U.S. patent application number 16/138944 was filed with the patent office on 2019-01-10 for antibody therapeutics that bind psma.
This patent application is currently assigned to Sorrento Therapeutics, Inc.. The applicant listed for this patent is Sorrento Therapeutics, Inc.. Invention is credited to Edwige Gros, Silpa Yalamanchili, Heyue Zhou.
Application Number | 20190010250 16/138944 |
Document ID | / |
Family ID | 56879307 |
Filed Date | 2019-01-10 |
United States Patent
Application |
20190010250 |
Kind Code |
A1 |
Gros; Edwige ; et
al. |
January 10, 2019 |
ANTIBODY THERAPEUTICS THAT BIND PSMA
Abstract
There is disclosed compositions and methods relating to or
derived from anti-PSMA antibodies. More specifically, there is
disclosed fully human antibodies that bind PSMA, PSMA-antibody
binding fragments and derivatives of such antibodies, and
PSMA-binding polypeptides comprising such fragments. Further still,
there is disclosed nucleic acids encoding such antibodies, antibody
fragments and derivatives and polypeptides, cells comprising such
polynucleotides, methods of making such antibodies, antibody
fragments and derivatives and polypeptides, and methods of using
such antibodies, antibody fragments and derivatives and
polypeptides, including methods of treating a disease. There is
further disclosed targeted therapies for treatment of prostate
cancer having the disclosed anti-PSMA antibodies and fragments
thereof targeting the therapeutic agent or cell.
Inventors: |
Gros; Edwige; (San Diego,
CA) ; Yalamanchili; Silpa; (San Diego, CA) ;
Zhou; Heyue; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sorrento Therapeutics, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Sorrento Therapeutics, Inc.
San Diego
CA
|
Family ID: |
56879307 |
Appl. No.: |
16/138944 |
Filed: |
September 21, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
15066806 |
Mar 10, 2016 |
10100126 |
|
|
16138944 |
|
|
|
|
62131227 |
Mar 10, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/00 20180101;
C07K 2317/92 20130101; A61P 15/00 20180101; C07K 16/40 20130101;
A61P 35/00 20180101; C07K 2317/55 20130101; C07K 2317/21 20130101;
A61P 11/00 20180101; A61P 19/00 20180101; C07K 16/3069 20130101;
A61K 47/6803 20170801; C07K 2317/73 20130101; A61K 39/39558
20130101; A61K 47/6829 20170801; A61K 47/6869 20170801; A61P 13/08
20180101; A61K 2039/505 20130101; C07K 2317/56 20130101; A61P 35/02
20180101; A61P 1/04 20180101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; C07K 16/30 20060101 C07K016/30; A61K 47/68 20170101
A61K047/68 |
Claims
1.-7. (canceled)
8. A method for treating prostate cancer, said method comprising
administering an effective amount of a fully human anti-PSMA
antibody or antigen-binding fragment thereof, that binds to a PSMA
epitope, to a subject in need thereof, such that the prostate
cancer is treated, wherein the antibody comprising: a heavy chain
variable domain sequence that is at least 95% identical to the
amino acid sequence SEQ ID NO. 1; and a light chain variable domain
sequence that is at least 95% identical to an amino acid sequence
selected from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 3,
SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.
8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ
ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, and SEQ ID
NO. 17.
9. (canceled)
10. The method for treating prostate cancer of claim 8, wherein the
cancer is selected from the group consisting of: ovarian cancer,
colon cancer, breast cancer, lung cancer, myelomas,
neuroblastic-derived CNS tumors, monocytic leukemias, B-cell
derived leukemias, T-cell derived leukemias, B-cell derived
lymphomas, T-cell derived lymphomas, and mast cell derived
tumors.
11. The method of claim 8, wherein the antibody comprises a heavy
chain/light chain variable domain sequence selected from the group
consisting of: SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO.
3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO.
1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 1/SEQ ID NO.
8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID NO. 1/SEQ ID NO. 10, SEQ ID
NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ ID NO. 12, SEQ ID NO. 1/SEQ
ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14, SEQ ID NO. 1/SEQ ID NO. 15,
SEQ ID NO. 1/SEQ ID NO. 16, and SEQ ID NO. 1/SEQ ID NO. 17.
12.-18. (canceled)
19. A method for treating cancer associated with PSMA expression in
a human subject in need thereof, comprising administering an
effective amount of the anti-PSMA antibody, or antigen-binding
fragment thereof, to the subject, such that the cancer is treated,
wherein the antibody comprises a heavy chain variable domain
comprising complementarity determining regions (CDRs) as set forth
in SEQ ID NO. 1; and comprises a light chain variable domain
comprising CDRs as set forth in a light chain variable region amino
acid sequence selected from the group consisting of: SEQ ID NO. 2,
SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID
NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16,
and SEQ ID NO. 17.
20. The method for treating cancer of claim 19, wherein the cancer
is selected from the group consisting of: prostate cancer, ovarian
cancer, colon cancer, breast cancer, lung cancer, myelomas,
neuroblastic-derived CNS tumors, monocytic leukemias, B-cell
derived leukemias, T-cell derived leukemias, B-cell derived
lymphomas, T-cell derived lymphomas, and mast cell derived
tumors.
21. The method of claim 19, wherein the antibody comprises a heavy
chain/light chain variable domain sequence selected from the group
consisting of: SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO.
3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO.
1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 1/SEQ ID NO.
8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID NO. 1/SEQ ID NO. 10, SEQ ID
NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ ID NO. 12, SEQ ID NO. 1/SEQ
ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14, SEQ ID NO. 1/SEQ ID NO. 15,
SEQ ID NO. 1/SEQ ID NO. 16, and SEQ ID NO. 1/SEQ ID NO. 17.
22. The method of claim 8, wherein the antigen-binding fragment is
a Fab fragment.
23. The method of claim 8, wherein the antibody is a single chain
antibody comprising a peptide linker connecting the heavy and light
chain variable domains.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 62/131,227, filed on Mar. 10, 2015, the entire
contents of which are incorporated by reference in its entirety
herein.
TECHNICAL FIELD
[0002] The present disclosure provides compositions and methods
relating to or derived from anti-PSMA (prostate-specific membrane
antigen) antibodies. More specifically, the present disclosure
provides fully human antibodies that bind PSMA, PSMA-antibody
binding fragments and derivatives of such antibodies, and
PSMA-binding polypeptides comprising such fragments. Further still,
the present disclosure provides nucleic acids encoding such
antibodies, antibody fragments and derivatives and polypeptides,
cells comprising such polynucleotides, methods of making such
antibodies, antibody fragments and derivatives and polypeptides,
and methods of using such antibodies, antibody fragments and
derivatives and polypeptides, including methods of treating a
disease. The present disclosure further provides targeted therapies
for treatment of prostate cancer having the disclosed anti-PSMA
antibodies and fragments thereof targeting the therapeutic agent or
cell.
BACKGROUND
[0003] Prostate cancer is the most prevalent type of cancer and the
second leading cause of death from cancer in American men (Landis,
S. H. et al. C A Cancer J. Clin. 48:6-29 (1998)). The number of men
diagnosed with prostate cancer is steadily increasing as a result
of the increasing population of older men as well as a greater
awareness of the disease leading to its earlier diagnosis (Parker
et al., 1997, CA Cancer J. Clin. 47:5-280). The life time risk for
men developing prostate cancer is about 1 in 5 for Caucasians, 1 in
6 for African Americans. High risk groups are represented by those
with a positive family history of prostate cancer or African
Americans.
[0004] Over a lifetime, more than 2/3 of the men diagnosed with
prostate cancer die of the disease (Wingo et al., 1996, CA Cancer
J. Clin. 46:113-25). Moreover, many patients who do not succumb to
prostate cancer require continuous treatment to ameliorate symptoms
such as pain, bleeding and urinary obstruction. Thus, prostate
cancer also represents a major cause of suffering and increased
health care expenditures.
[0005] Radiation therapy has also been used as an alternative to
radical prostatectomy. Patients generally treated by radiation
therapy are those who are older and less healthy and those with
higher-grade, more clinically advanced tumors. Particularly
preferred procedures are external-beam therapy which involves three
dimensional, confocal radiation therapy where the field of
radiation is designed to conform to the volume of tissue treated;
interstitial-radiation therapy where seeds of radioactive compounds
are implanted using ultrasound guidance; and a combination of
external-beam therapy and interstitial-radiation therapy.
[0006] For treatment of patients with locally advanced disease,
hormonal therapy before or following radical prostatectomy or
radiation therapy has been utilized. Hormonal therapy is the main
form of treating men with disseminated prostate cancer. Orchiectomy
reduces serum testosterone concentrations, while estrogen treatment
is similarly beneficial. Diethylstilbestrol from estrogen is
another useful hormonal therapy which has a disadvantage of causing
cardiovascular toxicity. When gonadotropin-releasing hormone
agonists are administered testosterone concentrations are
ultimately reduced. Flutamide and other nonsteroidal, anti-androgen
agents block binding of testosterone to its intracellular
receptors. As a result, it blocks the effect of testosterone,
increasing serum testosterone concentrations and allows patients to
remain potent, a significant problem after radical prostatectomy
and radiation treatments.
[0007] Cytotoxic chemotherapy is largely ineffective in treating
prostate cancer. Its toxicity makes such therapy unsuitable for
elderly patients. In addition, prostate cancer is relatively
resistant to cytotoxic agents.
[0008] Relapsed or more advanced disease is also treated with
anti-androgen therapy. Unfortunately, almost all tumors become
hormone-resistant and progress rapidly in the absence of any
effective therapy.
[0009] Accordingly, there is a need for effective therapeutics for
prostate cancer which are not overwhelmingly toxic to normal
tissues of a patient, and which are effective in selectively
eliminating prostate cancer cells.
SUMMARY OF THE INVENTION
[0010] This invention pertains to binding proteins capable of
binding to prostate-specific membrane antigen (PSMA), (e.g., human
PSMA), including anti-PSMA antibodies, and antigen-binding
fragments thereof.
[0011] In one aspect, the present disclosure provides an isolated
fully human anti-PSMA antibody of an IgG class that binds to a PSMA
epitope, said antibody comprising a heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequence
SEQ ID NO. 1; and a light chain variable domain sequence that is at
least 95% identical to an amino acid sequence selected from the
group consisting of: SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ
ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 8, SEQ ID NO. 9,
SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID
NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, and SEQ ID NO. 17.
[0012] In another aspect, the present disclosure provides a fully
human antibody of an IgG class that binds to a PSMA epitope with a
binding affinity of at least 10.sup.-6M, and comprises a heavy
chain variable domain sequence that is at least 95% identical to
the amino acid sequence SEQ ID NO. 1, and comprises a light chain
variable domain sequence that is at least 95% identical to the
amino acid sequence consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ
ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 8,
SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID
NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17.
In one embodiment, the fully human antibody comprises both a heavy
chain and a light chain wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2 (called PSA11 herein), SEQ
ID NO. 1/SEQ ID NO. 3 (called PSGB11 herein), SEQ ID NO. 1/SEQ ID
NO. 4 (called PSGB12 herein), SEQ ID NO. 1/SEQ ID NO. 5 (called
PSGC8 herein), SEQ ID NO. 1/SEQ ID NO. 6 (called PSGC9 herein), SEQ
ID NO. 1/SEQ ID NO. 7 (called PSGC12 herein), SEQ ID NO. 1/SEQ ID
NO. 8 (called PSGD3 herein), SEQ ID NO. 1/SEQ ID NO. 9 (called
PSGD4 herein), SEQ ID NO. 1/SEQ ID NO. 10 (called PSGD6 herein),
SEQ ID NO. 1/SEQ ID NO. 11 (called PSGE10 herein), SEQ ID NO. 1/SEQ
ID NO. 12 (called PSGE11 herein), SEQ ID NO. 1/SEQ ID NO. 13
(called PSGF9 herein), SEQ ID NO. 1/SEQ ID NO. 14 (called PSGF11
herein), SEQ ID NO. 1/SEQ ID NO. 15 (called PSGG6 herein), SEQ ID
NO. 1/SEQ ID NO. 16 (called PSGH3 herein), SEQ ID NO. 1/SEQ ID NO.
17 (called PSGH8 herein).
[0013] In another aspect, the present disclosure provides an
anti-PSMA Fab fully human antibody fragment, having a variable
domain region from a heavy chain and a variable domain region from
a light chain, wherein the heavy chain variable domain comprises an
amino acid sequence that is at least 95% identical to the amino
acid sequence of SEQ ID NO. 1, and comprises a light chain variable
domain sequence that is at least 95% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID
NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ
ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO.
12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ
ID NO. 17. In one embodiment, the fully human antibody Fab fragment
comprises both a heavy chain variable domain region and a light
chain variable domain region wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3,
SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO.
1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 1/SEQ ID NO.
8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID NO. 1/SEQ ID NO. 10, SEQ ID
NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ ID NO. 12, SEQ ID NO. 1/SEQ
ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14, SEQ ID NO. 1/SEQ ID NO. 15,
SEQ ID NO. 1/SEQ ID NO. 16, SEQ ID NO. 1/SEQ ID NO. 17.
[0014] In another aspect, the present disclosure provides an
anti-PSMA single chain human antibody, comprising a variable domain
region from a heavy chain and a variable domain region from a light
chain and a peptide linker connection the heavy chain and light
chain variable domain regions, wherein the heavy chain variable
domain comprises an amino acid sequence that is at least 95%
identical to the amino acid sequence of SEQ ID NO. 1, and comprises
a light chain variable domain sequence that is at least 95%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.
5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID
NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14,
SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17. In one embodiment, the
fully human single chain antibody comprises both a heavy chain
variable domain region and a light chain variable domain region,
wherein the single chain fully human antibody comprises a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3,
SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO.
1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 1/SEQ ID NO.
8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID NO. 1/SEQ ID NO. 10, SEQ ID
NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ ID NO. 12, SEQ ID NO. 1/SEQ
ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14, SEQ ID NO. 1/SEQ ID NO. 15,
SEQ ID NO. 1/SEQ ID NO. 16, and SEQ ID NO. 1/SEQ ID NO. 17.
[0015] In one embodiment, the invention features an isolated
anti-PSMA antibody, or an antigen-binding fragment thereof,
comprising a heavy chain variable domain comprising complementarity
determining regions (CDRs) as set forth in SEQ ID NO. 1; and
comprising a light chain variable domain comprising CDRs as set
forth in a light chain variable region amino acid sequence selected
from the group consisting of: SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID
NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 8, SEQ
ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.
13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, and SEQ ID NO. 17.
In one embodiment, the isolated anti-PSMA antibody, or
antigen-binding fragment thereof, is human.
[0016] The present disclosure further provides a method for
treating a prostate cancer, comprising administering an anti-PSMA
antibody used to target a chemical or cellular therapeutic payload,
wherein the anti-PSMA antibody has a heavy chain variable domain
sequence that is at least 95% identical to the amino acid sequence
SEQ ID NO. 1, and that has a light chain variable domain sequence
that is at least 95% identical to the amino acid consisting of SEQ
ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6,
SEQ ID NO. 8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO.
11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ
ID NO. 16, SEQ ID NO. 17, combinations thereof.
[0017] The present disclosure further provides a method for
treating a prostate cancer, comprising administering an anti-PSMA
antibody Fab fully human fragment used to target a chemical or
cellular therapeutic payload, wherein the Fab fully human antibody
fragment comprises a heavy chain variable domain sequence that is
at least 95% identical to the amino acid sequence SEQ ID NO. 1, and
comprises a light chain variable domain sequence that is at least
95% identical to an amino acid sequence selected from the group
consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO.
5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID
NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14,
SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17.
[0018] The present disclosure also provides a method for treating a
prostate cancer, comprising administering an anti-PSMA single chain
human antibody used to target a chemical or cellular therapeutic
payload, wherein the anti-PSMA antibody single chain human antibody
comprises a heavy chain variable domain sequence that is at least
95% identical to the amino acid sequence SEQ ID NO. 1, and
comprises a light chain variable domain sequence that is at least
95% identical to the amino acid sequence consisting of SEQ ID NO.
2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID
NO. 8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11,
SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID
NO. 16, SEQ ID NO. 17.
[0019] In one embodiment, the fully human antibody comprises both a
heavy chain and a light chain, wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2 (called PSA11 herein), SEQ
ID NO. 1/SEQ ID NO. 3 (called PSGB11 herein), SEQ ID NO. 1/SEQ ID
NO. 4 (called PSGB12 herein), SEQ ID NO. 1/SEQ ID NO. 5 (called
PSGC8 herein), SEQ ID NO. 1/SEQ ID NO. 6 (called PSGC9 herein), SEQ
ID NO. 1/SEQ ID NO. 7 (called PSGC12 herein), SEQ ID NO. 1/SEQ ID
NO. 8 (called PSGD3 herein), SEQ ID NO. 1/SEQ ID NO. 9 (called
PSGD4 herein), SEQ ID NO. 1/SEQ ID NO. 10 (called PSGD6 herein),
SEQ ID NO. 1/SEQ ID NO. 11 (called PSGE10 herein), SEQ ID NO. 1/SEQ
ID NO. 12 (called PSGE11 herein), SEQ ID NO. 1/SEQ ID NO. 13
(called PSGF9 herein), SEQ ID NO. 1/SEQ ID NO. 14 (called PSGF11
herein), SEQ ID NO. 1/SEQ ID NO. 15 (called PSGG6 herein), SEQ ID
NO. 1/SEQ ID NO. 16 (called PSGH3 herein), and SEQ ID NO. 1/SEQ ID
NO. 17 (called PSGH8 herein). In one embodiment, the fully human
single chain antibody has both a heavy chain variable domain region
and a light chain variable domain region, wherein the single chain
fully human antibody has a heavy chain/light chain variable domain
sequence selected from the group consisting of SEQ ID NO. 1/SEQ ID
NO. 2, SEQ ID NO. 1/SEQ ID NO. 3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID
NO. 1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID
NO. 7, SEQ ID NO. 1/SEQ ID NO. 8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID
NO. 1/SEQ ID NO. 10, SEQ ID NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ
ID NO. 12, SEQ ID NO. 1/SEQ ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14,
SEQ ID NO. 1/SEQ ID NO. 15, SEQ ID NO. 1/SEQ ID NO. 16, and SEQ ID
NO. 1/SEQ ID NO. 17.
[0020] The present invention further provides in another aspect, a
method for treating cancer, said method comprising administering an
anti-PSMA antibody, or antibody fragment of any one of the aspects
or embodiments described herein, to a subject in need thereof. In
one embodiment, the cancer is selected from the group consisting of
ovarian cancer, colon cancer, breast cancer, lung cancer, myeloma,
neuroblastic-derived CNS tumor, monocytic leukemia, B-cell derived
leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell
derived lymphoma, and mast cell derived tumor.
[0021] In certain embodiments, the anti-PSMA antibody is used to
treat a broad spectrum of mammalian cancers, including, for
example, ovarian, colon, breast, lung cancers, myelomas,
neuroblastic-derived CNS tumors, monocytic leukemias, B-cell
derived leukemias, T-cell derived leukemias, B-cell derived
lymphomas, T-cell derived lymphomas, mast cell derived tumors, and
combinations thereof.
[0022] The present invention also provides in another aspect, a
method for treating prostate cancer, said method comprising
administering an anti-PSMA antibody, or antibody fragment of any
one of the aspects or embodiments described herein, to a subject in
need thereof.
[0023] In certain embodiments, the antibody, or antigen-binding
fragment thereof, of the invention has a binding affinity (K.sub.D)
of at least 1.times.10.sup.-6M. In other embodiments, the antibody,
or antigen-binding fragment thereof, of the invention has a K.sub.D
of at least 1.times.10.sup.-7 M. In other embodiments, the
antibody, or antigen-binding fragment thereof, of the invention has
a K.sub.D of at least 1.times.10.sup.-8M.
[0024] In certain embodiments, the antibody is an IgG1 isotype. In
other embodiments, the antibody is an IgG4 isotype.
[0025] In certain embodiments, the antibody, or antigen-binding
fragment, described herein is recombinant.
[0026] In certain embodiments, the antibody, or antigen-binding
fragment, described herein is a human antibody, or antigen binding
fragment of an antibody.
[0027] The invention also provides pharmaceutical compositions
comprising an effective amount of an anti-PSMA antibody or fragment
disclosed herein, and a pharmaceutically acceptable carrier.
DESCRIPTION OF THE DRAWINGS
[0028] FIGS. 1A to 1D graphically depict binding of anti-PSMA
antibodies to PSMA-positive cells (LNCaP) (as described in FIGS. 1A
and 1B), and PSMA-negative normal human fibroblast cells (PC3), (as
described in FIGS. 1C and 1D).
[0029] FIGS. 2A to 2C illustrate cytotoxic potential of anti-PSMA
antibodies PSGF9, PSGC9, PSGD6, (shown in FIG. 2A); PSGD3, PSGE10,
PSGH3, PSGB11, (shown in FIG. 2B); and PSGD4 and PSGB12 (shown in
FIG. 2C) complexed with Protein G (PG)-MMAF molecules on
PSMA-overexpressing LNCaP cancer cells.
[0030] FIG. 2D illustrates the non-specific cell killing effect
observed on normal human fibroblasts (PC3) with various anti-PSMA
antibodies complexed with Protein G-MMAF molecules.
[0031] FIG. 3 graphically depicts affinity binding characteristics
of anti-PSMA antibody PSA11.
[0032] FIG. 4 graphically depicts affinity binding characteristics
of anti-PSMA antibody PSGB11.
DETAILED DESCRIPTION
Definitions
[0033] The terms "peptide," "polypeptide" and "protein" each refers
to a molecule comprising two or more amino acid residues joined to
each other by peptide bonds. These terms encompass, e.g., native
and artificial proteins, protein fragments and polypeptide analogs
(such as muteins, variants, and fusion proteins) of a protein
sequence as well as post-translationally, or otherwise covalently
or non-covalently, modified proteins. A peptide, polypeptide, or
protein may be monomeric or polymeric.
[0034] A "variant" of a polypeptide (for example, a variant of an
antibody) comprises an amino acid sequence wherein one or more
amino acid residues are inserted into, deleted from and/or
substituted into the amino acid sequence relative to another
polypeptide sequence. Disclosed variants include, for example,
fusion proteins.
[0035] A "derivative" of a polypeptide is a polypeptide (e.g., an
antibody) that has been chemically modified, e.g., via conjugation
to another chemical moiety (such as, for example, polyethylene
glycol or albumin, e.g., human serum albumin), phosphorylation, and
glycosylation. Unless otherwise indicated, the term "antibody"
includes, in addition to antibodies comprising two full-length
heavy chains and two full-length light chains, derivatives,
variants, fragments, and muteins thereof, examples of which are
described below.
[0036] An "antigen binding protein" is a protein comprising a
portion that binds to an antigen and, optionally, a scaffold or
framework portion that allows the antigen binding portion to adopt
a conformation that promotes binding of the antigen binding protein
to the antigen. Examples of antigen binding proteins include
antibodies, antibody fragments (e.g., an antigen binding portion of
an antibody), antibody derivatives, and antibody analogs. The
antigen binding protein can comprise, for example, an alternative
protein scaffold or artificial scaffold with grafted CDRs or CDR
derivatives. Such scaffolds include, but are not limited to,
antibody-derived scaffolds comprising mutations introduced to, for
example, stabilize the three-dimensional structure of the antigen
binding protein as well as wholly synthetic scaffolds comprising,
for example, a biocompatible polymer. See, for example, Korndorfer
et al., 2003, Proteins: Structure, Function, and Bioinformatics,
Volume 53, Issue 1:121-129; Roque et al., 2004, Biotechnol. Prog.
20:639-654. In addition, peptide antibody mimetics ("PAMs") can be
used, as well as scaffolds based on antibody mimetics utilizing
fibronection components as a scaffold.
[0037] An antigen binding protein can have, for example, the
structure of an immunoglobulin. An "immunoglobulin" is a tetrameric
molecule composed of two identical pairs of polypeptide chains,
each pair having one "light" (about 25 kDa) and one "heavy" chain
(about 50-70 kDa). The amino-terminal portion of each chain
includes a variable region of about 100 to 110 or more amino acids
primarily responsible for antigen recognition. The carboxy-terminal
portion of each chain defines a constant region primarily
responsible for effector function. Human light chains are
classified as kappa or lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
Preferably, the anti-PSMA antibodies disclosed herein are
characterized by their variable domain region sequences in the
heavy V.sub.H and light V.sub.L amino acid sequences. Within light
and heavy chains, the variable and constant regions are joined by a
"J" region of about 12 or more amino acids, with the heavy chain
also including a "D" region of about 10 more amino acids. See
generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed.
Raven Press, N.Y. (1989)). The variable regions of each light/heavy
chain pair form the antibody binding site such that an intact
immunoglobulin has two binding sites.
[0038] The variable regions of immunoglobulin chains exhibit the
same general structure of relatively conserved framework regions
(FR) joined by three hypervariable regions, also called
complementarity determining regions or CDRs. From N-terminus to
C-terminus, both light and heavy chains comprise the domains FR1,
CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids
to each domain is in accordance with the definitions of Kabat et
al. in Sequences of Proteins of Immunological Interest, 5.sup.th
Ed., US Dept. of Health and Human Services, PHS, NIH, NIH
Publication no. 91-3242, 1991. Other numbering systems for the
amino acids in immunoglobulin chains include IMGT.RTM.
(international ImMunoGeneTics information system; Lefranc et al,
Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and
Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).
[0039] An "antibody" refers to an intact immunoglobulin or to an
antigen binding portion thereof that competes with the intact
antibody for specific binding, unless otherwise specified. In one
embodiment, an antibody comprises two identical heavy chains each
comprising a heavy chain variable domain and heavy chain constant
regions C.sub.H1, C.sub.H2 and C.sub.H3; and comprises two
identical light chains each comprising a light chain variable
domain and a light chain constant region (C.sub.L). In one
embodiment, an antibody of the invention comprises a heavy and
light chain variable domain sequence selected from those described
herein in Table 3.
[0040] In certain embodiments, antibodies can be obtained from
sources such as serum or plasma that contain immunoglobulins having
varied antigenic specificity. If such antibodies are subjected to
affinity purification, they can be enriched for a particular
antigenic specificity. Such enriched preparations of antibodies
usually are made of less than about 10% antibody having specific
binding activity for the particular antigen. Subjecting these
preparations to several rounds of affinity purification can
increase the proportion of antibody having specific binding
activity for the antigen. Antibodies prepared in this manner are
often referred to as "monospecific."
[0041] The term "monospecific", as used herein, refers to an
antibody that displays an affinity for one particular epitope.
Monospecific antibody preparations can be made up of about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or
99.9% antibody having specific binding activity for the particular
antigen.
[0042] In certain embodiments, an antigen binding protein, such as
an antibody, may have one or more binding sites. If there is more
than one binding site, the binding sites may be identical to one
another or may be different. For example, a naturally occurring
human immunoglobulin typically has two identical binding sites,
while a "bispecific" or "bifunctional" antibody has two different
binding sites.
[0043] An "antibody fragment" or "antigen binding fragment of an
antibody" comprises a portion of an intact antibody, and preferably
comprises the antibody antigen binding or variable domains.
Examples of an antibody fragment include a Fab, an Fab', an
F(ab')2, an Fv fragment, and a linear antibody.
[0044] Antigen binding portions (or fragments) of an antibody may
be produced by recombinant DNA techniques or by enzymatic or
chemical cleavage of intact antibodies. Antigen binding portions
include, inter alia, Fab, Fab', F(ab').sub.2, Fv, domain antibodies
(dAbs), and complementarity determining region (CDR) fragments,
single-chain antibodies (scFv), chimeric antibodies, diabodies,
triabodies, tetrabodies, and polypeptides that contain at least a
portion of an immunoglobulin that is sufficient to confer specific
antigen binding to the polypeptide.
[0045] A Fab fragment is a monovalent fragment having the V.sub.L,
V.sub.H, C.sub.L and C.sub.H1 domains; a F(ab').sub.2 fragment is a
bivalent fragment having two Fab fragments linked by a disulfide
bridge at the hinge region; a Fd fragment has the V.sub.H and
C.sub.H1 domains; an Fv fragment has the V.sub.L and V.sub.H
domains of a single arm of an antibody; and a dAb fragment has a
V.sub.H domain, a V.sub.L domain, or an antigen-binding fragment of
a V.sub.H or V.sub.L domain (U.S. Pat. Nos. 6,846,634; 6,696,245,
US App. Pub. 20/0202512; 2004/0202995; 2004/0038291; 2004/0009507;
20 03/0039958, and Ward et al., Nature 341:544-546, 1989).
[0046] A single-chain antibody (scFv) is an antibody in which a
V.sub.L and a V.sub.H region are joined via a linker (e.g., a
synthetic sequence of amino acid residues) to form a continuous
protein chain wherein the linker is long enough to allow the
protein chain to fold back on itself and form a monovalent antigen
binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and
Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).
[0047] Diabodies are bivalent antibodies comprising two polypeptide
chains, wherein each polypeptide chain comprises V.sub.H and
V.sub.L domains joined by a linker that is too short to allow for
pairing between two domains on the same chain, thus allowing each
domain to pair with a complementary domain on another polypeptide
chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA
90:6444-48, and Poljak et al., 1994, Structure 2:1121-23). If the
two polypeptide chains of a diabody are identical, then a diabody
resulting from their pairing will have two identical antigen
binding sites. Polypeptide chains having different sequences can be
used to make a diabody with two different antigen binding sites.
Similarly, triabodies and tetrabodies are antibodies comprising
three and four polypeptide chains, respectively, and forming three
and four antigen binding sites, respectively, which can be the same
or different.
[0048] The term "human antibody" includes antibodies that have one
or more variable and constant regions derived from human
immunoglobulin sequences. In one embodiment, all of the variable
and constant domains of the antibody are derived from human
immunoglobulin sequences (referred to as "a fully human antibody").
These antibodies may be prepared in a variety of ways, examples of
which are described below, including through the immunization with
an antigen of interest of a mouse that is genetically modified to
express antibodies derived from human heavy and/or light
chain-encoding genes. In a preferred embodiment, a fully human
antibody is made using recombinant methods such that the
glycosylation pattern of the antibody is different than an antibody
having the same sequence if it were to exist in nature.
[0049] A "humanized antibody" has a sequence that differs from the
sequence of an antibody derived from a non-human species by one or
more amino acid substitutions, deletions, and/or additions, such
that the humanized antibody is less likely to induce an immune
response, and/or induces a less severe immune response, as compared
to the non-human species antibody, when it is administered to a
human subject. In one embodiment, certain amino acids in the
framework and constant domains of the heavy and/or light chains of
the non-human species antibody are mutated to produce the humanized
antibody. In another embodiment, the constant domain(s) from a
human antibody are fused to the variable domain(s) of a non-human
species. In another embodiment, one or more amino acid residues in
one or more CDR sequences of a non-human antibody are changed to
reduce the likely immunogenicity of the non-human antibody when it
is administered to a human subject, wherein the changed amino acid
residues either are not critical for immunospecific binding of the
antibody to its antigen, or the changes to the amino acid sequence
that are made are conservative changes, such that the binding of
the humanized antibody to the antigen is not significantly worse
than the binding of the non-human antibody to the antigen. Examples
of how to make humanized antibodies may be found in U.S. Pat. Nos.
6,054,297, 5,886,152 and 5,877,293.
[0050] The term "chimeric antibody" refers to an antibody that
contains one or more regions from one antibody and one or more
regions from one or more other antibodies. In one embodiment, one
or more of the CDRs are derived from a human anti-PSMA antibody. In
another embodiment, all of the CDRs are derived from a human
anti-PSMA antibody. In another embodiment, the CDRs from more than
one human anti-PSMA antibodies are mixed and matched in a chimeric
antibody. For instance, a chimeric antibody may comprise a CDR1
from the light chain of a first human anti-PAR-2 antibody, a CDR2
and a CDR3 from the light chain of a second human anti-PSMA
antibody, and the CDRs from the heavy chain from a third anti-PSMA
antibody. Other combinations are possible.
[0051] Further, the framework regions may be derived from one of
the same anti-PSMA antibodies, from one or more different
antibodies, such as a human antibody, or from a humanized antibody.
In one example of a chimeric antibody, a portion of the heavy
and/or light chain is identical with, homologous to, or derived
from an antibody from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is/are identical with, homologous to, or derived from an
antibody (-ies) from another species or belonging to another
antibody class or subclass. Also included are fragments of such
antibodies that exhibit the desired biological activity (i.e., the
ability to specifically bind PSMA).
[0052] A "neutralizing antibody" or an "inhibitory antibody" is an
antibody that inhibits the proteolytic activation of PSMA when an
excess of the anti-PSMA antibody reduces the amount of activation
by at least about 20% using an assay such as those described herein
in the Examples. In various embodiments, the antigen binding
protein reduces the amount of amount of proteolytic activation of
PSMA by at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
97%, 99%, and 99.9%.
[0053] A "CDR grafted antibody" is an antibody comprising one or
more CDRs derived from an antibody of a particular species or
isotype and the framework of another antibody of the same or
different species or isotype.
[0054] A "multi-specific antibody" is an antibody that recognizes
more than one epitope on one or more antigens. A subclass of this
type of antibody is a "bi-specific antibody" which recognizes two
distinct epitopes on the same or different antigens.
[0055] An antigen binding protein "specifically binds" to an
antigen (e.g., PSMA) if it binds to the antigen with a dissociation
constant of 1 nanomolar or less.
[0056] An "antigen binding domain, "antigen binding region," or
"antigen binding site" is a portion of an antigen binding protein
that contains amino acid residues (or other moieties) that interact
with an antigen and contribute to the antigen binding protein's
specificity and affinity for the antigen. For an antibody that
specifically binds to its antigen, this will include at least part
of at least one of its CDR domains.
[0057] The term "Fc polypeptide" includes native and mutein forms
of polypeptides derived from the Fc region of an antibody.
Truncated forms of such polypeptides containing the hinge region
that promotes dimerization also are included. Fusion proteins
comprising Fc moieties (and oligomers formed therefrom) offer the
advantage of facile purification by affinity chromatography over
Protein A or Protein G columns.
[0058] An "epitope" is the portion of a molecule that is bound by
an antigen binding protein (e.g., by an antibody). An epitope can
comprise non-contiguous portions of the molecule (e.g., in a
polypeptide, amino acid residues that are not contiguous in the
polypeptide's primary sequence but that, in the context of the
polypeptide's tertiary and quaternary structure, are near enough to
each other to be bound by an antigen binding protein).
[0059] The "percent identity" or "percent homology" of two
polynucleotide or two polypeptide sequences is determined by
comparing the sequences using the GAP computer program (a part of
the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego,
Calif.)) using its default parameters.
[0060] The terms "polynucleotide," "oligonucleotide" and "nucleic
acid" are used interchangeably throughout and include DNA molecules
(e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of
the DNA or RNA generated using nucleotide analogs (e.g., peptide
nucleic acids and non-naturally occurring nucleotide analogs), and
hybrids thereof. The nucleic acid molecule can be single-stranded
or double-stranded. In one embodiment, the nucleic acid molecules
of the invention comprise a contiguous open reading frame encoding
an antibody, or a fragment, derivative, mutein, or variant
thereof.
[0061] Two single-stranded polynucleotides are "the complement" of
each other if their sequences can be aligned in an anti-parallel
orientation such that every nucleotide in one polynucleotide is
opposite its complementary nucleotide in the other polynucleotide,
without the introduction of gaps, and without unpaired nucleotides
at the 5' or the 3' end of either sequence. A polynucleotide is
"complementary" to another polynucleotide if the two
polynucleotides can hybridize to one another under moderately
stringent conditions. Thus, a polynucleotide can be complementary
to another polynucleotide without being its complement.
[0062] A "vector" is a nucleic acid that can be used to introduce
another nucleic acid linked to it into a cell. One type of vector
is a "plasmid," which refers to a linear or circular double
stranded DNA molecule into which additional nucleic acid segments
can be ligated. Another type of vector is a viral vector (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), wherein additional DNA segments can be
introduced into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g., bacterial vectors comprising a bacterial origin
of replication and episomal mammalian vectors). Other vectors
(e.g., non-episomal mammalian vectors) are integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. An "expression
vector" is a type of vector that can direct the expression of a
chosen polynucleotide.
[0063] A nucleotide sequence is "operably linked" to a regulatory
sequence if the regulatory sequence affects the expression (e.g.,
the level, timing, or location of expression) of the nucleotide
sequence. A "regulatory sequence" is a nucleic acid that affects
the expression (e.g., the level, timing, or location of expression)
of a nucleic acid to which it is operably linked. The regulatory
sequence can, for example, exert its effects directly on the
regulated nucleic acid, or through the action of one or more other
molecules (e.g., polypeptides that bind to the regulatory sequence
and/or the nucleic acid). Examples of regulatory sequences include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Further examples of regulatory sequences
are described in, for example, Goeddel, 1990, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.
[0064] A "host cell" is a cell that can be used to express a
nucleic acid, e.g., a nucleic acid of the invention. A host cell
can be a prokaryote, for example, E. coli, or it can be a
eukaryote, for example, a single-celled eukaryote (e.g., a yeast or
other fungus), a plant cell (e.g., a tobacco or tomato plant cell),
an animal cell (e.g., a human cell, a monkey cell, a hamster cell,
a rat cell, a mouse cell, or an insect cell) or a hybridoma.
Examples of host cells include the COS-7 line of monkey kidney
cells (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L
cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary
(CHO) cells or their derivatives such as Veggie CHO and related
cell lines which grow in serum-free media (see Rasmussen et al.,
1998, Cytotechnology 28:31) or CHO strain DX-B11, which is
deficient in DHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci.
USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the
CV1/EBNA cell line derived from the African green monkey kidney
cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J.
10:2821), human embryonic kidney cells such as 293,293 EBNA or MSR
293, human epidermal A431 cells, human Colo205 cells, other
transformed primate cell lines, normal diploid cells, cell strains
derived from in vitro culture of primary tissue, primary explants,
HL-60, U937, HaK or Jurkat cells. In one embodiment, a host cell is
a mammalian host cell. In one embodiment, a host cell is a
mammalian host cell, but is not a human host cell. Typically, a
host cell is a cultured cell that can be transformed or transfected
with a polypeptide-encoding nucleic acid, which can then be
expressed in the host cell. The phrase "recombinant host cell" can
be used to denote a host cell that has been transformed or
transfected with a nucleic acid to be expressed. A host cell also
can be a cell that comprises the nucleic acid but does not express
it at a desired level unless a regulatory sequence is introduced
into the host cell such that it becomes operably linked with the
nucleic acid. It is understood that the term host cell refers not
only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to, e.g., mutation or
environmental influence, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0065] The term "recombinant antibody" refers to an antibody that
is prepared according to standard recombinant expression methods. A
recombinant antibody can, for example, be expressed from a cell or
cell line transfected with an expression vector (or possibly more
than one expression vector) comprising the coding sequence of the
antibody, where said coding sequence is not naturally associated
with the cell. In one embodiment, a recombinant antibody has a
glycosylation pattern that is different than the glycosylation
pattern of an antibody having the same sequence if it were to exist
in nature. In one embodiment, a recombinant antibody is expressed
in a mammalian host cell which is not a human host cell. Notably,
individual mammalian host cells have unique glycosylation
patterns.
[0066] The term "effective amount" as used herein, refers to that
amount of an antibody, or an antigen binding portion thereof that
binds PSMA, which is sufficient to effect treatment of a disease
when administered to a subject. A therapeutically effective amount
of an antibody, or fragment, provided herein will vary depending
upon the relative activity of the antibodies and depending upon the
subject and disease condition being treated, the weight and age of
the subject, the severity of the disease condition, the manner of
administration and the like, which can readily be determined by one
of ordinary skill in the art.
[0067] The term "isolated" refers to a protein (e.g., an antibody)
that is substantially free of other cellular material. In one
embodiment, an isolated antibody is substantially free of other
proteins from the same species. In one embodiment, an isolated
antibody is expressed by a cell from a different species and is
substantially free of other proteins from the different species. A
protein may be rendered substantially free of naturally associated
components (or components associated with the cellular expression
system used to produce the antibody) by isolation, using protein
purification techniques well known in the art. In one embodiment,
the anti-PSMA antibodies, or antigen binding fragments, of the
invention are isolated.
PSMA Antigen Binding Proteins
[0068] The present invention pertains to PSMA binding proteins,
particularly anti-PSMA antibodies, or antigen-binding portions
thereof, that bind PSMA, e.g., human PSMA, and uses thereof.
Various aspects of the invention relate to antibodies and antibody
fragments, pharmaceutical compositions, nucleic acids, recombinant
expression vectors, and host cells for making such antibodies and
fragments. Methods of using the antibodies of the invention to
detect human PSMA, to inhibit PSMA activity, either in vitro or in
vivo, and to prevent or treat disorders such as cancer are also
encompassed by the invention. In one embodiment, the antibody of
the invention is a human antibody.
[0069] As described in Table 3 below, included in the invention are
novel human antibody heavy and light chain variable regions that
are specific to PSMA. In one embodiment, the invention provides an
anti-PSMA antibody, or an antigen-binding fragment thereof, that
comprises a heavy chain having a variable domain comprising an
amino acid sequence as set forth in SEQ ID NO:1. In one embodiment,
the invention provides an anti-PSMA antibody, or an antigen-binding
fragment thereof, that comprises a light chain having a variable
domain comprising an amino acid sequence as set forth in any one of
SEQ ID Nos: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and
17. In one embodiment, the invention provides an anti-PSMA
antibody, or an antigen-binding fragment thereof, that comprises a
light chain having a variable domain comprising an amino acid
sequence as set forth in any one of SEQ ID Nos: 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16 and 17; and a heavy chain having a
variable domain comprising an amino acid sequence as set forth in
SEQ ID NO: 1.
[0070] Complementarity determining regions (CDRs) are known as
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework (FR). Complementarity determining
regions (CDRs) and framework regions (FR) of a given antibody may
be identified using the system described by Kabat et al. supra;
Lefranc et al., supra and/or Honegger and Pluckthun, supra. Also
familiar to those in the art is the numbering system described in
Kabat et al. (1991, NIH Publication 91-3242, National Technical
Information Service, Springfield, Va.). In this regard Kabat et al.
defined a numbering system for variable domain sequences that is
applicable to any antibody. One of ordinary skill in the art can
unambiguously assign this system of "Kabat numbering" to any
variable domain amino acid sequence, without reliance on any
experimental data beyond the sequence itself.
[0071] In certain embodiments, the present invention provides an
anti-PSMA antibody comprising the CDRs of the heavy and light chain
variable domains described in Table 3 (SEQ ID Nos: 1 to 17). For
example, the invention provides an anti-PSMA antibody, or
antigen-binding fragment thereof, comprising a heavy chain variable
region having the CDRs described in an amino acid sequence as set
forth in SEQ ID NO:1. In one embodiment, the invention provides an
anti-PSMA antibody, or antigen-binding fragment thereof, comprising
a light chain variable region having the CDRs described in an amino
acid sequence as set forth in any one of SEQ ID Nos: 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17. In one embodiment, the
invention provides an anti-PSMA antibody, or antigen-binding
fragment thereof, comprising a light chain variable region having
the CDRs described in an amino acid sequence as set forth in any
one of SEQ ID Nos: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16 and 17; and a heavy chain variable region having the CDRs
described in an amino acid sequence as set forth in SEQ ID
NO:1.
[0072] In one embodiment, the present disclosure provides an
isolated anti-PSMA antibody, or antigen binding fragment thereof,
that comprises a heavy chain comprising a variable domain
comprising the amino acid sequence of SEQ ID NO: 1, and a light
chain comprising a variable domain comprising an amino acid
sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID
NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ
ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO.
12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ
ID NO. 17.
[0073] In one embodiment, the present disclosure provides a fully
human antibody of an IgG class that binds to a PSMA epitope with a
binding affinity of 10.sup.-6M or less, that has a heavy chain
variable domain sequence that is at least 95% identical, at least
96% identical, at least 97% identical, at least 98% identical, or
at least 99% identical, to the amino acid sequence SEQ ID NO. 1,
and that has a light chain variable domain sequence that is at
least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical, to
the amino acid sequence consisting of SEQ ID NO. 2, SEQ ID NO. 3,
SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.
8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ
ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO.
17, and combinations thereof.
[0074] In one embodiment, the fully human antibody has both a heavy
chain and a light chain wherein the antibody has a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2 (called PSA11 herein), SEQ
ID NO. 1/SEQ ID NO. 3 (called PSGB11 herein), SEQ ID NO. 1/SEQ ID
NO. 4 (called PSGB12 herein), SEQ ID NO. 1/SEQ ID NO. 5 (called
PSGC8 herein), SEQ ID NO. 1/SEQ ID NO. 6 (called PSGC9 herein), SEQ
ID NO. 1/SEQ ID NO. 7 (called PSGC12 herein), SEQ ID NO. 1/SEQ ID
NO. 8 (called PSGD3 herein), SEQ ID NO. 1/SEQ ID NO. 9 (called
PSGD4 herein), SEQ ID NO. 1/SEQ ID NO. 10 (called PSGD6 herein),
SEQ ID NO. 1/SEQ ID NO. 11 (called PSGE10 herein), SEQ ID NO. 1/SEQ
ID NO. 12 (called PSGE11 herein), SEQ ID NO. 1/SEQ ID NO. 13
(called PSGF9 herein), SEQ ID NO. 1/SEQ ID NO. 14 (called PSGF11
herein), SEQ ID NO. 1/SEQ ID NO. 15 (called PSGG6 herein), SEQ ID
NO. 1/SEQ ID NO. 16 (called PSGH3 herein), SEQ ID NO. 1/SEQ ID NO.
17 (called PSGH8 herein), and combinations thereof.
[0075] In one embodiment, the invention provides an anti-PSMA
antibody, or an antigen-binding fragment thereof, comprising a
heavy chain comprising a CDR3 domain as set forth in SEQ ID NO: 1,
and comprising a variable domain comprising an amino acid sequence
that is at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% identical to a sequence as set forth in SEQ ID NO: 1.
In one embodiment, the invention provides an anti-PSMA antibody, or
an antigen-binding fragment thereof, comprising a light chain
comprising a CDR3 domain as set forth in any one of SEQ ID Nos: 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17 and
comprising a light chain variable domain comprising an amino acid
sequence that has at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99% identical to a sequence as set forth in
any one of SEQ ID Nos: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16 and 17. Thus, in certain embodiments, the CDR3 domain is
held constant, while variability may be introduced into the
remaining CDRs and/or framework regions of the heavy and/or light
chains, while the antibody, or antigen binding fragment thereof,
retains the ability to bind to PSMA and retains the functional
characteristics, e.g., binding affinity, of the parent.
[0076] In one embodiment, the substitutions made within a heavy or
light chain that is at least 95% identical (or at least 96%
identical, or at least 97% identical, or at least 98% identical, or
at least 99% identical) are conservative amino acid substitutions.
A "conservative amino acid substitution" is one in which an amino
acid residue is substituted by another amino acid residue having a
side chain (R group) with similar chemical properties (e.g., charge
or hydrophobicity). In general, a conservative amino acid
substitution will not substantially change the functional
properties of a protein. In cases where two or more amino acid
sequences differ from each other by conservative substitutions, the
percent sequence identity or degree of similarity may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well-known to those of skill
in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:
307-331, herein incorporated by reference. Examples of groups of
amino acids that have side chains with similar chemical properties
include (1) aliphatic side chains: glycine, alanine, valine,
leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine
and threonine; (3) amide-containing side chains: asparagine and
glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and
tryptophan; (5) basic side chains: lysine, arginine, and histidine;
(6) acidic side chains: aspartate and glutamate, and (7)
sulfur-containing side chains are cysteine and methionine.
[0077] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having the
antigen binding regions of any of the antibodies described in Table
3.
[0078] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSA11. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 2. In one embodiment, the invention is directed
to an antibody having a heavy chain variable domain comprising the
CDRs of SEQ ID NO: 1, and a light chain variable domain comprising
the CDRs of SEQ ID NO: 2. In one embodiment, the invention features
an isolated human antibody, or antigen-binding fragment thereof,
that comprises a heavy chain variable region having an amino acid
sequence that is at least 95% identical, at least 96% identical, at
least 97% identical, at least 98% identical, or at least 99%
identical to the sequence set forth in SEQ ID NO: 1 and comprises a
light chain variable region having an amino acid sequence that is
at least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical to the
sequence set forth in SEQ ID NO: 2. The antibody may further be an
IgG1 or an IgG4 isotype.
[0079] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGB11. In one embodiment, the
invention provides an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable domain sequence as set
forth in SEQ ID NO: 1, and a light chain variable domain sequence
as set forth in SEQ ID NO: 3. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 3. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 3. The
antibody may further be an IgG1 or an IgG4 isotype.
[0080] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGB12. In one embodiment, the
invention provides an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable domain sequence as set
forth in SEQ ID NO: 1, and a light chain variable domain sequence
as set forth in SEQ ID NO: 4. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO:4. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 4. The
antibody may further be an IgG1 or an IgG4 isotype.
[0081] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGC8. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 5. In one embodiment, the invention is directed
to an antibody having a heavy chain variable domain comprising the
CDRs of SEQ ID NO: 1, and a light chain variable domain comprising
the CDRs of SEQ ID NO: 5. In one embodiment, the invention features
an isolated human antibody, or antigen-binding fragment thereof,
that comprises a heavy chain variable region having an amino acid
sequence that is at least 95% identical, at least 96% identical, at
least 97% identical, at least 98% identical, or at least 99%
identical to the sequence set forth in SEQ ID NO: 1 and comprises a
light chain variable region having an amino acid sequence that is
at least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical to the
sequence set forth in SEQ ID NO: 5. The antibody may further be an
IgG1 or an IgG4 isotype.
[0082] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGC9. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 6. In one embodiment, the invention is directed
to an antibody having a heavy chain variable domain comprising the
CDRs of SEQ ID NO: 1, and a light chain variable domain comprising
the CDRs of SEQ ID NO: 6. In one embodiment, the invention features
an isolated human antibody, or antigen-binding fragment thereof,
that comprises a heavy chain variable region having an amino acid
sequence that is at least 95% identical, at least 96% identical, at
least 97% identical, at least 98% identical, or at least 99%
identical to the sequence set forth in SEQ ID NO: 1 and comprises a
light chain variable region having an amino acid sequence that is
at least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical to the
sequence set forth in SEQ ID NO: 6. The antibody may further be an
IgG1 or an IgG4 isotype.
[0083] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGC12. In one embodiment, the
invention provides an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable domain sequence as set
forth in SEQ ID NO: 1, and a light chain variable domain sequence
as set forth in SEQ ID NO: 7. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 7. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 7. The
antibody may further be an IgG1 or an IgG4 isotype.
[0084] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGD3. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 8. In one embodiment, the invention is directed
to an antibody having a heavy chain variable domain comprising the
CDRs of SEQ ID NO: 1, and a light chain variable domain comprising
the CDRs of SEQ ID NO: 8. In one embodiment, the invention features
an isolated human antibody, or antigen-binding fragment thereof,
that comprises a heavy chain variable region having an amino acid
sequence that is at least 95% identical, at least 96% identical, at
least 97% identical, at least 98% identical, or at least 99%
identical to the sequence set forth in SEQ ID NO: 1 and comprises a
light chain variable region having an amino acid sequence that is
at least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical to the
sequence set forth in SEQ ID NO: 8. The antibody may further be an
IgG1 or an IgG4 isotype.
[0085] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGD4. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 9. In one embodiment, the invention is directed
to an antibody having a heavy chain variable domain comprising the
CDRs of SEQ ID NO: 1, and a light chain variable domain comprising
the CDRs of SEQ ID NO: 9. In one embodiment, the invention features
an isolated human antibody, or antigen-binding fragment thereof,
that comprises a heavy chain variable region having an amino acid
sequence that is at least 95% identical, at least 96% identical, at
least 97% identical, at least 98% identical, or at least 99%
identical to the sequence set forth in SEQ ID NO: 1 and comprises a
light chain variable region having an amino acid sequence that is
at least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical to the
sequence set forth in SEQ ID NO: 9. The antibody may further be an
IgG1 or an IgG4 isotype.
[0086] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGD6. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 10. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 10. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 10. The
antibody may further be an IgG1 or an IgG4 isotype.
[0087] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGE10. In one embodiment, the
invention provides an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable domain sequence as set
forth in SEQ ID NO: 1, and a light chain variable domain sequence
as set forth in SEQ ID NO: 11. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 11. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 11. The
antibody may further be an IgG1 or an IgG4 isotype.
[0088] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGE11. In one embodiment, the
invention provides an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable domain sequence as set
forth in SEQ ID NO: 1, and a light chain variable domain sequence
as set forth in SEQ ID NO: 12. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 12. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 12. The
antibody may further be an IgG1 or an IgG4 isotype.
[0089] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGF9. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 13. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 13. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 13. The
antibody may further be an IgG1 or an IgG4 isotype.
[0090] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGF11. In one embodiment, the
invention provides an antibody, or antigen-binding fragment
thereof, comprising a heavy chain variable domain sequence as set
forth in SEQ ID NO: 1, and a light chain variable domain sequence
as set forth in SEQ ID NO: 14. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 14. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 14. The
antibody may further be an IgG1 or an IgG4 isotype.
[0091] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGG6. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 15. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 15. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 15. The
antibody may further be an IgG1 or an IgG4 isotype.
[0092] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGH3. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 16. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 16. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 16. The
antibody may further be an IgG1 or an IgG4 isotype.
[0093] In one embodiment, the present invention is directed to an
antibody, or an antigen binding fragment thereof, having antigen
binding regions of antibody PSGH8. In one embodiment, the invention
provides an antibody, or antigen-binding fragment thereof,
comprising a heavy chain variable domain sequence as set forth in
SEQ ID NO: 1, and a light chain variable domain sequence as set
forth in SEQ ID NO: 17. In one embodiment, the invention is
directed to an antibody having a heavy chain variable domain
comprising the CDRs of SEQ ID NO: 1, and a light chain variable
domain comprising the CDRs of SEQ ID NO: 17. In one embodiment, the
invention features an isolated human antibody, or antigen-binding
fragment thereof, that comprises a heavy chain variable region
having an amino acid sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical to the sequence set forth in
SEQ ID NO: 1 and comprises a light chain variable region having an
amino acid sequence that is at least 95% identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at
least 99% identical to the sequence set forth in SEQ ID NO: 17. The
antibody may further be an IgG1 or an IgG4 isotype.
[0094] Antigen binding proteins (e.g., antibodies, antibody
fragments, antibody derivatives, antibody muteins, and antibody
variants) are polypeptides that bind to PSMA.
[0095] Antigen-binding fragments of antigen binding proteins of the
invention may be produced by conventional techniques. Examples of
such fragments include, but are not limited to, Fab and
F(ab').sub.2 fragments.
[0096] Single chain antibodies may be formed by linking heavy and
light chain variable domain (Fv region) fragments via an amino acid
bridge (short peptide linker), resulting in a single polypeptide
chain. Such single-chain Fvs (scFvs) have been prepared by fusing
DNA encoding a peptide linker between DNAs encoding the two
variable domain polypeptides (V.sub.L and V.sub.H). The resulting
polypeptides can fold back on themselves to form antigen-binding
monomers, or they can form multimers (e.g., dimers, trimers, or
tetramers), depending on the length of a flexible linker between
the two variable domains (Kortt et al., 1997, Prot. Eng. 10:423;
Kortt et al., 2001, Biomol. Eng. 18:95-108). By combining different
V.sub.L and V.sub.H-comprising polypeptides, one can form
multimeric scFvs that bind to different epitopes (Kriangkum et al.,
2001, Biomol. Eng. 18:31-40). Techniques developed for the
production of single chain antibodies include those described in
U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et
al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989,
Nature 334:544, de Graaf et al., 2002, Methods Mol. Biol.
178:379-87.
[0097] In certain embodiments, the present disclosure provides a
Fab fully human antibody fragment, having a variable domain region
from a heavy chain and a variable domain region from a light chain,
wherein the heavy chain variable domain sequence that is at least
95% identical, at least 96% identical, at least 97% identical, at
least 98% identical, or at least 99% identical, to the amino acid
sequence SEQ ID NO. 1, and that has a light chain variable domain
sequence that is at least 95% identical, at least 96% identical, at
least 97% identical, at least 98% identical, or at least 99%
identical, to the amino acid sequence consisting of SEQ ID NO. 2,
SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO.
8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID
NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16,
SEQ ID NO. 17, and combinations thereof. In one embodiment, the
fully human antibody Fab fragment has both a heavy chain variable
domain region and a light chain variable domain region wherein the
antibody has a heavy chain/light chain variable domain sequence
selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2,
SEQ ID NO. 1/SEQ ID NO. 3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO.
1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ ID NO. 6, SEQ ID NO.
[0098] 1/SEQ ID NO. 7, SEQ ID NO. 1/SEQ ID NO. 8, SEQ ID NO. 1/SEQ
ID NO. 9, SEQ ID NO. 1/SEQ ID NO. 10, SEQ ID NO. 1/SEQ ID NO. 11,
SEQ ID NO. 1/SEQ ID NO. 12, SEQ ID NO. 1/SEQ ID NO. 13, SEQ ID NO.
1/SEQ ID NO. 14, SEQ ID NO. 1/SEQ ID NO. 15, SEQ ID NO. 1/SEQ ID
NO. 16, SEQ ID NO. 1/SEQ ID NO. 17, and combinations thereof.
[0099] In one embodiment, the present disclosure provides a single
chain human antibody, having a variable domain region from a heavy
chain and a variable domain region from a light chain and a peptide
linker connection the heavy chain and light chain variable domain
regions, wherein the heavy chain variable domain sequence is at
least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical, to
the amino acid sequence of SEQ ID NO. 1, and wherein the light
chain variable domain sequence is at least 95% identical, at least
96% identical, at least 97% identical, at least 98% identical, or
at least 99% identical, to an amino acid sequence selected from the
group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ
ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 8, SEQ ID NO. 9,
SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID
NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, and
combinations thereof. In one embodiment, the fully human single
chain antibody has both a heavy chain variable domain region and a
light chain variable domain region, wherein the single chain fully
human antibody has a heavy chain/light chain variable domain
sequence selected from the group consisting of SEQ ID NO. 1/SEQ ID
NO. 2, SEQ ID NO. 1/SEQ ID NO. 3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID
NO. 1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID
NO. 7, SEQ ID NO. 1/SEQ ID NO. 8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID
NO. 1/SEQ ID NO. 10, SEQ ID NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ
ID NO. 12, SEQ ID NO. 1/SEQ ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14,
SEQ ID NO. 1/SEQ ID NO. 15, SEQ ID NO. 1/SEQ ID NO. 16, SEQ ID NO.
1/SEQ ID NO. 17, and combinations thereof.
[0100] Techniques are known for deriving an antibody of a different
subclass or isotype from an antibody of interest, i.e., subclass
switching. Thus, IgG antibodies may be derived from an IgM
antibody, for example, and vice versa. Such techniques allow the
preparation of new antibodies that possess the antigen-binding
properties of a given antibody (the parent antibody), but also
exhibit biological properties associated with an antibody isotype
or subclass different from that of the parent antibody. Recombinant
DNA techniques may be employed. Cloned DNA encoding particular
antibody polypeptides may be employed in such procedures, e.g., DNA
encoding the constant domain of an antibody of the desired isotype
(Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover, if
an IgG4 is desired, it may also be desired to introduce a point
mutation (CPSC->CPPC) (SEQ ID NOS 18 and 19, respectively) in
the hinge region (Bloom et al., 1997, Protein Science 6:407) to
alleviate a tendency to form intra-H chain disulfide bonds that can
lead to heterogeneity in the IgG4 antibodies. Thus, in one
embodiment, the antibody of the invention is a human IgG1 antibody.
Thus, in one embodiment, the antibody of the invention is a human
IgG4 antibody.
[0101] The present disclosure provides a number of antibodies
structurally characterized by the amino acid sequences of their
variable domain regions. However, the amino acid sequences can
undergo some changes while retaining their high degree of binding
to their specific targets. More specifically, many amino acids in
the variable domain region can be changed with conservative
substitutions and it is predictable that the binding
characteristics of the resulting antibody will not differ from the
binding characteristics of the wild type antibody sequence. There
are many amino acids in an antibody variable domain that do not
directly interact with the antigen or impact antigen binding and
are not critical for determining antibody structure. For example, a
predicted nonessential amino acid residue in any of the disclosed
antibodies is preferably replaced with another amino acid residue
from the same class. Methods of identifying amino acid conservative
substitutions which do not eliminate antigen binding are well-known
in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187
(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and
Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). Near et
al. Mol. Immunol. 30:369-377, 1993 explains how to impact or not
impact binding through site-directed mutagenesis. Near et al. only
mutated residues that they thought had a high probability of
changing antigen binding. Most had a modest or negative effect on
binding affinity (Near et al. Table 3) and binding to different
forms of digoxin (Near et al. Table 2). Thus, the invention also
includes, in certain embodiments, variable sequences having at
least 95% identity, at least 96% identity, at least 97% identity,
at least 98% identity, or at least 99% identity to those sequences
disclosed herein.
[0102] In certain embodiments, an antibody, or antigen-binding
fragment thereof, provided herein has a binding affinity (K.sub.D)
of 1.times.10.sup.-6 M or less; 5.times.10.sup.-7 M or less'
1.times.10.sup.-7 M or less; 5.times.10.sup.-8 M or less;
1.times.10.sup.-8 M or less; 5.times.10.sup.-9 M or less; or
1.times.10.sup.-9 M or less. In one embodiment, the antibody, or
antigen-binding fragment thereof, of the invention as a K.sub.D
from 1.times.10.sup.-7 M to 1.times.10.sup.-10 M. In one
embodiment, the antibody, or antigen-binding fragment thereof, of
the invention as a K.sub.D from 1.times.10.sup.-8 M to
1.times.10.sup.-10 M.
[0103] Those of ordinary skill in the art will appreciate standard
methods known for determining the K.sub.D of an antibody, or
fragment thereof. For example, in one embodiment, K.sub.D is
measured by a radiolabeled antigen binding assay (RIA). In one
embodiment, an RIA is performed with the Fab version of an antibody
of interest and its antigen. For example, solution binding affinity
of Fabs for antigen is measured by equilibrating Fab with a minimal
concentration of (.sup.125I)-labeled antigen in the presence of a
titration series of unlabeled antigen, then capturing bound antigen
with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J.
Mol. Biol. 293:865-881(1999)).
[0104] According to another embodiment, K.sub.D is measured using a
BIACORE surface plasmon resonance assay. The term "surface plasmon
resonance", as used herein, refers to an optical phenomenon that
allows for the analysis of real-time interactions by detection of
alterations in protein concentrations within a biosensor matrix,
for example using the BIACORE system (Biacore Life Sciences
division of GE Healthcare, Piscataway, N.J.).
[0105] In particular embodiments, antigen binding proteins of the
present invention have a K.sub.a for PSMA of at least 10.sup.6. In
other embodiments, the antigen binding proteins exhibit a K.sub.a
of at least 10.sup.7, at least 10.sup.8, at least 10.sup.9, or at
least 10.sup.10. In another embodiment, the antigen binding protein
exhibits a K.sub.a substantially the same as that of an antibody
described herein in the Examples.
[0106] In another embodiment, the present disclosure provides an
antigen binding protein that has a low dissociation rate from PSMA.
In one embodiment, the antigen binding protein has a K.sub.off of
1.times.10.sup.-4 to .sup.-1 or lower. In another embodiment, the
K.sub.off is 5.times.10.sup.-5 to .sup.-1 or lower. In another
embodiment, the K.sub.off is substantially the same as an antibody
described herein. In another embodiment, the antigen binding
protein binds to PSMA with substantially the same K.sub.off as an
antibody described herein.
[0107] In another aspect, the present disclosure provides an
antigen binding protein that inhibits an activity of PSMA. In one
embodiment, the antigen binding protein has an IC.sub.50 of 1000 nM
or lower. In another embodiment, the IC.sub.50 is 100 nM or lower;
in another embodiment, the IC.sub.50 is 10 nM or lower. In another
embodiment, the IC.sub.50 is substantially the same as that of an
antibody described herein in the Examples. In another embodiment,
the antigen binding protein inhibits an activity of PSMA with
substantially the same IC.sub.50 as an antibody described
herein.
[0108] In another aspect, the present disclosure provides an
antigen binding protein that binds to PSMA expressed on the surface
of a cell and, when so bound, inhibits PSMA signaling activity in
the cell without causing a significant reduction in the amount of
PSMA on the surface of the cell. Any method for determining or
estimating the amount of PSMA on the surface and/or in the interior
of the cell can be used. In other embodiments, binding of the
antigen binding protein to the PSMA-expressing cell causes less
than about 75%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 1%, or 0.1% of
the cell-surface PSMA to be internalized.
[0109] In another aspect, the present disclosure provides an
antigen binding protein having a half-life of at least one day in
vitro or in vivo (e.g., when administered to a human subject). In
one embodiment, the antigen binding protein has a half-life of at
least three days. In another embodiment, the antigen binding
protein has a half-life of four days or longer. In another
embodiment, the antigen binding protein has a half-life of eight
days or longer. In another embodiment, the antigen binding protein
is derivatized or modified such that it has a longer half-life as
compared to the underivatized or unmodified antigen binding
protein. In another embodiment, the antigen binding protein
contains one or more point mutations to increase serum half life,
such as described in WO00/09560, incorporated by reference
herein.
[0110] The present disclosure further provides multi-specific
antigen binding proteins, for example, bispecific antigen binding
protein, e.g., antigen binding protein that bind to two different
epitopes of PSMA, or to an epitope of PSMA and an epitope of
another molecule, via two different antigen binding sites or
regions. Moreover, bispecific antigen binding protein as disclosed
herein can comprise a PSMA binding site from one of the
herein-described antibodies and a second PSMA binding region from
another of the herein-described antibodies, including those
described herein by reference to other publications. Alternatively,
a bispecific antigen binding protein may comprise an antigen
binding site from one of the herein described antibodies and a
second antigen binding site from another PSMA antibody that is
known in the art, or from an antibody that is prepared by known
methods or the methods described herein.
[0111] Numerous methods of preparing bispecific antibodies are
known in the art. Such methods include the use of hybrid-hybridomas
as described by Milstein et al., 1983, Nature 305:537, and chemical
coupling of antibody fragments (Brennan et al., 1985, Science
229:81; Glennie et al., 1987, J. Immunol. 139:2367; U.S. Pat. No.
6,010,902). Moreover, bispecific antibodies can be produced via
recombinant means, for example by using leucine zipper moieties
(i.e., from the Fos and Jun proteins, which preferentially form
heterodimers; Kostelny et al., 1992, J. Immunol. 148:1547) or other
lock and key interactive domain structures as described in U.S.
Pat. No. 5,582,996. Additional useful techniques include those
described in U.S. Pat. Nos. 5,959,083; and 5,807,706.
[0112] In another aspect, the antigen binding protein comprises a
derivative of an antibody. The derivatized antibody can comprise
any molecule or substance that imparts a desired property to the
antibody, such as increased half-life in a particular use. The
derivatized antibody can comprise, for example, a detectable (or
labeling) moiety (e.g., a radioactive, colorimetric, antigenic or
enzymatic molecule, a detectable bead (such as a magnetic or
electrodense (e.g., gold) bead), or a molecule that binds to
another molecule (e.g., biotin or streptavidin), a therapeutic or
diagnostic moiety (e.g., a radioactive, cytotoxic, or
pharmaceutically active moiety), or a molecule that increases the
suitability of the antibody for a particular use (e.g.,
administration to a subject, such as a human subject, or other in
vivo or in vitro uses). Examples of molecules that can be used to
derivatize an antibody include albumin (e g, human serum albumin)
and polyethylene glycol (PEG). Albumin-linked and PEGylated
derivatives of antibodies can be prepared using techniques well
known in the art. In one embodiment, the antibody is conjugated or
otherwise linked to transthyretin (TTR) or a TTR variant. The TTR
or TTR variant can be chemically modified with, for example, a
chemical selected from the group consisting of dextran,
poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene
glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols and polyvinyl alcohols.
[0113] An alternative approach to antibody-targeted therapy is to
utilize anti-PSMA antibodies of the invention for delivery of
cytotoxic drugs specifically to PSMA antigen-expressing cancer
cells. In one embodiment, an anti-PSMA antibody, or fragment, of
the invention is conjugated to a cytotoxic agent via a linker, to
form an anti-PSMA Antibody Drug Conjugate (ADC). An alternative
approach to antibody-targeted therapy is to utilize anti-PSMA
antibodies for delivery of cytotoxic drugs specifically to PSMA
antigen-expressing cancer cells. Various cytotoxic drugs are known
in the art which can be conjugated with any of the antibodies
disclosed herein to form an ADC, including, but not limited to, an
auristatin. Auristatins, such as Monomethyl auristatin E (MMAE) and
Monomethyl auristatin F (MMAF) are antimitotic agents that inhibit
cell division by blocking the polymerization of tubulin (Francisco
et al. Blood. 2003 Aug. 15; 102(4):1458-65; Smith et al. Mol Cancer
Ther Jun. 2006 5; 1474-82). Antibody-drug conjugates (ADCs)
composed of the auristatin MMAF linked to an anti-PSMA antibody, as
described in Example 2 below, showed potent anti-tumor activity in
PSMA-expressing tumor cell lines. Thus, in one embodiment, an
anti-PSMA antibody, or fragment thereof, of the invention is
conjugated to an auristatin, e.g., MMAF.
[0114] Oligomers that contain one or more antigen binding proteins
may be employed as PSMA antagonists. Oligomers may be in the form
of covalently-linked or non-covalently-linked dimers, trimers, or
higher oligomers. Oligomers comprising two or more antigen binding
protein are contemplated for use, with one example being a
homodimer. Other oligomers include heterodimers, homotrimers,
heterotrimers, homotetramers, heterotetramers, etc.
[0115] One embodiment is directed to oligomers comprising multiple
antigen binding proteins joined via covalent or non-covalent
interactions between peptide moieties fused to the antigen binding
proteins. Such peptides may be peptide linkers (spacers), or
peptides that have the property of promoting oligomerization.
Leucine zippers and certain polypeptides derived from antibodies
are among the peptides that can promote oligomerization of antigen
binding proteins attached thereto, as described in more detail
below.
[0116] In particular embodiments, the oligomers comprise from two
to four antigen binding proteins. The antigen binding proteins of
the oligomer may be in any form, such as any of the forms described
above, e.g., variants or fragments. Preferably, the oligomers
comprise antigen binding proteins that have PSMA binding
activity.
[0117] In one embodiment, an oligomer is prepared using
polypeptides derived from immunoglobulins. Preparation of Fusion
Proteins Comprising Certain Heterologous Polypeptides Fused to
Various Portions of antibody-derived polypeptides (including the Fc
domain) has been described, e.g., by Ashkenazi et al., 1991, Proc.
Natl. Acad. Sci. USA 88:10535; Byrn et al., 1990, Nature 344:677;
and Hollenbaugh et al., 1992 "Construction of Immunoglobulin Fusion
Proteins", in Current Protocols in Immunology, Suppl. 4, pages
10.19.1-10.19.11.
[0118] One embodiment is directed to a dimer comprising two fusion
proteins created by fusing a PSMA binding fragment of an anti-PSMA
antibody to the Fc region of an antibody. The dimer can be made by,
for example, inserting a gene fusion encoding the fusion protein
into an appropriate expression vector, expressing the gene fusion
in host cells transformed with the recombinant expression vector,
and allowing the expressed fusion protein to assemble much like
antibody molecules, whereupon interchain disulfide bonds form
between the Fc moieties to yield the dimer.
[0119] Another method for preparing oligomeric antigen binding
proteins involves use of a leucine zipper. Leucine zipper domains
are peptides that promote oligomerization of the proteins in which
they are found. Leucine zippers were originally identified in
several DNA-binding proteins (Landschulz et al., 1988, Science
240:1759), and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurring
peptides and derivatives thereof that dimerize or trimerize.
Examples of leucine zipper domains suitable for producing soluble
oligomeric proteins are described in WO 94/10308, and the leucine
zipper derived from lung surfactant protein D (SPD) described in
Hoppe et al., 1994, FEBS Letters 344:191. The use of a modified
leucine zipper that allows for stable trimerization of a
heterologous protein fused thereto is described in Fanslow et al.,
1994, Semin. Immunol. 6:267-78. In one approach, recombinant fusion
proteins comprising an anti-PSMA antibody fragment or derivative
fused to a leucine zipper peptide are expressed in suitable host
cells, and the soluble oligomeric anti-PSMA antibody fragments or
derivatives that form are recovered from the culture
supernatant.
[0120] Antigen binding proteins directed against PSMA can be used,
for example, in assays to detect the presence of PSMA polypeptides,
either in vitro or in vivo. The antigen binding proteins also may
be employed in purifying PSMA proteins by immunoaffinity
chromatography. Blocking antigen binding proteins can be used in
the methods disclosed herein. Such antigen binding proteins that
function as PSMA antagonists may be employed in treating any
PSMA-induced condition, including but not limited to various
cancers.
[0121] Antigen binding proteins may be employed in an in vitro
procedure, or administered in vivo to inhibit PSMA-induced
biological activity. Disorders caused or exacerbated (directly or
indirectly) by the proteolytic activation of PSMA, examples of
which are provided herein, thus may be treated. In one embodiment,
the present invention provides a therapeutic method comprising in
vivo administration of a PSMA blocking antigen binding protein to a
mammal in need thereof in an amount effective for reducing a
PSMA-induced biological activity.
[0122] In certain embodiments of the invention, antigen binding
proteins include fully human monoclonal antibodies that inhibit a
biological activity of PSMA.
[0123] Antigen binding proteins, including antibodies and antibody
fragments described herein, may be prepared by any of a number of
conventional techniques. For example, they may be purified from
cells that naturally express them (e.g., an antibody can be
purified from a hybridoma that produces it), or produced in
recombinant expression systems, using any technique known in the
art. See, for example, Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Kennet et al. (eds.), Plenum
Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow
and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1988).
[0124] Any expression system known in the art can be used to make
the recombinant polypeptides, including antibodies and antibody
fragments described herein, of the invention. In general, host
cells are transformed with a recombinant expression vector that
comprises DNA encoding a desired polypeptide. Among the host cells
that may be employed are prokaryotes, yeast or higher eukaryotic
cells. Prokaryotes include gram negative or gram positive
organisms, for example E. coli or bacilli. Higher eukaryotic cells
include insect cells and established cell lines of mammalian
origin. Examples of suitable mammalian host cell lines include the
COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,
1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC
CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC
CRL 10) cell lines, and the CV1/EBNA cell line derived from the
African green monkey kidney cell line CV1 (ATCC CCL 70) as
described by McMahan et al., 1991, EMBO J. 10: 2821. Appropriate
cloning and expression vectors for use with bacterial, fungal,
yeast, and mammalian cellular hosts are described by Pouwels et al.
(Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).
[0125] The transformed cells can be cultured under conditions that
promote expression of the polypeptide, and the polypeptide
recovered by conventional protein purification procedures. One such
purification procedure includes the use of affinity chromatography,
e.g., over a matrix having all or a portion (e.g., the
extracellular domain) of PSMA bound thereto. Polypeptides
contemplated for use herein include substantially homogeneous
recombinant mammalian anti-PSMA antibody polypeptides substantially
free of contaminating endogenous materials.
[0126] Antigen binding proteins may be prepared, and screened for
desired properties, by any of a number of known techniques. Certain
of the techniques involve isolating a nucleic acid encoding a
polypeptide chain (or portion thereof) of an antigen binding
protein of interest (e.g., an anti-PSMA antibody), and manipulating
the nucleic acid through recombinant DNA technology. The nucleic
acid may be fused to another nucleic acid of interest, or altered
(e.g., by mutagenesis or other conventional techniques) to add,
delete, or substitute one or more amino acid residues, for
example.
[0127] Polypeptides of the present disclosure can be produced using
any standard methods known in the art. In one example, the
polypeptides are produced by recombinant DNA methods by inserting a
nucleic acid sequence (e.g., a cDNA) encoding the polypeptide into
a recombinant expression vector and expressing the DNA sequence
under conditions promoting expression.
[0128] Nucleic acids encoding any of the various polypeptides
disclosed herein may be synthesized chemically. Codon usage may be
selected so as to improve expression in a cell. Such codon usage
will depend on the cell type selected. Specialized codon usage
patterns have been developed for E. coli and other bacteria, as
well as mammalian cells, plant cells, yeast cells and insect cells.
See for example: Mayfield et al., Proc. Natl. Acad. Sci. USA. 2003
100(2):438-42; Sinclair et al. Protein Expr. Purif. 2002
(1):96-105; Connell N D. Curr. Opin. Biotechnol. 2001 12(5):446-9;
Makrides et al. Microbiol. Rev. 1996 60(3):512-38; and Sharp et al.
Yeast. 1991 7(7):657-78.
[0129] In one embodiment, the invention features nucleic acids
encoding the antibodies or antibody fragments described herein. For
example, in one embodiment, the invention includes a nucleic acid
encoding a heavy chain variable domain as set forth in SEQ ID NO: 1
and/or a light chain variable domain as set forth in any one of SEQ
ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6,
SEQ ID NO. 8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO.
11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ
ID NO. 16, and SEQ ID NO. 17.
[0130] General techniques for nucleic acid manipulation are
described for example in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press,
2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular
Biology (Green Publishing and Wiley-Interscience: New York, 1987)
and periodic updates, herein incorporated by reference. The DNA
encoding the polypeptide is operably linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, viral, or insect genes. Such regulatory elements include
a transcriptional promoter, an optional operator sequence to
control transcription, a sequence encoding suitable mRNA ribosomal
binding sites, and sequences that control the termination of
transcription and translation. The ability to replicate in a host,
usually conferred by an origin of replication, and a selection gene
to facilitate recognition of transformants is additionally
incorporated.
[0131] The recombinant DNA can also include any type of protein tag
sequence that may be useful for purifying the protein. Examples of
protein tags include but are not limited to a histidine tag, a FLAG
tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and
expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts can be found in Cloning Vectors: A
Laboratory Manual, (Elsevier, N.Y., 1985).
[0132] The expression construct is introduced into the host cell
using a method appropriate to the host cell. A variety of methods
for introducing nucleic acids into host cells are known in the art,
including, but not limited to, electroporation; transfection
employing calcium chloride, rubidium chloride, calcium phosphate,
DEAE-dextran, or other substances; microprojectile bombardment;
lipofection; and infection (where the vector is an infectious
agent). Suitable host cells include prokaryotes, yeast, mammalian
cells, or bacterial cells.
[0133] Suitable bacteria include gram negative or gram positive
organisms, for example, E. coli or Bacillus spp. Yeast, preferably
from the Saccharomyces species, such as S. cerevisiae, may also be
used for production of polypeptides. Various mammalian or insect
cell culture systems can also be employed to express recombinant
proteins. Baculovirus systems for production of heterologous
proteins in insect cells are reviewed by Luckow and Summers,
(Bio/Technology, 6:47, 1988). Examples of suitable mammalian host
cell lines include endothelial cells, COS-7 monkey kidney cells,
CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human
embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines.
Purified polypeptides are prepared by culturing suitable
host/vector systems to express the recombinant proteins. In some
embodiments, the small size of many of the polypeptides disclosed
herein would make expression in E. coli as the preferred method for
expression. The protein is then purified from culture media or cell
extracts.
[0134] Proteins disclosed herein can also be produced using
cell-translation systems. For such purposes the nucleic acids
encoding the polypeptide must be modified to allow in vitro
transcription to produce mRNA and to allow cell-free translation of
the mRNA in the particular cell-free system being utilized
(eukaryotic such as a mammalian or yeast cell-free translation
system or prokaryotic such as a bacterial cell-free translation
system. PSMA-binding polypeptides can also be produced by chemical
synthesis (e.g., by the methods described in Solid Phase Peptide
Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.).
Modifications to the protein can also be produced by chemical
synthesis.
[0135] The polypeptides of the present disclosure can be purified
by isolation/purification methods for proteins generally known in
the field of protein chemistry. Non-limiting examples include
extraction, recrystallization, salting out (e.g., with ammonium
sulfate or sodium sulfate), centrifugation, dialysis,
ultrafiltration, adsorption chromatography, ion exchange
chromatography, hydrophobic chromatography, normal phase
chromatography, reversed-phase chromatography, gel filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, countercurrent distribution or any combinations of
these. After purification, polypeptides may be exchanged into
different buffers and/or concentrated by any of a variety of
methods known to the art, including, but not limited to, filtration
and dialysis. The purified polypeptide is preferably at least 85%
pure, more preferably at least 95% pure, and most preferably at
least 98% pure. Regardless of the exact numerical value of the
purity, the polypeptide is sufficiently pure for use as a
pharmaceutical product.
[0136] In certain embodiments, the present disclosure provides
monoclonal antibodies that bind to PSMA. Monoclonal antibodies may
be produced using any technique known in the art, e.g., by
immortalizing spleen cells harvested from the transgenic animal
after completion of the immunization schedule. The spleen cells can
be immortalized using any technique known in the art, e.g., by
fusing them with myeloma cells to produce hybridomas. Myeloma cells
for use in hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency, and enzyme
deficiencies that render them incapable of growing in certain
selective media which support the growth of only the desired fused
cells (hybridomas). Examples of suitable cell lines for use in
mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4
1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0
Bul; examples of cell lines used in rat fusions include R210.RCY3,
Y3-Ag 1.2.3, IR983F and 48210. Other cell lines useful for cell
fusions are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.
[0137] Fragments or analogs of antibodies can be readily prepared
by those of ordinary skill in the art following the teachings of
this specification and using techniques known in the art. Preferred
amino- and carboxy-termini of fragments or analogs occur near
boundaries of functional domains. Structural and functional domains
can be identified by comparison of the nucleotide and/or amino acid
sequence data to public or proprietary sequence databases.
Computerized comparison methods can be used to identify sequence
motifs or predicted protein conformation domains that occur in
other proteins of known structure and/or function. Methods to
identify protein sequences that fold into a known three-dimensional
structure are known. See, Bowie et al., 1991, Science 253:164.
Post-Translational Modifications of Polypeptides
[0138] In certain embodiments, the binding polypeptides of the
invention may further comprise post-translational modifications.
Exemplary post-translational protein modifications include
phosphorylation, acetylation, methylation, ADP-ribosylation,
ubiquitination, glycosylation, carbonylation, sumoylation,
biotinylation or addition of a polypeptide side chain or of a
hydrophobic group. As a result, the modified soluble polypeptides
may contain non-amino acid elements, such as lipids, poly- or
mono-saccharide, and phosphates. A preferred form of glycosylation
is sialylation, which conjugates one or more sialic acid moieties
to the polypeptide. Sialic acid moieties improve solubility and
serum half-life while also reducing the possible immunogeneticity
of the protein. See Raju et al. Biochemistry. 2001 31;
40(30):8868-76.
[0139] In one embodiment, modified forms of the subject soluble
polypeptides comprise linking the subject soluble polypeptides to
nonproteinaceous polymers. In one embodiment, the polymer is
polyethylene glycol ("PEG"), polypropylene glycol, or
polyoxyalkylenes, in the manner as set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0140] PEG is a water soluble polymer that is commercially
available or can be prepared by ring-opening polymerization of
ethylene glycol according to methods well known in the art (Sandler
and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,
pages 138-161). The term "PEG" is used broadly to encompass any
polyethylene glycol molecule, without regard to size or to
modification at an end of the PEG, and can be represented by the
formula: X--O(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2OH (1),
where n is 20 to 2300 and X is H or a terminal modification, e.g.,
a C.sub.1-4 alkyl. In one embodiment, the PEG of the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or
CH.sub.3 ("methoxy PEG"). A PEG can contain further chemical groups
which are necessary for binding reactions; which results from the
chemical synthesis of the molecule; or which is a spacer for
optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked
together. PEGs with more than one PEG chain are called multi-armed
or branched PEGs. Branched PEGs can be prepared, for example, by
the addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEG are described in, for example, EP-A 0 473 084
and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEG
side-chains (PEG2) linked via the primary amino groups of a lysine
(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).
[0141] The serum clearance rate of PEG-modified polypeptide may be
decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even
90%, relative to the clearance rate of the unmodified binding
polypeptide. The PEG-modified polypeptide may have a half-life
(t.sub.1/2) which is enhanced relative to the half-life of the
unmodified protein. The half-life of PEG-binding polypeptide may be
enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by
1000% relative to the half-life of the unmodified binding
polypeptide. In some embodiments, the protein half-life is
determined in vitro, such as in a buffered saline solution or in
serum. In other embodiments, the protein half-life is an in vivo
half life, such as the half-life of the protein in the serum or
other bodily fluid of an animal.
Therapeutic Methods, Formulations and Modes of Administration
[0142] The present disclosure provides a method for treating
cancer, comprising administering an anti-PSMA antibody, or antibody
fragment, as described herein. The present disclosure further
provides a method for treating a prostate cancer, comprising
administering an anti-PSMA antibody, or an antibody fragment as
described herein.
[0143] Studies have demonstrated PSMA expression in many types of
prostate tissue and increased PSMA expression in cancer tissue
(Chang, S. Rev Urol. 2004; 6(Suppl 10): S13-S18; Silver et al. Clin
Cancer Res. 1997; 3:81-85; Troyer et al. Int J Cancer. 1995;
62:552-558; Haffner et al. Hum Pathol. 2009 December;
40(12):1754-61). Accordingly, in one embodiment, the anti-PSMA
antibodies and antibody fragments of the invention are used to
treat cancer associated with increased PSMA expression.
[0144] Any of the antibodies disclosed herein may be used in such
methods. For example, the methods may be performed using a fully
human antibody of an IgG class that binds to a PSMA epitope with a
binding affinity of at least 10.sup.-6M, a Fab fully human antibody
fragment, having a variable domain region from a heavy chain and a
variable domain region from a light chain, a single chain human
antibody, having a variable domain region from a heavy chain and a
variable domain region from a light chain and a peptide linker
connection the heavy chain and light chain variable domain regions,
including the heavy and light chain variable regions (and CDRs
within said sequences) described in SEQ ID Nos. 1-17 (Table 3).
[0145] In one embodiment, the antibodies disclosed herein are used
to target a chemical or cellular therapeutic payload, wherein the
anti-PSMA antibody comprises a heavy chain variable domain sequence
that is at least 95% identical, at least 96% identical, at least
97% identical, at least 98% identical, or at least 99% identical,
to the amino acid sequence SEQ ID NO. 1, and comprising a light
chain variable domain sequence that is at least 95% identical, at
least 96% identical, at least 97% identical, at least 98%
identical, or at least 99% identical, to an amino acid sequences
selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3,
SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.
8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ
ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, and SEQ ID
NO. 17.
[0146] In one embodiment, the methods described herein include the
use of a fully human Fab antibody fragment comprising a heavy chain
variable domain sequence that is at least 95% identical, at least
96% identical, at least 97% identical, at least 98% identical, or
at least 99% identical, to the amino acid sequence SEQ ID NO. 1,
and comprising a light chain variable domain sequence that is at
least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical, to an
amino acid sequence selected from the group consisting SEQ ID NO.
2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID
NO. 8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11,
SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID
NO. 16, and SEQ ID NO. 17.
[0147] In one embodiment, the methods described herein include the
use of a single chain human antibody comprising a heavy chain
variable domain sequence that is at least 95% identical, at least
96% identical, at least 97% identical, at least 98% identical, or
at least 99% identical, to the amino acid sequence SEQ ID NO. 1,
and comprising a light chain variable domain sequence that is at
least 95% identical, at least 96% identical, at least 97%
identical, at least 98% identical, or at least 99% identical, to an
amino acid sequence selected from the group consisting of SEQ ID
NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ
ID NO. 8, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11,
SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID
NO. 16, and SEQ ID NO. 17.
[0148] In one embodiment, the fully human antibody has both a heavy
chain and a light chain wherein the antibody comprises a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3,
SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO.
1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 1/SEQ ID NO.
8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID NO. 1/SEQ ID NO. 10, SEQ ID
NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ ID NO. 12, SEQ ID NO. 1/SEQ
ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14, SEQ ID NO. 1/SEQ ID NO. 15,
SEQ ID NO. 1/SEQ ID NO. 16, and SEQ ID NO. 1/SEQ ID NO. 17.
[0149] In one embodiment, the fully human antibody Fab fragment
comprises both a heavy chain variable domain region and a light
chain variable domain region wherein the antibody comprises a heavy
chain/light chain variable domain sequence selected from the group
consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 1/SEQ ID NO. 3,
SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID NO. 1/SEQ ID NO. 5, SEQ ID NO.
1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID NO. 7, SEQ ID NO. 1/SEQ ID NO.
8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID NO. 1/SEQ ID NO. 10, SEQ ID
NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ ID NO. 12, SEQ ID NO. 1/SEQ
ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14, SEQ ID NO. 1/SEQ ID NO. 15,
SEQ ID NO. 1/SEQ ID NO. 16, and SEQ ID NO. 1/SEQ ID NO. 17.
[0150] In one embodiment, the fully human single chain antibody
comprises both a heavy chain variable domain region and a light
chain variable domain region, wherein the single chain fully human
antibody comprises a heavy chain/light chain variable domain
sequence selected from the group consisting of SEQ ID NO. 1/SEQ ID
NO. 2, SEQ ID NO. 1/SEQ ID NO. 3, SEQ ID NO. 1/SEQ ID NO. 4, SEQ ID
NO. 1/SEQ ID NO. 5, SEQ ID NO. 1/SEQ ID NO. 6, SEQ ID NO. 1/SEQ ID
NO. 7, SEQ ID NO. 1/SEQ ID NO. 8, SEQ ID NO. 1/SEQ ID NO. 9, SEQ ID
NO. 1/SEQ ID NO. 10, SEQ ID NO. 1/SEQ ID NO. 11, SEQ ID NO. 1/SEQ
ID NO. 12, SEQ ID NO. 1/SEQ ID NO. 13, SEQ ID NO. 1/SEQ ID NO. 14,
SEQ ID NO. 1/SEQ ID NO. 15, SEQ ID NO. 1/SEQ ID NO. 16, and SEQ ID
NO. 1/SEQ ID NO. 17.
[0151] In one embodiment, cancer which can be treated using the
antibodies and fragments disclosed herein include, but are not
limited to, ovarian cancer, colon cancer, breast cancer, lung
cancer, myelomas, neuroblastic-derived CNS tumors, monocytic
leukemias, B-cell derived leukemias, T-cell derived leukemias,
B-cell derived lymphomas, T-cell derived lymphomas, and mast cell
derived tumors.
[0152] In one embodiment, the anti-PSMA antibodies and antibody
fragments of the invention are used to treat prostate cancer.
[0153] The PSMA antibodies, and antibody fragments, described
herein are useful in treating, delaying the progression of,
preventing relapse of or alleviating a symptom of a cancer or other
neoplastic condition, including, hematological malignancies and/or
PSMA+tumors. The PSMA antibodies described herein are useful in
treating a cancer selected from the group consisting of
non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL),
acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL),
chronic myelogenous leukemia (CML), multiple myeloma (MM), breast
cancer, ovarian cancer, head and neck cancer, bladder cancer,
melanoma, colorectal cancer, pancreatic cancer, lung cancer,
leiomyoma, leiomyosarcoma, glioma, glioblastoma, and solid tumors,
wherein solid tumors are selected from the group consisting of
breast tumors, ovarian tumors, lung tumors, pancreatic tumors,
prostate tumors, melanoma tumors, colorectal tumors, lung tumors,
head and neck tumors, bladder tumors, esophageal tumors, liver
tumors, and kidney tumors.
[0154] As used herein, "hematological cancer" refers to a cancer of
the blood, and includes leukemia, lymphoma and myeloma among
others. "Leukemia" refers to a cancer of the blood in which too
many white blood cells that are ineffective in fighting infection
are made, thus crowding out the other parts that make up the blood,
such as platelets and red blood cells. It is understood that cases
of leukemia are classified as acute or chronic. As such, antibodies
and fragments of the invention may be used to treat a subject
having a hematological cancer.
[0155] Certain forms of leukemia include, acute lymphocytic
leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic
leukemia (CLL); chronic myelogenous leukemia (CML);
Myeloproliferative disorder/neoplasm (MPDS); and myelodysplasia
syndrome. "Lymphoma" may refer to a Hodgkin's lymphoma, both
indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma,
and follicular lymphoma (small cell and large cell), among others.
Myeloma may refer to multiple myeloma (MM), giant cell myeloma,
heavy-chain myeloma, and light chain or Bence-Jones myeloma.
[0156] The present disclosure features methods for treating or
preventing the S. aureus infection comprising administering an
anti-PSMA polypeptide. Techniques and dosages for administration
vary depending on the type of specific polypeptide and the specific
condition being treated but can be readily determined by the
skilled artisan. In general, regulatory agencies require that a
protein reagent to be used as a therapeutic is formulated so as to
have acceptably low levels of pyrogens. Accordingly, therapeutic
formulations will generally be distinguished from other
formulations in that they are substantially pyrogen free, or at
least contain no more than acceptable levels of pyrogen as
determined by the appropriate regulatory agency (e.g., FDA).
[0157] Therapeutic compositions of the present disclosure may be
administered with a pharmaceutically acceptable diluent, carrier,
or excipient, in unit dosage form. Administration may be parenteral
(e.g., intravenous, subcutaneous), oral, or topical, as
non-limiting examples. In addition, any gene therapy technique,
using nucleic acids encoding the polypeptides of the invention, may
be employed, such as naked DNA delivery, recombinant genes and
vectors, cell-based delivery, including ex vivo manipulation of
patients' cells, and the like.
[0158] The composition can be in the form of a pill, tablet,
capsule, liquid, or sustained release tablet for oral
administration; or a liquid for intravenous, subcutaneous or
parenteral administration; gel, lotion, ointment, cream, or a
polymer or other sustained release vehicle for local
administration.
[0159] In certain embodiments, the disclosed antibodies are
administered by inhalation, but aerosolization of full IgG
antibodies may prove limiting due to their molecular size
(.about.150 kDa).
[0160] Methods well known in the art for making formulations are
found, for example, in "Remington: The Science and Practice of
Pharmacy" (20th ed., ed. A. R. Gennaro A R., 2000, Lippincott
Williams & Wilkins, Philadelphia, Pa.). Formulations for
parenteral administration may, for example, contain excipients,
sterile water, saline, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, or hydrogenated napthalenes.
Biocompatible, biodegradable lactide polymer, lactide/glycolide
copolymer, or polyoxyethylene-polyoxypropylene copolymers may be
used to control the release of the compounds. Nanoparticulate
formulations (e.g., biodegradable nanoparticles, solid lipid
nanoparticles, liposomes) may be used to control the
biodistribution of the compounds. Other potentially useful
parenteral delivery systems include ethylene-vinyl acetate
copolymer particles, osmotic pumps, implantable infusion systems,
and liposomes. The concentration of the compound in the formulation
varies depending upon a number of factors, including the dosage of
the drug to be administered, and the route of administration.
[0161] The polypeptide may be optionally administered as a
pharmaceutically acceptable salt, such as non-toxic acid addition
salts or metal complexes that are commonly used in the
pharmaceutical industry. Examples of acid addition salts include
organic acids such as acetic, lactic, pamoic, maleic, citric,
malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic,
tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic
acids or the like; polymeric acids such as tannic acid,
carboxymethyl cellulose, or the like; and inorganic acid such as
hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid,
or the like. Metal complexes include zinc, iron, and the like. In
one example, the polypeptide is formulated in the presence of
sodium acetate to increase thermal stability.
[0162] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose and sorbitol), lubricating
agents, glidants, and anti-adhesives (e.g., magnesium stearate,
zinc stearate, stearic acid, silicas, hydrogenated vegetable oils,
or talc).
[0163] Formulations for oral use may also be provided as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent, or as soft gelatin capsules
wherein the active ingredient is mixed with water or an oil
medium.
[0164] A therapeutically effective dose refers to a dose that
produces the therapeutic effects for which it is administered. The
exact dose will depend on the disorder to be treated, and may be
ascertained by one skilled in the art using known techniques. In
general, the polypeptide is administered at about 0.01 .mu.g/kg to
about 50 mg/kg per day, preferably 0.01 mg/kg to about 30 mg/kg per
day, most preferably 0.1 mg/kg to about 20 mg/kg per day. The
polypeptide may be given daily (e.g., once, twice, three times, or
four times daily) or preferably less frequently (e.g., weekly,
every two weeks, every three weeks, monthly, or quarterly). In
addition, as is known in the art, adjustments for age as well as
the body weight, general health, sex, diet, time of administration,
drug interaction, and the severity of the disease may be necessary,
and will be ascertainable with routine experimentation by those
skilled in the art.
[0165] A PSMA binding polypeptide, as disclosed herein, can be
administered alone or in combination with one or more additional
therapies such as chemotherapy radiotherapy, immunotherapy,
surgical intervention, or any combination of these. Long-term
therapy is equally possible as is adjuvant therapy in the context
of other treatment strategies, as described above.
[0166] In certain embodiments of such methods, one or more
polypeptide therapeutic agents can be administered, together
(simultaneously) or at different times (sequentially). In addition,
polypeptide therapeutic agents can be administered with another
type of compounds for treating cancer or for inhibiting
angiogenesis.
[0167] In certain embodiments, the subject anti-PSMA antibodies
agents of the invention can be used alone.
[0168] In certain embodiments, the binding polypeptides of
fragments thereof can be labeled or unlabeled for diagnostic
purposes. Typically, diagnostic assays entail detecting the
formation of a complex resulting from the binding of a binding
polypeptide to PSMA. The binding polypeptides or fragments can be
directly labeled, similar to antibodies. A variety of labels can be
employed, including, but not limited to, radionuclides,
fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme
inhibitors and ligands (e.g., biotin, haptens). Numerous
appropriate immunoassays are known to the skilled artisan (see, for
example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and
4,098,876). When unlabeled, the binding polypeptides can be used in
assays, such as agglutination assays. Unlabeled binding
polypeptides can also be used in combination with another (one or
more) suitable reagent which can be used to detect the binding
polypeptide, such as a labeled antibody reactive with the binding
polypeptide or other suitable reagent (e.g., labeled protein
A).
[0169] In one embodiment, the binding polypeptides of the present
invention can be utilized in enzyme immunoassays, wherein the
subject polypeptides are conjugated to an enzyme. When a biological
sample comprising a PSMA protein is combined with the subject
binding polypeptides, binding occurs between the binding
polypeptides and the PSMA protein. In one embodiment, a sample
containing cells expressing a PSMA protein (e.g., endothelial
cells) is combined with the subject antibodies, and binding occurs
between the binding polypeptides and cells bearing a PSMA protein
recognized by the binding polypeptide. These bound cells can be
separated from unbound reagents and the presence of the binding
polypeptide-enzyme conjugate specifically bound to the cells can be
determined, for example, by contacting the sample with a substrate
of the enzyme which produces a color or other detectable change
when acted on by the enzyme. In another embodiment, the subject
binding polypeptides can be unlabeled, and a second, labeled
polypeptide (e.g., an antibody) can be added which recognizes the
subject binding polypeptide.
[0170] In certain aspects, kits for use in detecting the presence
of a PSMA protein in a biological sample can also be prepared. Such
kits will include a PSMA binding polypeptide which binds to a PSMA
protein or portion of said receptor, as well as one or more
ancillary reagents suitable for detecting the presence of a complex
between the binding polypeptide and the receptor protein or
portions thereof. The polypeptide compositions of the present
invention can be provided in lyophilized form, either alone or in
combination with additional antibodies specific for other epitopes.
The binding polypeptides and/or antibodies, which can be labeled or
unlabeled, can be included in the kits with adjunct ingredients
(e.g., buffers, such as Tris, phosphate and carbonate, stabilizers,
excipients, biocides and/or inert proteins, e.g., bovine serum
albumin). For example, the binding polypeptides and/or antibodies
can be provided as a lyophilized mixture with the adjunct
ingredients, or the adjunct ingredients can be separately provided
for combination by the user. Generally these adjunct materials will
be present in less than about 5% weight based on the amount of
active binding polypeptide or antibody, and usually will be present
in a total amount of at least about 0.001% weight based on
polypeptide or antibody concentration. Where a second antibody
capable of binding to the binding polypeptide is employed, such
antibody can be provided in the kit, for instance in a separate
vial or container. The second antibody, if present, is typically
labeled, and can be formulated in an analogous manner with the
antibody formulations described above.
[0171] Polypeptide sequences are indicated using standard one- or
three-letter abbreviations. Unless otherwise indicated, each
polypeptide sequence has amino termini at the left and a carboxy
termini at the right; each single-stranded nucleic acid sequence,
and the top strand of each double-stranded nucleic acid sequence,
has a 5' termini at the left and a 3' termini at the right. A
particular polypeptide sequence also can be described by explaining
how it differs from a reference sequence.
[0172] Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included for purposes of illustration only and
are not intended to be limiting of the invention.
Example 1
[0173] Human anti-PSMA antibodies were identified, and the amino
acid sequences of the light chain and heavy chain variable regions
are described in Table 3 below.
[0174] This example illustrates the binding of anti-PSMA antibodies
to endogenous human PSMA expressed on LNCaP human prostate cancer
cells, as assayed by flow cytometry. EC.sub.50 values for
antibodies were determined as follows.
[0175] PSMA expressing LNCaP cells were harvested with enzyme-free
Cell Dissociation Buffer (GIBCO) and transferred to V-Bottom 96
well-plates (50,000 cells/well). Cells were incubated on ice for 45
min with serial dilutions of anti-PSMA antibodies PSGB11, PSGB12,
PSGC12, PSGD4, PSGD6 and PSA11 in FACS buffer (PBS+2%
FBS)+NaN.sub.3. The control antibody (clg) was a negative control,
and was an isotype matched, nonspecific (i.e., does not bind to
PSMA and also did not bind the cells) antibody. After 1 wash in
FACS buffer, a 1:1000 dilution of Phycoerythrin conjugated
anti-Human IgG (y-chain specific) was added and incubated for 30
min Following a final wash, fluorescence intensity was measured on
an Intellicyt High Throughput Flow Cytometer (HTFC).
[0176] Data were analyzed using Graphpad Prism software and
non-linear regression fit. Data points are shown as the median
fluorescence intensity (MFI) of positively labeled cells+/-Standard
Error. EC.sub.50 values are reported as the concentration of
antibody to achieve 50% of maximal PSMA antibodies binding to PSMA
expressing cells.
[0177] In parallel, cell binding was also assessed with the same
method on PC3, a prostate cancer cell line that does not express
human PSMA endogenously, and thus served as a negative control.
[0178] The results from the binding assays for both cancer,
PSMA-specific LNCaP cells and negative control PC3 cells are
provided in FIG. 1 (A-D). As shown in FIGS. 1A and B, anti-PSMA
antibodies PSGB11, PSGB12, PSGC12, PSGD4, PSGD6 and PSA11 strongly
bound PSMA-expressing LNCaP cells relative to the negative IgG
control (see FIG. 1A). Furthermore, as described in Figures (C) and
(D), anti-PSMA antibodies PSGB11, PSGB12, PSGC12, PSGD4, PSGD6 and
PSA11 showed little to no binding to cells not expressing human
PSMA (PC3 cells), as the cells showed similar results to the
negative control antibody (see FIG. 1(C)). Thus, the results in
FIG. 1(A-D) show that the antibodies were specific to PSMA
expressing cells, including PSMA expressing cancer cells.
[0179] In addition, the anti-PSMA antibodies identified herein
displayed EC.sub.50 values in the nanomolar range for LNCaP PSMA
expressing cells, as described below in Table 1. These data
demonstrate strong and specific binding of the antibodies described
in Table 3 to endogenous PSMA.
TABLE-US-00001 TABLE 1 Anti-PSMA Binding to LNCaP Antibodies cells
(EC.sub.50, nM) PSA11 2.4 PSGF9 2.08 PSGC9 2.05 PSGD6 1.92 PSGD3
3.15 PSGE10 1.92 PSGH3 3.72 PSGD4 1.59 PSGB12 3.46 PSGB11 1.98
PSGE11 553.70 PSGC8 3.84 PSG6 6.95 PSGH8 2.13 PSGG6 1.3 PSGB11 1.83
PSGC12 2.84
Example 2
[0180] This example illustrates in vitro data showing the
assessment of anti-PSMA antibodies in a cytotoxicity assay using
secondary antibody-drug conjugate technique ("Secondary
Antibody-Drug Conjugates as Tools for ADC Discovery". Helen Mao,
Poster, IBC 24.sup.th Annual, 2013). This example demonstrates the
potential of anti-PSMA antibodies to be used as antibody drug
conjugates.
[0181] PSMA-expressing prostate cancer cells (LNCaP, ATCC
CRL-1740.TM.) were harvested with enzyme-free Cell Dissociation
Buffer (GIBCO), seeded into white 96-Well Clear Bottom plates
(2,000 cells/well in 90 .mu.l) and allowed to adhere overnight at
37.degree. C. Anti-PSMA antibodies PSGF9, PSGC9, PSGD6, PSGD3,
PSGE10, PSGH3, PSGB11, PSGD4, PSGB12 and PSA11 were used in the
experiment. Antibodies were pre-complexed with Protein G(PG)-MMAF
(Monomethyl auristatin F, Concortis Biosystems) in cell culture
media, at a 1:4 molar ratio. The control antibody (cIg) was an
isotype matched, nonspecific (i.e., does not bind to PSMA)
antibody. PGMMAF alone was used as a negative control. After 10 min
at room temperature, serial dilutions of the antibody-ProteinG-MMAF
complex were prepared in cell culture media, incubated 10 more
minutes at room temperature, and added to cells (10 .mu.l/well) in
triplicate. In some experiments, plates were incubated at
37.degree. C. for 4 days ("No wash method"). In other experiments
("2-hours-wash method"), plates were incubated at 37.degree. C. for
two hours, media was aspirated. Then cells were washed once with
full media, and left in fresh full media before being incubated at
37.degree. C. for 4 days. For all experiments, cell proliferation
was then analyzed as follows: 100 .mu.l of Cell Titer Glo buffer
(Promega) was added to each well. Plates were incubated with
shaking at room temperature for 20 min Luminescence signal was then
measured on a Flexstation 3 plate reader (Molecular Device). Data
were reported as relative Luminescent Units. Dose-response curves
were generated in GraphPad prism, and IC.sub.50 values were
calculated using non-linear regression fit (Log (inhibitor) vs.
response--Variable slope equation).
[0182] The results are shown in FIG. 2(A-C) and below in Table 2.
The results described in FIG. 2A-C show that anti-PSMA ADCs
comprising antibodies PSGF9, PSGC9, PSGD6, (FIG. 2A); PSGD3,
PSGE10, PSGH3, PSGB11, (FIG. 2B); PSGD4, PSGB12 and PSA11 (FIG.
2C), each conjugated to MMAF can induce cell killing in LNCaP
cancer cells. In contrast, MMAF alone and the IgG control (see FIG.
2A) had little to no effect on cell death and had high rates of
cell survival.
[0183] The same methods were used with PSMA-negative prostate
cancer cells (PC-3, ATCC CRL-1435) to assess the non-specificity of
cell killing of anti-PSMA antibodies (PSGF9, PSGC9, PSGD6, PSGD3,
PSGE10, PSGH3, PSGB11, PSGD4, PSGB12 and PSA11)/Protein G-MMAF
complexes. The results, shown in FIG. 2D, indicate that anti-PSMA
antibodies do not induce cell killing when complexed with a
cytotoxin such as MMAF in PSMA negative cancer cells. Thus, FIGS. 1
and 2 show that little to no non-specific cell killing is observed
on cells not overexpressing PSMA, suggesting a good selectivity
index for future PSMA antibody drug conjugate (ADC). Altogether,
this illustrates the potential of PSMA antibodies as antibody-drug
conjugates.
[0184] IC.sub.50 values for the anti-PSMA-MMAF ADCs were determined
and are described below in Table 2. To verify that the cell killing
observed for these types of secondary conjugates was due to the
specifically internalized conjugates tested, unbound conjugates
were washed out (2-hours-wash method in Table 2). As described in
Table 2, the IC.sub.50 values were in the nanomolar range.
TABLE-US-00002 TABLE 2 Cell killing, No wash Cell killing,
2-hours-wash Antibodies method (IC.sub.50, nM) method (IC.sub.50,
nM) PSA11 0.08 1.55 PSGB11 0.04 1.60 PSGB12 0.05 1.66 PSGC9 0.06
1.22 PSGD3 0.12 2.24 PSGD4 0.06 2.09 PSGD6 0.04 1.13 PSGE10 0.08
1.58 PSGF9 0.13 1.33 PSGH3 0.06 1.51
Example 3
[0185] In addition to the ability of the anti-PSMA antibodies
described herein to bind to PSMA and also to induce cell death when
conjugated to a toxin, the affinity values for antibodies PSA11 and
PSGB11 were determined on Octet. As described in FIG. 3, anti-PSMA
antibody PSA11 had an K.sub.D of about 6.7.times.10.sup.-10 M, and
antibody PSA11 (described in FIG. 4) had a K.sub.D of about
6.8.times.10.sup.-10 M.
[0186] Specifically, an amine Reactive Second-Generation (AR2G)
sensor was coated with antibody (15 ug/ml in acetic buffer, pH5.0)
using
Sulfo-N-hydroxysuccinimide/N-Ethyl-N'-(3-dimethylaminopropyl)carbodiimide
hydrochloride (Sulfo-NHS/EDC) coupling methodology. The sensors
were quenched with 1M pH 8.5 ethanolamine Data was collected by
moving sensors to serially diluted recombinant human PSMA/His (in
PBS buffer) wells, then the data was dissociated by transferring
the sensors to PBS wells. A 1:1 binding model was used to fit the
data. Results from the assay are provided in FIGS. 3 and 4.
TABLE-US-00003 TABLE 3 Amino Acid Heavy and Light Chain Variable
Domains Heavy chain variable domain Light chain variable domain
PSA11 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS
VIWMTQSPSSVSASVGDRVTITCRASQGISS YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD
WLAWYQQKPGKAPKLLIYAASNLQSGVPSRF SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
SGSGSGTDFTLTISSLQPEDFATYYCQQANS YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS
FPLTFGGGTKVDIK SEQ ID NO. 1 SEQ ID NO. 2 PSGB11
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS EIVLTQSPSTLSASVGDRVTITCRASQSISS
YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD WLAWYQQKPGKAPRLLIYAASILQRGVPSRF
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV SGSGSETDFTLTISSLQPEDLATYYCQETYS
YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS NLFTFGPGTKVDIK SEQ ID NO. 1 SEQ ID
NO. 3 PSGB12 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS
DVVMTQSPSTLSASVGDRVTITCRASQSISS YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD
WLAWYQQKPGKAPKLLIFAASSLQSGVPSRF SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
SGSGSGTDFALTISSLQPEDFATYYCQESYS YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS
IPWTFGQGTKVEIK SEQ ID NO. 1 SEQ ID NO. 4 PSGC8
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS AIRMTQSPSSVSASVGDRVTITCRASQGISS
YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD WLAWYQQKPGKAPKLLIYAASSLQSGVPSRF
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV SGSGSGTDFTLTISSLKPEDFATYYCQQANS
YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS FPRALTFGGGTKVEIK SEQ ID NO. 1 SEQ
ID NO. 5 PSGC9 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS
AIRMTQSPSTLSASVGDRVTITCRASQNIYG YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD
WLAWYQQKPGKAPELLIYAASSLQSGYPSRF SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
SGSGSGTDFTLTINSLQPEDFATYYCQQSYT YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS
IPFTFGPGTKVDIK SEQ ID NO. 1 SEQ ID NO. 6 PSGC12
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS DIVMTQSPSSVSASVGDRVTITCRASQDVGT
YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD WLAWYQQKPGRAPKLLIYVASSLQSGYPSRF
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV SGSGSGTDFTLTISSLQPEDSATYYCQQAKG
YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS IPYTFGQGTKLEIK SEQ ID NO. 1 SEQ ID
NO. 7 PSGD3 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS
DIVMTQSPSSVSASVGDRVTITCRASQDINN YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD
WLAWYQQKAGKAPKLLIYVATKLQNGVPSRF SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
SGSGSGTDFTLSISNLQPEDFATYYCQQAKS YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS
FPYTFGQGTKLEIK SEQ ID NO. 1 SEQ ID NO. 8 PSGD4
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS DIVMTQSPSSLSASVGDRVSITCRASQGIST
YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD WLAWYQQKPGKAPDLLIYAASNLQSGVPSRF
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV SGSGSGTDFTLTISSLQPEDFATYYCQQANS
YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS FPLTFGGGTKVEIK SEQ ID NO. 1 SEQ ID
NO. 9 PSGD6 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS
DIVMTQSPSSVSASVGDRVTITCRASQAISS YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD
WLAWYQQKPGKAPKLLIYAASSLQSGVPSRF SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
SGSGSGTDFTLTISSLQPEDFATYYCQQAYS YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS
FPVTFGPGTKVDIK SEQ ID NO. 1 SEQ ID NO. 10 PSGE10
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS DVVMTQSPSSVSASVGDRVTITCRASQGISS
YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD WLAWYQQKPGKAPKLLIYAASTLQSGYPSRF
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV SGSGSGTDFTLTINNLQPEDFATYYCQQTAS
YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS FPINFGGGTKVEIK SEQ ID NO. 1 SEQ ID
NO. 11 PSGE11 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS
DIVMTQSPSTLSASVGDRVTITCRASQSISN YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD
WLAWYQQKPGKPPKLLIYAASSLQSGVPSRF SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
SGSGSGTDFTLTISSLQPEDFATYYCQQSYR YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS
SLTFAGGTKVEIK SEQ ID NO. 1 SEQ ID NO. 12 PSGF9
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS AIQMTQSPSSVSASVGDRVTITCRASQGISS
YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD WLAWYQQKPGKAPKLLIYAASSLQSGVPSRF
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV SGSGSGTDFTLTISSLQPEDIATYYCQESYS
YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS TPFTFGPGTKVDIK SEQ ID NO. 1 SEQ ID
NO. 13 PSGF11 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS
DIQMTQSPSYVSASVGDRVTITCRASQGVSH YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD
WLAWYQQKPGKAPKLLIYAASRLQSGVPSRF SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
SGSGSGTDFTLTISSLQPEDFATYYCQQAYS YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS
FPLTFGQGTKLEIK SEQ ID NO. 1 SEQ ID NO. 14 PSGG6
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS EIVLTQSPSSLSASVGDRVTITCRASQGISS
YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD YLAWYQQKPGKAPKLLIYAASTLQSGVPSRF
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV SGSGSGTDFTLTISSLQPEDFATYYCQQLNS
YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS YPRGITFGQGTKLEIK SEQ ID NO. 1 SEQ
ID NO. 15 PSGH3 QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS
DIVMTQSPSSVSASVGDRVTITCRASQGISN YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD
WLAWYQQKPGKAPKLLIYVASSLQSGVPSRF SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV
SGSGSGTDFTLTISSLQPEDFATYYCQQANS YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS
FPITFGQGTRLEIK SEQ ID NO. 1 SEQ ID NO. 16 PSGH8
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSS DIVMTQSPSSLSASVGDRVTITCRASQGISS
YWMSWVRQAPGKGLEWVANIKQDGSEKYYVD WLAWYQQKPGKAPKLLIYAASSLQSGVPSRF
SVKGRFTISRDNAKNSLYLQMNSLRAEDTAV SGSGSGTDFTLTINSLQPEDFATYYCQQASG
YYCARVWDYYYDSSGDAFDIWGQGTMVTVSS FPFTFGPGTKVDIK SEQ ID NO. 1 SEQ ID
NO. 17
INCORPORATION BY REFERENCE
[0187] The contents of all references, patents, pending patent
applications and published patents, cited throughout this
application are hereby expressly incorporated by reference.
Sequence CWU 1
1
191124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Val Trp Asp Tyr Tyr Tyr Asp Ser Ser Gly Asp Ala Phe Asp
100 105 110 Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
2107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 2Val Ile Trp Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Asn
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100 105
3107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Glu Ile Val Leu Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ile
Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Glu Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Leu Ala Thr Tyr Tyr Cys Gln Glu Thr Tyr Ser Asn Leu Phe 85 90
95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
4107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Asp Val Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Glu Ser Tyr Ser Ile Pro Trp 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
5109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Ala Ile Arg Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Lys Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Arg 85 90
95 Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
6107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 6Ala Ile Arg Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asn Ile Tyr Gly Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Ile Pro Phe 85 90
95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
7107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Val Gly Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Arg Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ala Lys Gly Ile Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
8107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 8Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Asn Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Ala Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Thr Lys
Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Ser Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Lys Ser Phe Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
9107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Asp Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Asn
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
10107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ala Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Phe Pro Val 85 90
95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
11107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Asp Val Val Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Ala Ser Phe Pro Ile 85 90
95 Asn Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
12106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 12Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Ser Leu Thr 85 90
95 Phe Ala Gly Gly Thr Lys Val Glu Ile Lys 100 105
13107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Ala Ile Gln Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Glu Ser Tyr Ser Thr Pro Phe 85 90
95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
14107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 14Asp Ile Gln Met Thr Gln Ser Pro Ser Tyr Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Val Ser His Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Arg
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Phe Pro Leu 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
15109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 15Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Arg 85 90
95 Gly Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
16107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Val Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Ile 85 90
95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105
17107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ser Gly Phe Pro Phe 85 90
95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
184PRTUnknownDescription of Unknown Hinge region sequence 18Cys Pro
Ser Cys 1 194PRTUnknownDescription of Unknown Hinge region sequence
19Cys Pro Pro Cys 1
* * * * *