include, but are not
limited to, the use of bifunctional conjugating agents (see e.g.,
U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;
5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560; and 5,808,003).
[0252] According to one specific embodiment, .beta.5.beta.1
polypeptide expression or overexpression is determined in a
diagnostic or prognostic assay after administration of a VEGF
antagonist therapeutic agent by evaluating levels of
.alpha.5.beta.1 present on the surface of a cell (e.g., via an
immunohistochemistry assay using anti-.alpha.5.beta.1 antibodies).
Alternatively, or additionally, one can measure levels of
.alpha.5.beta.1 polypeptide-encoding nucleic acid or mRNA in the
cell, e.g., via fluorescent in situ hybridization using a nucleic
acid based probe corresponding to an .alpha.5.beta.1-encoding
nucleic acid or the complement thereof; (FISH; see WO98/45479
published October, 1998), Southern blotting, Northern blotting, or
polymerase chain reaction (PCR) techniques, such as real time
quantitative PCR (RT-PCR). One can also study .beta.5.beta.1
overexpression by measuring shed antigen in a biological fluid such
as serum, e.g., using antibody-based assays (see also, e.g., U.S.
Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr.
18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et
al., J. Immunol. Methods 132:73-80 (1990)). Aside from the above
assays, various in vivo and ex vivo assays are available to the
skilled practitioner. For example, one can expose cells within the
body of the mammal to an antibody which is optionally labeled with
a detectable label, e.g., a radioactive isotope, and binding of the
antibody to the can be evaluated, e.g., by external scanning for
radioactivity or by analyzing a sample (e.g., a biopsy or other
biological sample) taken from a mammal previously exposed to the
antibody.
VII. Articles of Manufacture and Kits
[0253] Another embodiment of the invention is an article of
manufacture containing materials useful for the treatment of cancer
(e.g. tumors), ocular disease (e.g., wet AMD) or autoimmune
diseases and related conditions. The article of manufacture can
comprise a container and a label or package insert on or associated
with the container. Suitable containers include, for example,
bottles, vials, syringes, etc. The containers may be formed from a
variety of materials such as glass or plastic. Generally, the
container holds a composition which is effective for treating the
condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is a VEGF antagonist or an
.beta.5.beta.1 antagonist or an VEGF agonist or an .beta.5.beta.1
agonist of the invention. The label or package insert indicates
that the composition is used for treating the particular condition.
The label or package insert will further comprise instructions for
administering the antibody composition to the patient. Articles of
manufacture and kits comprising combinatorial therapies described
herein are also contemplated.
[0254] Package insert refers to instructions customarily included
in commercial packages of therapeutic products, that contain
information about the indications, usage, dosage, administration,
contraindications and/or warnings concerning the use of such
therapeutic products.
[0255] Additionally, the article of manufacture may further
comprise a second container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0256] Kits are also provided that are useful for various purposes,
e.g., for isolation or detection of .alpha.5.beta.1 and/or VEGF in
patients, optionally in combination with the articles of
manufacture. For isolation and purification of .alpha.5.beta.1, the
kit can contain an anti-.alpha.5.beta.1 antibody coupled to beads
(e.g., sepharose beads). Kits can be provided which contain the
antibodies for detection and quantitation of .alpha.5.beta.1 and/or
VEGF in vitro, e.g. in an ELISA or a Western blot. As with the
article of manufacture, the kit comprises a container and a label
or package insert on or associated with the container. For example,
the container holds a composition comprising at least one
anti-.beta.5.beta.1 antibody of the invention. Additional
containers may be included that contain, e.g., diluents and
buffers, control antibodies. The label or package insert may
provide a description of the composition as well as instructions
for the intended in vitro or diagnostic use.
[0257] Commercially available reagents referred to in the Examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
Examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Unless otherwise noted, the present invention uses standard
procedures of recombinant DNA technology, such as those described
hereinabove and in the following textbooks: Sambrook et al., supra;
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green
Publishing Associates and Wiley Interscience, N.Y., 1989); Innis et
al., PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (Academic
Press, Inc.: N.Y., 1990); Harlow et al., ANTIBODIES: A LABORATORY
MANUAL (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait,
OLIGONUCLEOTIDE SYNTHESIS (IRL Press: Oxford, 1984); Freshney,
ANIMAL CELL CULTURE, 1987; Coligan et al., CURRENT PROTOCOLS IN
IMMUNOLOGY, 1991.
EXAMPLES
[0258] The following examples are offered to illustrate, but not to
limit the claimed invention. Those of skill in the art will readily
recognize a variety of noncritical parameters which can be changed
or modified to yield essentially the same results.
Example 1
Materials and Methods
[0259] A. BIAcore Analysis:
[0260] To determine binding kinetics and affinities of the
antibodies described herein, Surface Plasmon Resonance (SRP)
measurement with a BIAcore.TM.-3000 instrument was used. Antibody
was first captured by pre-immobilized rabbit anti-human Fc CM5
biosensor chip on the different flow cell to achieve approximately
150 RU (Response Units). For kinetics measurements, 2-fold serial
dilutions of human integrin .alpha.5.beta.1 (300 nM to 1.2 nM) were
injected in PBT buffer (PBS with 0.05% Tween 20) at 25.degree. C.
with a flow rate of 30 .mu.l/min. Association rates (k.sub.on) and
dissociation rates (k.sub.off) were calculated using a simple
one-to-one Langmuir binding model (BIAcore Evaluation Software
version 3.2). The equilibrium dissociation constant (K.sub.D) was
calculated as the ratio k.sub.off/k.sub.on.
[0261] B. Fibronectin Binding Assay with U937 Cells
[0262] 100 .mu.l of 10 .mu.g/ml Fibronectin (R & D Systems)
diluted in PBS pH 7.4 was added to the wells of 96-well plates
(Nuen MaxiSorp) and incubated overnight at 2-8.degree. C. Plates
were washed 3 times with 200 .mu.L PBS pH 7.4. 200 .mu.l PBS pH
7.4/1% BSA was added to all wells and the plates were incubated for
at least 60 minutes at room temperature with gentle agitation.
Plates were washed 3 times with 200 .mu.L PBS pH 7.4.
[0263] 50 .mu.l of diluted anti-.beta.5.beta.1 antibody was added
to each well (10 .mu.g/mL serially diluted 1:3 10 times),
immediately followed by 50 .mu.L U937 cells (ATCC, Cat#CRL-1593.2)
at a concentration of 1.times.10.sup.6 cells/mL in Assay Media
(RPMI 1640 (Media Prep A0806), 5 mg/mL BSA, 1 mM L-glutamine, 1 mM
MgCl.sub.2).
[0264] Plates were incubated for 30 min at 37.degree. C., unbound
cells were discarded and the plates were washed with twice with 150
.mu.L Assay Media. 100 .mu.l of Assay Media and 100 .mu.L of
Cell-Titer Glow (Promega Cat#G7573) were added to each well. The
plates were incubated for 10-15 minutes at room temperature,
covered. Luminescence was detected on a VICTOR .sup.2V
(Perkin-Elmer) plate reader and the luminescence units were
analyzed relative to the concentration of the antibodies used in
the assay, using a four-parameter nonlinear least squares fit, to
obtain the IC.sub.50 values.
[0265] C. Fibronectin Binding Assay with .alpha.5.beta.1
[0266] 25 .mu.l of 1 .mu.g/ml Fibronectin (R & D Systems)
diluted in PBS, was added to the wells of 384-well plates (Nucn
MaxiSorp) and incubated overnight at 2-8.degree. C. Plates were
washed 3 times with 80 .mu.L PBT buffer. 50 .mu.l PBS/1% BSA was
added to all wells, the plates were incubated for at least 60
minutes at room temperature with gentle agitation, then washed 3
times with 80 .mu.L PBT buffer.
[0267] anti-.beta.5.beta.1 antibody (30 .mu.g/mL) was serially
diluted 1:3 11 times. 50 .mu.l of the serially diluted
anti-.beta.5.beta.1 antibody was added to 0.65 mL micro tubes and
premixed (1:1) with 50 .mu.L of 200 ng/mL .beta.5.beta.1 (R & D
Systems) in ELISA buffer (150 mM NaCl, 10 mM Tris, pH 7.5, 5 mg/mL
BSA, 1 mM MnCl.sub.2) for 60 minutes at room temperature with
gentle agitation. 25 .mu.L of the premixed .alpha.5.beta.1 and
anti-.beta.5.beta.1 antibody was added to the Fibronectin coated
wells and the plates were incubated at room temperature for 60
minutes with gentle agitation. Plates were washed 6 times with PBT
buffer, 100 ng/mL anti-.beta.1-biotin in ELISA buffer was added to
each well and the plates were incubated for 60 minutes at room
temperature with gentle agitation. Plates were washed 6 times with
80 .mu.L PBT buffer, 25 .mu.L of Streptadvidin-HRP (1:50,000 in
ELISA buffer, GE Healthcare) was added to each well, and the plates
were incubated for 60 minutes at room temperature with gentle
agitation. Plates were washed 6 times with 80 .mu.L PBT buffer, 25
.mu.l of TMB substrate (Kirkgaard and Perry Laboratories) was added
to each well, plates were incubated about 6 minutes at room
temperature, and the reaction was stopped with 25 .mu.L of 1 M
Phosphoric Acid. Absorbance was read at 450 nm and IC.sub.50 values
were analyzed as described in section B above.
[0268] D. HUVEC Migration Assay
[0269] HUVEC cells were grown in standard media to 80% confluency.
The cells were trypsinized, counted, and resuspended in EBM-2 media
with 0.1% BSA at a concentration of 5.times.10.sup.5 cells/ml. For
the assay, 100 .mu.l of cells (5.times.10.sup.4 cells/well) were
plated per well in a BD Falcon HTS Multiwell System 24 well (BD
Ref: 351185, Pore size 8 .mu.m). The HTS plates were pre-coated
with Fibronectin (1-2 .mu.g/ml) overnight. The plates were washed
with PBS and 500 .mu.l (EBM with 0.1% BSA) solution was added to
the bottom chamber. For each sample, 3-6 wells were used. No
migration stimulus (e.g., VEGF) was added to the negative control
well.
[0270] To start the assay, 100 .mu.l of cells (5.times.10.sup.4
cells/well) were added to the upper chamber. The lower chamber
contained 500 .mu.l of media (EBM with 0.1% BSA). Anti-integrin
.alpha.5.beta.1 antibodies and isotype controls are generally added
to the upper chamber with the cells at a final concentration of 0
.mu.g/ml, 1 .mu.g/ml or 5 .mu.g/ml, and incubated for 15 minutes
before the migration stimulus (VEGF) was added to the lower
chamber.
[0271] Stimulus was added to the lower chamber (10 ng/ml VEGF-A),
and plates were incubated 4-6 hours, or overnight. Cells were
scraped from the upper chamber using a sponge swab, PBS was added,
then the cells from the upper chamber were scraped again. The media
was drained from the lower chamber and the cells were fixed with
500 .mu.l of 100% methanol for 5 minutes.
[0272] The cells were then stained with 500 .mu.l SYTOX.RTM. Green
nuclei acid stain (1:5000 or 1:10000 dilution in PBS) (Molecular
Probes 57020) for at least 10 min (in the dark). Then, a microscope
was used to take individual pictures of each well. Pictures were
taking using an AxioVision AC program and a 5.times. objective.
[0273] ImageJ was used to analyze the results (minimum 5 pixels,
bin 5). The number of cells per well was recorded and analyzed
using Microsoft Excel.
Example 2
Generation of Anti-.alpha.5.beta.1 Antibodies
[0274] Anti .alpha.5.beta.1 antibodies were generated by immunizing
hamsters with recombinant human .alpha.5.beta.1 extracellular
domain polypeptides. Hamster monoclonal antibody clone 18C12
comprising the VH and VL sequences set forth in FIGS. 1 and 2 was
selected.
[0275] Two versions of chimeric hamster 18C12, h18C12.v1.1 and
h18C12.v2.1, were generated by cloning the light chain from the
hamster 18C12 into two consensus VL.sub.kappa1/VH.sub.III
frameworks: h18C12.v1.1 and h18C12.v2.1 which differ only in the
CDR-H3 contact region. Specifically, the CDR-H3 contact region of
h18C12.v1.1 comprises the following residues:
C.sup.92A.sup.93R.sup.94 and the CDR-H3 contact region of hl
8C12.v2.1 comprises the following residues:
C.sup.92T.sup.93S.sup.94. Both h18C12.v1.1 and h18C12.v2.1 were
successfully displayed on phage in Fab format and binding was
observed by phage competition ELISA. No significant differences in
binding were observed between h18C12.v1.1 and h18C12.v2.1.
h18C12.v1.1 was selected for further humanization. Humanized
18C12.v3 was generated by changing most of the light chain
framework of h18C12.v1.1 into the human consensus lambda IV
framework with the exception of the following residues in the light
chain: Y36, A43, and Y49. In addition, the following residues in
the human VH.sub.III framework: G49 and D73 were retained.
[0276] h18C12.v3 was affinity matured and the following clones were
generated: h18C12.v6; h18C12.v7; h18C12.v9; h18C12.v15; h18C12.v16;
h18C12.v28; h18C12.v30; h18C12.v51; h18C12.v54; h18C12.v70;
h18C12.v78. CDR sequences for each of the clones are set forth in
FIG. 3. CDR sequences for the h18C12.v6 clone were modified as
follows: CDR-L2: D50cS and D56S; CDR-H1: N31A; and CDR-H2: N53A.
The modified CDR sequences were then inserted into a modified human
.lamda.III//VHIII framework to generate h18C12.v6.1Lam3
(h18C12.v6.1) and h18C12.v6.2Lam3 (h18C12.v6.2). The modified human
.lamda.III framework for h18C12.v6.1 contained the following
modifications: L46Y; V47L; I48M; N69A; A71R; and G77N. The modified
human .lamda.III framework for h18C12.v6.2 contained the following
modifications: N69A; A71R; and G77N. The modified VHIII framework
contained the following modifications: S49G and N73D.
[0277] Clone h18C12.v6.1 was further modified to remove Asparagine
(N) in CDR-L2 (N.sup.50aS.sup.50bS.sup.50cG.sup.50d). The following
clones were generated: h18C12.v6.1.1; h18C12.v6.1.2; h18C12.v6.1.3;
h18C12.v6.1.4; and h18C12.v6.1.5. The CDR-L2 sequence at positions
50a, 50b, 50c, and 50d for each of the h18C12.v6.1 clones is set
forth in FIG. 7.
Example 3
Phage Competition ELISA
[0278] MAXISORP.TM. microtiter plates were coated with recombinant
human integrin .beta.5.beta.1 (R&D) at 5 .mu.g/ml in PBS
overnight and then blocked with PBST buffer (0.5% BSA and 0.05%
Tween 20 in PBS) for an hour at room temperature. Phage from
culture supernatants were incubated with serially diluted human
integrin .alpha.5.beta.1 in PBST buffer in a tissue-culture
microtiter plate for an hour, after which 80 .mu.l of the mixture
was transferred to the target-coated wells for 15 minutes to
capture unbound phage. The plate was washed with PBT buffer (0.05%
Tween 20 in PBS), and HRP-conjugated anti-M13 (Amersham Pharmacia
Biotech) was added (1:5000 in PBST buffer) for 40 minutes. The
plate was washed with PBT buffer and developed by adding
tetramethylbenzidine substrate (Kirkegaard and Perry Laboratories,
Gaithersburg, Md.). The absorbance at 450 nm was plotted as a
function of target concentration in solution to determine phage
IC.sub.50. This was used as an affinity estimate for the Fab clone
displayed on the surface of the phage. FIG. 4 depicts results from
a phage competition assay demonstrating the binding of the affinity
matured 18C12 variants h18C12.v3; h18C12.v6; h18C12.v7; h18C12.v9;
h18C12.v15; h18C12.v16; h18C12.v28; h18C12.v30; h18C12.v51;
h18C12.v54; h18C12.v70; and h18C12.v78 to human .beta.5.beta.1
integrin.
Example 4
Antibody Affinity Determinations by BIAcore
[0279] To determine binding kinetics and affinities of chimeric
18C12, h18C12.v6.1, h18C12.v6.1.1, h18C12.v6.1.2, h18C12.v6.1.3,
h18C12.v6.1.4, h18C12.v6.1.5 IgGs, Surface Plasmon Resonance (SRP)
measurement with a BIAcore.TM.-3000 instrument as described in
Example 1 was used.
[0280] FIG. 5 depicts results from BIAcore analysis of the binding
of affinity matured 18C12 variants to human .beta.5.beta.1
integrin. FIG. 6 depicts results from BIACore analysis of the
binding of chimeric 18C12 (18C12 comprising the hamster VL and VH
sequences set forth in FIG. 1 and the Fc portion of IgG1) and
h18C12.v6.1 to human .alpha.5.beta.1 integrin. The data demonstrate
that h18C12.v6.1 has a binding affinity for human .alpha.5.beta.1
integrin that is approximately 2 times higher than the binding
affinity of hamster 18C12 for human .beta.5.beta.1 integrin. FIG. 7
depicts results from BIAcore analysis for each of the h18C12.v6.1
clones.
Example 5
18C12 Antibodies Inhibit Binding to Fibronectin
[0281] To compare the effects of chimeric 18C12 and h18C12.v6.1 or
h18C12.v6.1.5 IgGs on binding to fibronectin, the fibronectin
binding assays described in Example 1 above was used.
[0282] FIG. 8 depicts the results of a fibronectin binding assay
comparing the ability of chimeric 18C12 and h18C12.v6.1 to
interfere with the binding of U937 cells to fibronectin. FIG. 9
depicts the results of a fibronectin binding assay comparing the
ability of hamster 18C12 and h18C12.v6.1.5 to interfere with the
binding of U937 cells to fibronectin. FIG. 10 depicts the results
of a fibronectin binding assay comparing the ability of hamster
18C12 and h18C12.v6.1.5 to interfere with the binding of
recombinant .alpha.5.beta.1 extracellular domain to fibronectin.
The data demonstrate that hamster 18C12, chimeric 18C12,
h18C12.v6.1, and h18C12.v6.1.5 inhibit binding of U937 cells or
recombinant .alpha.5.beta.1 extracellular domain to
fibrinectin.
Example 6
18C12 Antibodies Inhibit HUVEC Migration
[0283] To compare the effects of chimeric 18C12 and h18C12.v6.1
IgGs on migration of HUVEC cells, the HUVEC migration assay
described in Example 1 above was used.
[0284] FIG. 11 depicts the results of a HUVEC migration assay
comparing the ability of chimeric 18C12 and h18C12.v6.1 to
interfere with migration of HUVEC cells on fibronectin. The data
demonstrate that chimeric 18C12 and h18C12.v6.1 inhibit migration
of HUVEC cells on fibrinectin.
Example 7
Embryonic Development Assays
[0285] .alpha.5 transgenic mice expressing a chimeric
.beta.5.beta.1 integrin (human .alpha.5 and murine .beta.1) were
used in embryonic development assays to demonstrate the efficacy of
the 18C12 antibodies described herein. Male transgenic mice
expressing human .alpha.5 and no murine .alpha.5 were mated to
female mice not expressing human .alpha.5 and heterozygous for
murine .alpha.5. Pregnant mice were injected i.p. with 10 mg/kg
anti-.beta.5.beta.1 antibody (h18C12.v6.1.5) or with a negative
control antibody 2.times./week starting at 9.5 days post-coitum. At
day 14.5 post-coitum, the embryos were collected, genotyped, and
embryonic vasculature was evaluated. The results are summarized in
the table below.
TABLE-US-00002 Integrin .alpha.5 Treatment Expression Vascular
Development h18C12.v6.1.5 only human .alpha.5 Hemorrhage, stunting,
and extensive systemic edema h18C12.v6.1.5 only murine .alpha.5
Normal negative control antibody only human .alpha.5 Normal
negative control antibody only murine .alpha.5 Normal
[0286] These results demonstrate that h18C12.v6.1.5 disrupts
.beta.5.beta.1 integrin function in vivo during vascular
development.
Example 8
Tumor Allograft Assays
[0287] C57/B16 allograft tumor models are used in studies to
demonstrate the efficacy of the 18C12 antibodies described herein.
C57/B16 tumor cells are implanted into murine as knockout::human
.alpha.5 transgenic mice and the mice are treated with
anti-.alpha.5.beta.1 antibodies.
[0288] Mice bearing established tumors are randomized into four
groups, so that all the groups have similar starting tumor volumes.
The dosing regimen is as follows:
[0289] (1) Negative control antibody (13.5 mg/kg,
2.times./week)
[0290] (2) Anti-VEGF antibody (3.5 mg/kg, 2.times./week)+Negative
control antibody (10 mg/kg, 2.times./week)
[0291] (3) Negative control antibody (3.5 mg/kg,
2.times./week)+anti-.beta.5.beta.1 antibody (18C12) (10 mg/kg,
2.times./week)
[0292] (4) Anti-VEGF antibody (3.5 mg/kg,
2.times./week)+anti-.beta.5.beta.1 antibody (18C12) (10 mg/kg,
2.times./week)
[0293] The length and width of tumors are measured twice per week
using calipers, and tumor volumes calculated using this formulus:
Tumor Volume (mm.sup.3)=(w.sup.2.times.1)/2, where w=width and
l=length in mm. Tumor volumes are plotted against time to reflect
tumor growth rates.
Example 9
Tumor Xenograft Assays
[0294] Tumor xenograft assays were used in studies to demonstrate
the efficacy of the 18C12 antibodies described herein. Human U87
glioma cells harbouring a luciferase reporter gene are implanted
into the brains of nude mice and the mice are treated with
anti-.alpha.5131 antibodies.
[0295] Mice bearing established tumors were imaged using
bioluminescence imaging and randomized into three groups, so that
all the groups had a similar starting tumor burden. The dosing
regimen was as follows:
[0296] (1) Negative control antibodies (ragweed) (5 mg/kg,
2.times./week)+anti-gD (10 mg/kg, 2.times./week)
[0297] (2) Anti-VEGF antibody (B20-4.1) (5 mg/kg,
2.times./week)+negative control antibody anti-gD (10 mg/kg,
2.times./week)
[0298] (3) Anti-VEGF antibody (B20-4A) (5 mg/kg,
2.times./week)+anti-.beta.5.beta.1 antibody (h18C12.v6.1.5) (10
mg/kg, 2.times./week)
[0299] The survival of these mice was monitored daily following
surgical implantation of tumor cells. Treatment with the
anti-.alpha.5.beta.1 antibody h18C12.v6.1.5 in combination with
anti-VEGF significantly enhanced the survival of mice relative to
anti-VEGF alone. The results are shown in FIG. 12. Tumor burdens
were also measured at weeks 1, 3, 4, and 5 following implantation
using bioluminescence imaging. Mice treated with the
anti-.alpha.5.beta.1 antibody h18C12.v6.1.5 in combination with
anti-VEGF had a reduced tumor burden relative to mice treated with
anti-VEGF alone. The results are shown in FIG. 13.
[0300] All publications (including, e.g., patents, published patent
applications, and Genbank Accession Nos.) cited herein are hereby
incorporated by reference in their entirety for all purposes as if
each reference were specifically and individually incorporated by
reference.
Sequence CWU 1
1
401110PRTHomo sapienVariant22-36X is any amino acid 1Ser Glu Leu
Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg
Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa
Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly Gln Ala 35 40 45Pro Val Leu
Val Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Gly
Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn 65 70 75Thr Ala Ser
Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala 80 85 90Asp Tyr Tyr
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 95 100 105Phe Gly
Gly Gly Thr 1102106PRTCricetulus griseus 2Gln Pro Val Leu Thr Gln
Ser Pro Ser Ala Ser Ala Ser Leu Gly1 5 10 15Asn Ser Val Lys Ile Thr
Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30Tyr Thr Ile Gly Trp Tyr
Gln Gln His Pro Asp Lys Ala Pro Lys 35 40 45Tyr Val Met Tyr Leu Asn
Ser Asp Gly Ser His Asn Lys Gly Asp 50 55 60Gly Ile Pro Asp Arg Phe
Ser Gly Ser Ser Ser Gly Ala His Arg 65 70 75Tyr Leu Ser Ile Ser Asn
Ile Gln Pro Glu Asp Glu Ala Asp Tyr 80 85 90Phe Cys Gly Ser Ser Tyr
Ser Ser Gly Tyr Val Phe Gly Ser Gly 95 100 105Thr3106PRTHomo sapien
3Asp Ile Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly1 5 10
15Asn Ser Val Lys Ile Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25
30Tyr Thr Ile Gly Trp Tyr Gln Gln His Pro Asp Lys Ala Pro Lys 35 40
45Tyr Val Met Tyr Leu Asn Ser Asp Gly Ser His Asn Lys Gly Asp 50 55
60Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala His Arg 65 70
75Tyr Leu Ser Ile Ser Asn Ile Gln Pro Glu Asp Glu Ala Asp Tyr 80 85
90Phe Cys Gly Ser Ser Tyr Ser Ser Gly Tyr Val Phe Gly Ser Gly 95
100 105Thr4106PRTHomo sapien 4Glu Pro Val Leu Thr Gln Ser Pro Ser
Ala Ser Ala Ser Leu Gly1 5 10 15Ala Ser Val Lys Leu Thr Cys Thr Leu
Ser Ser Gln His Ser Thr 20 25 30Tyr Thr Ile Gly Trp Tyr Gln Gln Gln
Pro Gly Lys Ala Pro Arg 35 40 45Tyr Leu Met Tyr Leu Asn Ser Asp Gly
Ser His Asn Lys Gly Asp 50 55 60Gly Ile Pro Asp Arg Phe Ser Gly Ser
Ser Ser Gly Ala Asp Arg 65 70 75Tyr Leu Thr Ile Ser Asn Leu Gln Ser
Glu Asp Glu Ala Asp Tyr 80 85 90Tyr Cys Gly Ser Ser Tyr Ser Ser Gly
Tyr Val Phe Gly Gly Gly 95 100 105Thr5106PRTHomo sapien 5Glu Pro
Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly1 5 10 15Ala Ser
Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30Tyr Thr
Ile Gly Trp Tyr Gln Gln Gln Pro Gly Lys Ala Pro Arg 35 40 45Tyr Leu
Met Tyr Leu Asn Ser Asp Gly Ser His Asn Lys Gly Asp 50 55 60Gly Ile
Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Asp Arg 65 70 75Tyr Leu
Thr Ile Ser Asn Leu Gln Ser Glu Asp Glu Ala Asp Tyr 80 85 90Tyr Cys
Ala Ala Tyr Tyr Ala Tyr Gly Tyr Val Phe Gly Gly Gly 95 100
105Thr6106PRTHomo sapien 6Ser Ser Glu Leu Thr Gln Asp Pro Ala Val
Ser Val Ala Leu Gly1 5 10 15Gln Thr Val Arg Ile Thr Cys Thr Leu Ser
Ser Gln His Ser Thr 20 25 30Tyr Thr Ile Gly Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val 35 40 45Tyr Leu Met Tyr Leu Asn Ser Ser Gly Ser
His Asn Lys Gly Ser 50 55 60Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser
Ser Gly Ala Thr Arg 65 70 75Ser Leu Thr Ile Thr Asn Ala Gln Ala Glu
Asp Glu Ala Asp Tyr 80 85 90Tyr Cys Ala Ala Tyr Tyr Ala Tyr Gly Tyr
Val Phe Gly Gly Gly 95 100 105Thr7106PRTHomo sapien 7Ser Ser Glu
Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly1 5 10 15Gln Thr Val
Arg Ile Thr Cys Thr Leu Ser Ser Gln His Ser Thr 20 25 30Tyr Thr Ile
Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val 35 40 45Leu Val Ile
Tyr Leu Asn Ser Ser Gly Ser His Asn Lys Gly Ser 50 55 60Gly Ile Pro
Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Thr Arg 65 70 75Ser Leu Thr
Ile Thr Asn Ala Gln Ala Glu Asp Glu Ala Asp Tyr 80 85 90Tyr Cys Ala
Ala Tyr Tyr Ala Tyr Gly Tyr Val Phe Gly Gly Gly 95 100
105Thr8106PRTHomo sapien 8Ser Ser Glu Leu Thr Gln Asp Pro Ala Val
Ser Val Ala Leu Gly1 5 10 15Gln Thr Val Arg Ile Thr Cys Thr Leu Ser
Ser Gln His Ser Thr 20 25 30Tyr Thr Ile Gly Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Val 35 40 45Tyr Leu Met Tyr Leu Asn Ser Asp Ser Ser
His Asn Lys Gly Ser 50 55 60Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser
Ser Gly Ala Thr Arg 65 70 75Ser Leu Thr Ile Thr Asn Ala Gln Ala Glu
Asp Glu Ala Asp Tyr 80 85 90Tyr Cys Ala Ala Tyr Tyr Ala Tyr Gly Tyr
Val Phe Gly Gly Gly 95 100 105Thr9114PRTCricetulus griseus 9Glu Val
His Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly1 5 10 15Ser Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Asn Arg
Trp Ile Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp
Val Gly Gly Ile Lys Thr Lys Pro Asn Ile Tyr Ala Thr 50 55 60Glu Tyr
Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 65 70 75Asp Ser
Lys Asn Ser Leu Tyr Leu Gln Met Asn Thr Leu Arg Val 80 85 90Asp Asp
Thr Ala Ile Tyr Tyr Cys Thr Ser Leu Thr Gly Met Arg 95 100 105Tyr
Phe Asp Tyr Trp Gly Gln Gly Thr 110 10116PRTHomo
sapienVariant26-35X is any amino acid 10Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Trp Asn
Trp Val Arg Gln Ala Pro Gly Lys 35 40 45Gly Leu Glu Trp Val Ser Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Arg Phe Thr Ile Ser 65 70 75Arg Asp Asn Ser Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser Leu 80 85 90Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Xaa Xaa Xaa 95 100 105Xaa Xaa Xaa Xaa Xaa Xaa
Trp Gly Gln Gly Thr 110 115 11114PRTHomo sapien 11Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Asn Arg Trp Ile
Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly
Gly Ile Lys Thr Lys Pro Asn Ile Tyr Ala Thr 50 55 60Glu Tyr Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 65 70 75Asp Ser Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 80 85 90Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Leu Thr Gly Met Arg 95 100 105Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr 110 12114PRTHomo sapien 12Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Asn Arg Trp Ile
Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly
Gly Ile Lys Thr Lys Pro Asn Ile Tyr Ala Thr 50 55 60Glu Tyr Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 65 70 75Asp Ser Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 80 85 90Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Leu Thr Gly Met Lys 95 100 105Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr 110 13114PRTHomo sapien 13Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ala Arg Trp Ile
Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly
Gly Ile Lys Thr Lys Pro Ala Ile Tyr Ala Thr 50 55 60Glu Tyr Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 65 70 75Asp Ser Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 80 85 90Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Leu Thr Gly Met Lys 95 100 105Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr 110 14114PRTHomo sapien 14Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30Ala Arg Trp Ile
Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly
Gly Ile Lys Thr Lys Pro Ala Ile Tyr Ala Thr 50 55 60Glu Tyr Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 65 70 75Asp Ser Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 80 85 90Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Leu Thr Gly Met Lys 95 100 105Tyr Phe Asp
Tyr Trp Gly Gln Gly Thr 110 1512PRTArtificial sequencesequence is
synthesized 15Thr Leu Xaa Xaa Xaa His Xaa Xaa Tyr Xaa Ile Xaa 5 10
1611PRTArtificial sequencesequence is synthesized 16Xaa Xaa Ser Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 5 10 179PRTArtificial sequencesequence
is synthesized 17Xaa Xaa Xaa Tyr Xaa Xaa Gly Tyr Xaa 5
1810PRTArtificial sequencesequence is synthesized 18Gly Phe Thr Phe
Ser Xaa Arg Trp Xaa Tyr 5 101919PRTArtificial sequencesequence is
synthesized 19Gly Ile Lys Thr Lys Pro Xaa Xaa Tyr Ala Thr Xaa Tyr
Ala Asp1 5 10 15Ser Val Lys Gly209PRTArtificial sequencesequence is
synthesized 20Xaa Thr Gly Xaa Xaa Tyr Phe Asp Tyr 5
2112PRTArtificial sequencesequence is synthesized 21Thr Leu Ser Ser
Gln His Ser Thr Tyr Thr Ile Gly 5 10 2212PRTArtificial
sequencesequence is synthesized 22Thr Leu Ser Pro Gln His Phe Thr
Tyr Lys Ile Asp 5 10 2312PRTArtificial sequencesequence is
synthesized 23Thr Leu Ser Ser Asn His Ser Ile Tyr Thr Ile Ser 5 10
2412PRTArtificial sequencesequence is synthesized 24Thr Leu Thr Thr
Gln His Ser Thr Tyr Thr Ile Gly 5 10 2511PRTArtificial
sequencesequence is synthesized 25Leu Asn Ser Asp Gly Ser His Asn
Lys Gly Asp 5 10 2611PRTArtificial sequencesequence is synthesized
26Ile Asn Ser Asp Gly Ser His Lys Lys Gly Val 5 10
2711PRTArtificial sequencesequence is synthesized 27Leu Thr Ser His
Gly Leu His Gln Lys Gly Val 5 10 2811PRTArtificial sequencesequence
is synthesized 28Leu Asn Ser Asp Ser Thr Tyr Ile Thr Ala Val 5 10
299PRTArtificial sequencesequence is synthesized 29Gly Ser Ser Tyr
Ser Ser Gly Tyr Val 5 309PRTArtificial sequencesequence is
synthesized 30Ala Ala Tyr Tyr Ala Tyr Gly Tyr Val 5
319PRTArtificial sequencesequence is synthesized 31Gly Tyr Ser Tyr
Tyr Ser Gly Tyr Val 5 329PRTArtificial sequencesequence is
synthesized 32Gly Ser Ser Tyr Ser Thr Gly Tyr Val 5
339PRTArtificial sequencesequence is synthesized 33Gly Ala Ser Tyr
Ser Ser Gly Tyr Ile 5 3410PRTArtificial sequencesequence is
synthesized 34Gly Phe Thr Phe Ser Asn Arg Trp Ile Tyr 5
103510PRTArtificial sequencesequence is synthesized 35Gly Phe Thr
Phe Ser Asn Arg Trp Val Tyr 5 103619PRTArtificial sequencesequence
is synthesized 36Gly Ile Lys Thr Lys Pro Asn Ile Tyr Ala Thr Glu
Tyr Ala Asp1 5 10 15Ser Val Lys Gly3719PRTArtificial
sequencesequence is synthesized 37Gly Ile Lys Thr Lys Pro Thr Arg
Tyr Ala Thr Gln Tyr Ala Asp1 5 10 15Ser Val Lys Gly389PRTArtificial
sequencesequence is synthesized 38Leu Thr Gly Met Arg Tyr Phe Asp
Tyr 5 399PRTArtificial sequencesequence is synthesized 39Leu Thr
Gly Met Lys Tyr Phe Asp Tyr 5 409PRTArtificial sequencesequence is
synthesized 40Val Thr Gly Met Arg Tyr Phe Asp Tyr 5
* * * * *