U.S. patent application number 15/252267 was filed with the patent office on 2017-02-02 for antibodies to human b7x for treatment of metastatic cancer.
This patent application is currently assigned to ALBERT EINSTEIN COLLEGE OF MEDICINE, INC.. The applicant listed for this patent is ALBERT EINSTEIN COLLEGE OF MEDICINE, INC., SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH. Invention is credited to James P. Allison, Xingxing Zang.
Application Number | 20170029525 15/252267 |
Document ID | / |
Family ID | 47041926 |
Filed Date | 2017-02-02 |
United States Patent
Application |
20170029525 |
Kind Code |
A1 |
Zang; Xingxing ; et
al. |
February 2, 2017 |
ANTIBODIES TO HUMAN B7X FOR TREATMENT OF METASTATIC CANCER
Abstract
Methods are provided for treating metastatic cancer in patients
having metastatic cancer or for preventing metastasis in cancer
patients at risk for metastasis comprising administering to the
patient an antibody to B7x, or an active antibody fragment that
binds B7x, in an amount effective to treat or prevent
metastasis.
Inventors: |
Zang; Xingxing; (New York,
NY) ; Allison; James P.; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC.
SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH |
Bronx
New York |
NY
NY |
US
US |
|
|
Assignee: |
ALBERT EINSTEIN COLLEGE OF
MEDICINE, INC.
Bronx
NY
SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH
New York
NY
|
Family ID: |
47041926 |
Appl. No.: |
15/252267 |
Filed: |
August 31, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14050512 |
Oct 10, 2013 |
9447186 |
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15252267 |
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PCT/US2012/034348 |
Apr 20, 2012 |
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14050512 |
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61477729 |
Apr 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/5011 20130101;
C07K 16/3015 20130101; A61K 39/3955 20130101; C07K 2317/732
20130101; C07K 2317/92 20130101; C07K 16/28 20130101; C07K 16/3023
20130101; C07K 2317/76 20130101; C07K 16/3053 20130101; C07K
2317/34 20130101; A61K 39/39558 20130101; C07K 16/30 20130101; C07K
16/303 20130101; C07K 16/3046 20130101; C07K 16/2827 20130101; A61K
2039/505 20130101; A61K 45/06 20130101; C07K 16/3069 20130101; C07K
16/3038 20130101; A61K 49/0008 20130101; A61K 9/0019 20130101 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 9/00 20060101 A61K009/00; A61K 49/00 20060101
A61K049/00; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
number DK083076 awarded by the National Institutes of Health and
grant number PC094137 awarded by the Department of Defense. The
government has certain rights in the invention.
Claims
1. A method for treating metastatic cancer in a patient having
metastatic cancer or for preventing metastasis in a cancer patient
at risk for metastasis comprising administering to the patient a
monoclonal antibody, or a fragment thereof, that binds to B7x, in
an amount effective to treat or prevent metastasis in a patient,
wherein the antibody or antibody fragment binds to amino acid
residues 35-148 of SEQ ID NO:1.
2. The method of claim 1 comprising determining the level of B7x
expression in a tumor sample from the patient, and if B7x is
over-expressed in the tumor sample compared to healthy tissue,
administering to the patient an antibody to B7x, or an antibody
fragment that binds B7x, in an amount effective to treat or prevent
metastasis in a patient.
3. The method of claim 1, wherein the patient has metastatic
cancer.
4. The method of claim 1, wherein the patient is a cancer patient
at risk for metastasis.
5. The method of claim 1, wherein the cancer is a cancer of the
skin, breast, pancreas, prostate, ovary, kidney, esophagus,
gastrointestional tract, colon, brain, liver, lung, head and/or
neck.
6-7. (canceled)
8. The method of claim 1, wherein the antibody is an IgG monoclonal
antibody.
9. The method of claim 1, wherein the antibody is an IgG1
monoclonal antibody.
10. The method of claim 1, wherein administration of the antibody
or antibody fragment decreases the number of tumor nodules in the
patient.
11. The method of claim 1, wherein administration of the antibody
or antibody fragment reduces the number of metastases.
12. The method of claim 1, wherein administration of the antibody
or antibody fragment prevents the occurrence or reoccurrence of
metastasis.
13. The method of claim 1, wherein administration of the antibody
or antibody fragment increases the patient's survival time.
14. The method of claim 1, wherein the antibody or antibody
fragment does not include an antibody-partner molecule
conjugate.
15. The method of claim 1, wherein the antibody or antibody
fragment is the sole therapeutic anti-cancer agent administered to
the patient.
16. The method of claim 1, wherein the antibody or antibody
fragment is administered in combination with another anti-cancer
agent.
17-18. (canceled)
19. The method of claim 1, wherein administration of the antibody
or antibody fragment prevents the reoccurrence of a tumor in the
patient.
20. A method for preventing reoccurrence of a tumor in a patient
comprising administering to the patient a monoclonal antibody, or a
fragment thereof, that binds B7x, in an amount effective to prevent
reoccurrence of a tumor in a patient, wherein the antibody or
antibody fragment binds to amino acid residues 35-148 of SEQ ID
NO:1.
21. A method of producing a monoclonal antibody to B7x comprising:
immunizing a B7x knockout mouse with a B7x-Ig fusion protein,
generating a hybridoma from spleen cells from the mouse, and
testing supernatant from the hybridoma for its ability to react
with immobilized B7x-Ig or a cell line expressing B7x, but not with
control Igs or cell lines expressing other B7 family members, to
identify a monoclonal antibody to B7x.
22. The method of claim 21 further comprising generating a B7x
knockout mouse.
23. The method of claim 21 further comprising purifying the
antibody from the supernatant.
24. A method of screening monoclonal antibodies to B7x to identify
an antibody that inhibits tumor growth in vivo, the method
comprising injecting tumor cells expressing B7x on their cell
surface into a mouse to induce a tumor in the mouse, wherein B7x is
stably expressed on the tumor cells using a retroviral expression
vector, and injecting a monclonal antibody to B7x into the mouse to
identify an antibody that inhibits tumor growth in vivo.
25-26. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/050,512, filed Oct. 10, 2013, now U.S. Pat. No.
9,447,186 B2, issued Sep. 20, 2016, which is a continuation-in-part
of and claims priority of PCT International Patent Application No.
PCT/US2012/034348, filed Apr. 20, 2012, which designates the United
States of America and which claims the benefit of U.S. Provisional
Patent Application No. 61/477,729, filed Apr. 21, 2011, the
contents of which are herein incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0003] Throughout this application various publications are
referred to in superscripts. Full citations for these references
may be found at the end of the specification. The disclosures of
these publications are hereby incorporated by reference in their
entirety into the subject application to more fully describe the
art to which the subject invention pertains.
[0004] Cancer is a serious public health problem in the U.S. and
other countries. More than 90% of cancer patient deaths result from
cancer metastasis rather than from a primary cancer. There are
about 924,310 new cancer cases and 339,150 cancer deaths in the
U.S. alone. According to Cancer Statistics 2010, in the U.S. in
2010 alone, there are an estimated 222,520 new cases and 157,300
deaths for lung cancer, 217,730 new cases and 32,050 deaths for
prostate cancer, 207,090 new cases and 39,840 deaths for breast
cancer, 145,500 new cases and 51,370 deaths for gut cancer, 58,240
new cases and 8,210 deaths for kidney cancer, and 51,350 new cases
and 36,800 deaths for pancreas cancer. While traditional therapies
such as surgery, chemotherapy, and radiation can often control
primary cancer growth, successful control of disseminated
metastases of cancer remains rare.
[0005] Cancer and the immune system have dynamic interactions,
which play crucial roles in determining tumor development and thus
clinical outcome. T cells of the immune system are the major
combatants against cancers. T cell activation, proliferation,
differentiation to effector function and memory generation are
determined by both positive costimulation and negative
coinhibition, generated mainly by the interaction between the B7
family and their receptor CD28 family (FIG. 1). In 2003, B7x was
discovered as a new member of the B7/CD28 family.sup.1. B7x
inhibits T cell function in vitro.sup.1. B7x is extremely
conservative with 87% amino acid identity between human and
mice.
[0006] A study of 823 prostatectomy patients for whom a minimum of
7 year follow-up data were available revealed that prostate cancer
patients with strong expression of B7x by tumor cells were
significantly more likely to have disease spread at time of
surgery, and were at significantly increased risk of clinical
cancer recurrence and cancer-specific death.sup.2. In addition, all
of 103 ovarian borderline tumors tested expressed B7x.sup.3. In
contrast, only scattered B7x-positive cells were detected in
non-neoplastic ovarian tissues. Other investigators have reported
that B7x is over-expressed in human cancers of the lung.sup.4,
ovary.sup.5, breast.sup.6,7, kidney.sup.8, brain.sup.9,
pancreas.sup.10, esophagus.sup.17, skin.sup.18, gut.sup.36,
stomach.sup.19 and thyroid.sup.35. In renal cell carcinoma,
patients with tumors expressing B7x were three times more likely to
die from renal cancer compared to patients lacking B7x.sup.8. In
human breast cancer, there was a significant association between a
high proportion of B7x positive cells in invasive ductal carcinomas
and decreased number of tumor infiltrating lymphocytes. In
esophageal squamous cell carcinoma, expression levels of B7x on
tumor cells were significantly correlated with distant metastasis,
tumor stage and poor survival, and were inversely correlated with
densities of CD3+ T cells in tumor nest and CD8+ T cells in tumor
stroma.
[0007] The present invention addresses the serious and long-felt
need for treatments for metastatic cancer.
SUMMARY OF THE INVENTION
[0008] Methods are provided for treating metastatic cancer in a
patient having metastatic cancer or for preventing metastasis in a
cancer patient at risk for metastasis comprising administering to
the patient an antibody to B7x, or an active antibody fragment that
binds B7x, in an amount effective to treat or prevent
metastasis.
[0009] Methods are also provided for treating metastatic cancer in
a patient having metastatic cancer or for preventing metastasis in
a cancer patient at risk for metastasis comprising determining the
level of B7x expression in a tumor sample from the patient, and if
B7x is over-expressed in the tumor sample compared to healthy
tissue, administering to the patient an antibody to B7x, or an
active antibody fragment that binds B7x, in an amount effective to
treat or prevent metastasis.
[0010] Methods are also provided for producing a monoclonal
antibody to B7x comprising immunizing a B7x knockout mouse with a
B7x-Ig fusion protein, generating a hybridoma from spleen cells
from the mouse, and testing supernatant from the hybridoma for its
ability to react with immobilized B7x-Ig or a cell line expressing
B7x, but not with control Igs or cell lines expressing other B7
family members, to identify a monoclonal antibody to B7x.
[0011] Methods are further provided for screening monoclonal
antibodies to B7x to identify an antibody that inhibits tumor
growth in vivo comprising injecting tumor cells expressing B7x on
their cell surface into a mouse to induce a tumor in the mouse, and
injecting a monclonal antibody to B7x into the mouse to identify an
antibody that inhibits tumor growth in vivo.
[0012] Methods are in addition provided for preventing reoccurrence
of a tumor in a patient comprising administering to the patient an
antibody to B7x, or an active antibody fragment that binds B7x, in
an amount effective to prevent reoccurrence of a tumor in a
patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. The B7 family/CD28 family. The members of the B7 and
CD28 families have been divided into three groups by phylogenetic
analysis: group I includes the pathway of B7-1/B7-2/CD28/CTLA-4,
and the pathway of B7h/ICOS; group II consists of the pathway of
PD-L1/PD-L2/PD-1; and group III contains B7-H3 and B7x, whose
receptors are currently unknown. Modified from Zang et
al..sup.1
[0014] FIG. 2A-2B. Expression of B7x on tumor CT26 (A) or MC38 (B)
accelerates disease progression in vivo. FACS analysis with
PE-anti-B7x (line) or control PE-Ab (solid) showed that tumor cell
lines CT26 and MC38 did not express B7x and that transfectants
B7x/CT26 and B7x/MC38 expressed abundant cell surface B7x. Survival
curves showed that syngeneic mice iv injected with B7x/CT26 or
B7x/MC38 died much faster than mice injected with CT26 or MC38. The
log-rank test was used for statistical analyses. P-values<0.05
were considered statistically significant.
[0015] FIG. 3. Syngeneic BLAB/c mice iv injected with CT26 or
B7x/CT26 and killed on day 20 for determining tumor nodules in
lungs. Mice receiving B7x/CT26 had more tumor nodules than mice
receiving CT26, P=0.0002.
[0016] FIG. 4A-4C. B7x deficiency protects mice from lung
metastasis of cancer. After intravenous injection with breast
cancer cell line 4T1, B7x knock-out (B7x-/-) mice had fewer tumor
nodules than Balb/c wildtype (B7x+/+) mice at day 18 (A); all
wildtype mice were dead within 36 days whereas 33% of B7x-/- mice
were still alive at day 72 (B). Surviving B7x-/- mice were still
alive at day 140 after rechallenge with 4T1(B), and no tumor was
found in lungs from surviving B7x-/- mice with 4T1
double-challenge. Wildtype mice had more regulatory T cells and
myeloid-derived suppressor cells in lungs than B7x-/- mice during
lung metastasis of cancer (C).
[0017] FIG. 5. Monoclonal antibody clones 12D11 and 1H3 reduced
more than 50% tumor nodules in a lung metastasis of cancer
model.
[0018] FIG. 6. BLAB/c mice iv injected with CT26 or hB7x/CT26 and
killed on day 17 for determining tumor nodules in lungs. Mice
receiving hB7x/CT26 had more tumor nodules than mice receiving
CT26, P=0.02.
[0019] FIG. 7. Anti-B7x monoclonal antibody 1H3 reduced more than
60% of tumor nodules in a human B7x-expressed lung metastasis of
cancer model.
[0020] FIG. 8. Antibody-dependent cell-mediated cytotoxicity (ADCC)
assay showed that three mAbs (1H3, 12D11, 15D2), but not 37G9, were
able to kill tumor cells by ADCC mechanism.
[0021] FIG. 9. Possible mechanisms by which treatment with anti-B7x
antibodies inhibit metastatic cancer progression in vivo (see
Experimental Details for discussion).
[0022] FIG. 10A-10D. The effect of anti-B7x monoclonal antibodies
on tumor growth and survival of mice. (A) BALB/c mice were iv
injected with CT26 cells expressing mouse B7x (B7x/CT26) at day 0
and then with anti-B7x mAbs 12D11 and 1H3 or control mouse IgG.
After sacrifice, tumor nodules in lungs were counted. Data were
pooled from three independent experiments (n=9 or 10). (B) BALB/c
females were injected with 4T1 cells expressing mouse B7x (B7x/4T1)
in the mammary fatpad. Mice were ip treated with mAb 1H3. After
mice were sacrificed, breast tumors induced tumor nodules on lungs
were counted (n=10). (C) BALB/c mice were iv injected with CT26
cells expressing human B7x (hB7x/CT26) at day 0 and then injected
ip with mAb 1H3 or control mouse IgG. After mice sacrifice, tumor
nodules in lungs were counted (n=9). Results were pooled from two
independent experiments. (D) BALB/c mice were iv injected with
B7x/CT26 at day 0 and then injected ip with 1H3 or control mouse
IgG. At day 60 post-injection, remaining mice were iv re-challenged
with B7x/CT26 (n=10).
[0023] FIG. 11A-11G Anti-B7x therapy alters the intratumor balance
of anti-tumor effector immune cells and immunosuppressive cells.
BALB/c mice were iv injected with B7x/CT26 and then treated with
1H3 or control mouse IgG. At day 17, single-cell suspensions from
tumor bearing lungs were FACS analyzed for percentage of
infiltrated CD45+ cells (A), CD8 T cells and NK cells (B), tumor
antigen AH1 (SPSYVYHQF (SEQ ID NO:5))-specific CD8 T cells (C), CD4
T cells that were Tim-3+PD-1+, Tim-3+ alone and PD-1+ alone (D),
and CD11b+Ly6C+ monocytic myeloid-derived suppressor cells (F).
Cell suspensions from tumor bearing lungs were stimulated with
1.times. cell stimulation cocktail for 5 hours and stained with
antibodies to CD3, CD4 and IFN-.gamma. or isotype controls (E).
Shown are the ratios of Treg (CD4+Foxp3+) and MDSCs to CD8 T cells,
CD4 T cells, and NK cells (G). Results are pooled from three
independent experiments; *p<0.05, **p<0.01,
***p<0.001.
[0024] FIG. 12A-12C. The effects of 1H3 on tumor microenvironments.
Paraffin sections of tumor bearing lungs from IgG- and 1H3-treated
mice were stained with anti-VEGF and anti-CD31 antibodies.
Hematoxylin was used for counter-staining. Total amount of VEGF and
TGF-.beta. from tumor bearing lungs were measured using ELISA. Each
group contained 5 mice. *p<0.05; **p<0.01.
[0025] FIG. 13A-13C. Anti-tumor mechanisms of 1H3. (A) 1H3 kills
tumor cells through antibody-dependent cellular cytotoxicity.
B7x/CT26 cells labeled with CFSE and PKH-26 were incubated with
splenocytes in the presence of various concentrations of mAb 1H3 or
IgG. Percentages of tumor cell death induced by 1H3-mediated ADCC
were normalized to those of control mouse IgG. Data are
representative of two independent experiments in triplicates and
shown as mean.+-.SE. (B) BALB/c mice were iv injected with B7x/CT26
and then treated with 1H3 or control mouse IgG. After mice
sacrifice at day 17, lung sections were subjected to the TUNEL
assay. Normalized apoptotic staining was measured and compared.
*p<0.05. (C) Comparison of therapeutic efficacies between 1H3
and its Fab. BALB/c mice were iv injected with B7x/CT26 cells at
day 0 and then injected ip 200 .mu.g/mouse with 1H3, Fab of 1H3, or
control mouse IgG at day 1, 3, 7, 11 and 14. At day 17, tumor
nodules in lungs were counted. Data were pooled from two
independent experiments (n=9 or 10). *p<0.05;
****p<0.0001.
[0026] FIG. 14A-14B. Anti-B7x antibody therapy is better than
anti-PD-1 antibody therapy in two lung metastasis of cancer models.
(A) BALB/c mice were iv injected with B7x/CT26 tumor on day 0 and
then ip injected with normal IgG (control), anti-B7x mAb 1H3, or
anti-PD-1 mAb RMP1-14 on day 1, 3, 7, 11, 14. On day 17, mice were
sacrificed and numbers of lung tumor nodules were evaluated.
Anti-B7x treatment reduced more than 58% of lung tumor nodules,
***P<0.001; whereas anti-PD-1 treatment reduced only 34% of lung
tumor nodules and did not reach statistical significance. (B)
BALB/c female mice were injected with B7x/4T1 tumor into the
mammary fatpad on day 0 and then ip injected with normal IgG
(control), anti-B7x mAb 1H3, or anti-PD-1 mAb RMP1-14 on day 8, 11,
13, 15, 18. On day 20, mice were sacrificed and numbers of lung
tumor nodules were evaluated. Anti-B7x treatment reduced more than
58% of lung tumor nodules, *P<0.05; whereas anti-PD-1 did not
have an effect.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention provides a method for treating
metastatic cancer in a patient having metastatic cancer or for
preventing metastasis in a cancer patient at risk for metastasis
comprising administering to the patient an antibody to B7x, or an
active antibody fragment that binds B7x, in an amount effective to
treat or prevent metastasis.
[0028] As used herein "metastasize" means, in regard to a cancer or
tumor, to spread from one organ or tissue of a patient to another
non-adjacent organ or tissue of the patient.
[0029] To "treat" a metastatic cancer means to reduce the number of
metastases in an organ or tissue, and/or to kill metastatic tumor
cells or tumor cells that are likely to metastasize, and/or to
prevent or reduce the spread of cancerous cells from an original
site in the body to another site in the body, and/or to inhibit the
progression of metastatic cancer, and/or to prevent the
reoccurrence of metastasis. Preferably, administration of the
antibody or antibody fragment decreases the number of tumor nodules
in the patient. Preferably, administration of the antibody or
antibody fragment increases the patient's survival time.
[0030] Various diagnostic procedures have been developed to
identify cancer patients at risk for metastasis. See, for example,
U.S. Patent Application Publication Nos. 2008/0138805, 2011/0059470
and 2011/0059470, the contents of which are herein incorporated by
reference.
[0031] The cancer can be, for example, a cancer of the skin,
breast, pancreas, prostate, ovary, kidney, espophagus,
gastrointestional tract, colon, brain, liver, lung, head and/or
neck. In a preferred embodiment, the cancer is lung cancer.
[0032] Preferably, the tumor in the patient expresses B7x or
overexpresses B7x compared to healthy tissue, for example, in
comparison to healthy tissue of the breast, pancreas, prostate,
ovary, kidney, gastrointestional tract, colon, brain, liver, lung,
head and/or neck. The level of B7x expression in the tumor can be
determined, for example, using immunohistocytochemistry on a tissue
sample obtained from the tumor (e.g., Zang et al..sup.2,3). B7x
expression can also be determined by measuring expression of B7x
mRNA in the tumor sample, for example by Northern blot
hybridization (e.g., Allison et al..sup.16).
[0033] Accordingly, the invention also provides a method for
treating metastatic cancer in a patient having metastatic cancer or
for preventing metastasis in a cancer patient at risk for
metastasis comprising determining the level of B7x expression in a
tumor sample from the patient, and if B7x is over-expressed in the
tumor sample compared to the level of B7x expression in healthy
tissue, administering to the patient an antibody to B7x, or an
active antibody fragment that binds B7x, in an amount effective to
treat or prevent metastasis. The method can further comprise
obtaining a tumor sample from the patient.
[0034] As used herein, the term "antibody" refers to complete,
intact antibodies. A "fragment" of an antibody refers to a fragment
that bind the antigen of interest, B7x. Antibody fragments include,
but are not limited to, F(ab').sub.2 and Fab' fragments and single
chain antibodies. F(ab').sub.2 is an antigen binding fragment of an
antibody molecule with deleted crystallizable fragment (Fc) region
and preserved binding region. Fab' is 1/2 of the F(ab').sub.2
molecule possessing only 1/2 of the binding region. Complete,
intact antibodies include, but are not limited to, polyclonal
antibodies, monoclonal antibodies such as murine monoclonal
antibodies, chimeric antibodies, human antibodies, and humanized
antibodies.
[0035] Various forms of antibodies may be produced using standard
recombinant DNA techniques. For example, "chimeric" antibodies may
be constructed, in which the antigen binding domain from an animal
antibody is linked to a human constant domain (an antibody derived
initially from a nonhuman mammal in which recombinant DNA
technology has been used to replace all or part of the hinge and
constant regions of the heavy chain and/or the constant region of
the light chain, with corresponding regions from a human
immunoglobulin light chain or heavy chain). Chimeric antibodies
reduce the immunogenic responses elicited by animal antibodies when
used in human clinical treatments. In addition, recombinant
"humanized" antibodies may be synthesized. Humanized antibodies are
antibodies initially derived from a nonhuman mammal in which
recombinant DNA technology has been used to substitute some or all
of the amino acids not required for antigen binding with amino
acids from corresponding regions of a human immunoglobulin light or
heavy chain. That is, they are chimeras comprising mostly human
immunoglobulin sequences into which the regions responsible for
specific antigen-binding have been inserted. For example, human IgG
Fc can be used to replace the mouse Fc part of a monoclonal
antibody.
[0036] The antibody can be, e.g., any of an IgA, IgD, IgE, IgG, or
IgM antibody. The IgA antibody can be, e.g., an IgA1 or an IgA2
antibody. The IgG antibody can be, e.g., an IgG1, IgG2, IgG2a,
IgG2b, IgG3 or IgG4 antibody. A combination of any of these
antibodies subtypes can also be used. One consideration in
selecting the type of antibody to be used is the desired serum
half-life of the antibody. IgG has a serum half-life of 23 days,
IgA 6 days, IgM 5 days, IgD 3 days, and IgE 2 days.sup.15. Another
consideration is the size of the antibody. For example, the size of
IgG is smaller than that of IgM allowing for greater penetration of
IgG into tumors. Preferred antibodies include IgG1 monclonal
antibodies.
[0037] The antibodies and antibody fragments of the present
invention are designed by humans and made outside of the human
body. The antibodies and antibody fragments are specific for B7x,
but not for other B7 family members (i.e., B7-1, B7-2, B7h, PD-L1,
PD-L2, and B7-H3).
[0038] Preferred antibodies include monoclonal antibodies 1H3 and
12D11. Preferred antibodies include monoclonal antibodies having a
light and/or heavy chain the same as the light and/or heavy chain
of 1H3 or 12D11. The heavy and/or light chains can contain
conservative amino acid sequence modifications that do not
significantly affect the binding characteristics of the antibody.
Preferred antibodies include monoclonal antibodies that bind to the
same epitope on B7x as 1H3 or 12D11. Preferred antibodies include
monoclonal antibodies that block the interaction between B7x and
its receptor. Preferred antibodies include antibodies that kill
tumor cells through antibody-dependent cell-mediated cytotoxicity.
Preferably, the antibody or antibody fragment blocks inhibition of
T cell function by B7x. Preferably, the antibody or antibody
fragment blocks interaction between cancer-expressed B7x and
activiated T cells. Preferably, the antibody or antibody fragment
binds cancer-expressed B7x and kill cancer cells.
[0039] The antibodies and antibody fragments used for therapy in
the present invention do not include antibody-partner molecule
conjugates, where the partner can be, for example, one or more of a
drug, toxin, radioisotope, therapeutic agent, or marker agent. The
antibodies and antibody fragments of the present invention are
therapeutically effective without the need for another therapeutic
agent conjugated to the antibody or fragment. Antibodies and
antibody fragments that are used for determining the expression of
B7x expression in a tumor sample from a patient can be conjugated
to a marker, such as, for example, a fluorescent marker or a
radioisotope marker.
[0040] In one embodiment, the antibody or antibody fragment is the
sole therapeutic anti-cancer agent administered to the patient. In
another embodiment, the antibody or antibody fragment can be
administered in combination with another anti-cancer agent that is
not bound to the antibody or antibody fragment. Anti-cancer agents
include, but are not limited to, an antibody against CTLA-4
(YERVOY.RTM.); an antibody against PD-1 (MDX-1106); an
anti-epidermal growth factor receptor (EGFR) agent such as, e.g.,
panitumumab, the anti-EGFR antibody cetuximab (Erbitux.RTM.), and
the EGFR tyrosine kinase (TK) inhibitors gefitinib (Iressa.RTM.)
and erlotinib (Tarceva.RTM.); an alkylating agent such as, e.g.,
cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin,
triplatin tetranitrate, mechlorethamine, cyclophosphamide,
chlorambucil and ifosfamide; paclitaxel and docetaxel; and
topoisomerase inhibitors such as, e.g., irinotecan, topotecan,
amsacrine, etoposide, etoposide phosphate and teniposide.
[0041] The antibody or antibody fragment can be screened for
efficacy using, e.g., procedures set forth herein in Experimental
Details.
[0042] The antibody or antibody fragment can be administered to the
subject in a pharmaceutical composition comprising a
pharmaceutically acceptable carrier. Examples of acceptable
pharmaceutical carriers include, but are not limited to, additive
solution-3 (AS-3), saline, phosphate buffered saline, Ringer's
solution, lactated Ringer's solution, Locke-Ringer's solution,
Krebs Ringer's solution, Hartmann's balanced saline solution, and
heparinized sodium citrate acid dextrose solution. The
pharmaceutical composition can be formulated for administration by
any method known in the art, including but not limited to, direct
administration to a tumor, parenteral administration, intravenous
administration, and intramuscular administration.
[0043] The patient can be a human or another animal.
[0044] Human and mouse B7x have the amino acid and nucleic
sequences indicated below.
TABLE-US-00001 Human B7x amino acid sequence (SEQ ID NO: 1):
MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVI
QWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGK
GNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVT
MKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHRQLLNSKASLCVSSFFAISWALLPLS
PYLMLK Mouse B7x amino acid sequence (SEQ ID NO: 2):
MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTSAGNIGEDGTLSCTFEPDIKLNGIVI
QWLKEGIKGLVHEFKEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNVQLTDAGTYTCYIRTSKGK
GNANLEYKTGAFSMPEINVDYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEVSNTSFELNSEVTM
KVVSVLYNVTINNTYSCMIENDIAKATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGWALLSLS
CCLMLR. Nucleic acid encoding Human B7x (SEQ ID NO: 3): atggcttccc
tggggcagat cctcttctgg agcataatta gcatcatcat tattctggct 60
ggagcaattg cactcatcat tggctttggt atttcaggga gacactccat cacagtcact
120 actgtcgcct cagctgggaa cattggggag gatggaatcc tgagctgcac
ttttgaacct 180 gacatcaaac tttctgatat cgtgatacaa tggctgaagg
aaggtgtttt aggcttggtc 240 catgagttca aagaaggcaa agatgagctg
tcggagcagg atgaaatgtt cagaggccgg 300 acagcagtgt ttgctgatca
agtgatagtt ggcaatgcct ctttgcggct gaaaaacgtg 360 caactcacag
atgctggcac ctacaaatgt tatatcatca cttctaaagg caaggggaat 420
gctaaccttg agtataaaac tggagccttc agcatgccgg aagtgaatgt ggactataat
480 gccagctcag agaccttgcg gtgtgaggct ccccgatggt tcccccagcc
cacagtggtc 540 tgggcatccc aagttgacca gggagccaac ttctcggaag
tctccaatac cagctttgag 600 ctgaactctg agaatgtgac catgaaggtt
gtgtctgtgc tctacaatgt tacgatcaac 660 aacacatact cctgtatgat
tgaaaatgac attgccaaag caacagggga tatcaaagtg 720 acagaatcgg
agatcaaaag gcggagtcac ctacagctgc taaactcaaa ggcttctctg 780
tgtgtctctt ctttctttgc catcagctgg gcacttctgc ctctcagccc ttacctgatg
840 ctaaaataa 849 Nucleic acid encoding mouse B7x (SEQ ID NO: 4):
atggcttcct tggggcagat catcttttgg agtattatta acatcatcat catcctggct
60 ggggccatcg cactcatcat tggctttggc atttcaggca agcacttcat
cacggtcacg 120 accttcacct cagctggaaa cattggagag gacgggaccc
tgagctgcac ttttgaacct 180 gacatcaaac tcaacggcat cgtcatccag
tggctgaaag aaggcatcaa aggtttggtc 240 cacgagttca aagaaggcaa
agacgacctc tcacagcagc atgagatgtt cagaggccgc 300 acagcagtgt
ttgctgatca ggtggtagtt ggcaatgctt ccctgagact gaaaaacgtg 360
cagctcacgg atgctggcac ctacacatgt tacatccgca cctcaaaagg caaagggaat
420 gcaaacctag agtataagac cggagccttc agtatgccag agataaatgt
ggactataat 480 gccagttcag agagtttacg ctgcgaggct cctcggtggt
tcccccagcc cacagtggcc 540 tgggcatctc aagtcgacca aggagccaac
ttctcagaag tctcgaacac cagctttgag 600 ttgaactctg agaatgtgac
catgaaggtc gtatctgtgc tctacaatgt cacaatcaac 660 aacacatact
cctgtatgat tgaaaatgac attgccaaag ccactgggga catcaaagtg 720
acagattcag aggtcaaaag gcggagtcag ctgcagctgc tcaactccgg gccttccccg
780 tgtgtttttt cttctgcctt tgcggctggc tgggcgctcc tatctctctc
ctgttgcctg 840 atgctaagat ga 852
[0045] The invention further provides an antibody to B7x, or an
active antibody fragment that binds B7x, for use in a method of
treatment of metastatic cancer in a patient having metastatic
cancer or for use in a method of prevention of metastasis in a
cancer patient at risk for metastasis. The invention still further
provides an antibody to B7x, or an active antibody fragment that
binds B7x, for use in a method of determining if B7x is
over-expressed in a tumor sample from a patient compared to healthy
tissue, and for use in a method of treatment of metastatic cancer
in a patient having metastatic cancer or for use in a method of
prevention of metastasis in a cancer patient at risk for
metastasis, where B7x is over-expressed in a tumor sample from the
patient.
[0046] Preferably, the antibody or antibody fragment binds to the
IgV domain of B7x and/or to amino acid residues 35-148 of B7x
(e.g., to amino acids 35-148 of SEQ ID NO:1).
[0047] Preferably, administration of the antibody or antibody
fragment prevents the reoccurrence of a tumor in the patient.
[0048] The invention also provides a method of producing a
monoclonal antibody to B7x comprising immunizing a B7x knockout
mouse with a B7x-Ig fusion protein, generating a hybridoma from
spleen cells from the mouse, and testing supernatant from the
hybridoma for its ability to react with immobilized B7x-Ig or a
cell line expressing B7x, but not with control Igs or cell lines
expressing other B7 family members, to identify a monoclonal
antibody to B7x. Control Igs include, for example, other B7-Igs
such as B7-1-Ig, B7-2-Ig, B7h-Ig, PD-L1-Ig, PD-L2-Ig and B7-H3-Ig.
Cell lines expressing other B7 family members include, for example,
cell lines expressing B7-1, B7-2, B7h, PD-L1, PD-L2 and/or B7-H3.
Supernatant from the hybridoma can be tested using, for example, an
enzyme-linked immunosorbent assay (ELISA) or a
fluorescence-activated cell sorter (FACS). The method can further
comprise purifying the antibody from the supernatant. The method
can further comprise generating a B7x knockout mouse. A B7x
knockout mouse can be generated, for example, as follows. 3.4- and
5.2-kb of B7x genomic fragments can be cloned into a knock-out
vector as 5' and 3' arms. The vector can then be electroporated
into embryonic stem cells. An embryonic stem cell clone
heterozygous for the mutant can then be microinjected into
blastocysts from normal mice. Chimeric males can be crossed with
females to give rise to the mutant B7x allele.
[0049] The invention further provides a method of screening
monoclonal antibodies to B7x to identify an antibody that inhibits
tumor growth in vivo, the method comprising injecting tumor cells
expressing B7x on their cell surface into a mouse to induce a tumor
in the mouse, and injecting a monclonal antibody to B7x into the
mouse to identify an antibody that inhibits tumor growth in vivo.
B7x can be stably expressed on tumor cells using, for example, a
retroviral expression vector. B7x expression on the surface of
tumor cells can be confirmed, for example, with antibody to B7x
using a fluorescence-activated cell sorter (FACS). Preferably, the
antibody inhibits metastasis.
[0050] The invention further provides a method for preventing
reoccurrence of a tumor in a patient comprising administering to
the patient an antibody to B7x, or an active antibody fragment that
binds B7x, in an amount effective to prevent reoccurrence of a
tumor in a patient. Therapy with the antibody or antibody fragment
may, for example, increase local infiltration of anti-tumor immune
cells such as CD8 T cells including tumor antigen-specific CD8 T
cells, NK cells, and IFN-.gamma.-producing CD4 T cells. In
addition, or instead, the therapy may, for example, reduce local
infiltration of immunosuppressive myeloid-derived suppressor cells
(MDSCs).
[0051] This invention will be better understood from the
Experimental Details, which follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims that follow thereafter.
EXPERIMENTAL DETAILS
Example I
Cancer-Expressed B7x Promotes Metastatic Cancer Progression
[0052] The over-expression of B7x by tumor cells raises the
possibility that it might provide a mechanism by which tumor cells
avoid destruction mediated by tumor-reactive T cells and other
immune cells. This is an interesting possibility for two reasons.
The first is that high levels of B7x expression might facilitate
tumor progression, and it may therefore provide a useful prognostic
marker to predict outcome. The second is that B7x may be useful
therapeutic target for checkpoint blockade with immunotherapy.
[0053] In order to develop new drugs with B7x as a target, a mouse
system was first developed in which cancer-expressed B7x can
promote cancer progression and drugs can be screened. Two lung
metastatic cancer models were developed in which the expression of
B7x on CT26 and MC38 cancer cells significantly increased mortality
in mice (FIG. 2).
[0054] Like most tumor cell lines which permanently lose endogenous
B7x protein expression after in vitro culture, two tumor lines CT26
and MC38 are B7x negative and are able to induce lung metastasis
when direct injected intravenously (iv) through the tail vein. A
retroviral expression vector B7x/MSCV was generated to make CT26
and MC38 stably expressing B7x. Syngeneic Balb/c mice were then iv
injected with 1.times.10.sup.5/per-mouse of CT26 or B7x/CT26, and
survival kinetics were monitored. Mice injected with CT26 started
to die at day 26 and half were still alive at day 50, whereas mice
injected with B7x/CT26 started to die at day 21 and were all dead
at day 33 (FIG. 2A). In a parallel study, syngeneic C57BL/6 started
to die at day 44 after iv injection with 1.times.10.sup.5/per-mouse
of MC38 and 37% were still alive at day 100, whereas mice iv
injected with 1.times.10.sup.5/per-mouse of B7x/MC38 started to die
at day 33 and were all dead at day 80 (FIG. 2B).
[0055] In a subsequent experiment, Balb/c mice were iv injected
with 1.times.10.sup.5/per-mouse of CT26 or B7x/CT26 and killed on
day 20 to determine tumor nodules in the lung. FIG. 3 showed that
B7x/CT26 resulted in much more tumor nodules in the lung than CT26.
Collectively, these results demonstrate that expression of B7x on
tumor cells accelerates disease progression in vivo.
Host Cell-Expressed B7x Promotes Expansion of Immune Suppressor
Cells within the Tumor Microenvironment
[0056] Some tissue cells express very low level of B7x, so B7x gene
knock-out mice were used to dissect the role of host B7x in cancer
immune responses. Naive B7x-/- mice were normal and healthy.
B7x-negative 4T1 cells (1.times.10.sup.5/per mouse) were injected
iv into both B7x-/- (on pure BALB/c background) and wildtype BALB/c
mice, and tumor progression and tumor nodules in lungs were
monitored. 4T1 was B7x negative in vitro and in vivo by RT-PCR
(data not shown). At day 18, B7x-/- mice had many fewer tumor
nodules in lungs than wildtype control (FIG. 4A). All wildtype mice
were dead at day 36, whereas 33% of B7x-/- mice were still alive at
day 72 (FIG. 4B). These results reveal that host B7x promotes lung
metastasis of cancer and that removal of host B7x protects mice
from this disease. As more than 30% of B7-/- mice survived lung
metastasis, these surviving B7x-/- mice were re-challenged with a
higher dose (2.times.10.sup.5/per mouse) of 4T1, and their survival
was monitored. Remarkably, none of these mice died (FIG. 4B). At
day 140 after primary injection, these mice were sacrificed and
histological examination demonstrated that lungs of these mice did
not have tumor. These results suggest that B7x deficiency not only
protects mice from lung metastasis but also helps to develop strong
immune memory of sufficient magnitude to completely eliminate the
tumor.
[0057] Flow cytometric analysis was used to identify host cells of
tumor infiltrates that are involved in anti-tumor responses.
Wildtype mice had significantly more Foxp3+CD4+ regulatory T cells
(Treg) and CD11b+Ly6G+ myeloid-derived suppressor cells (MDSC) in
the lung than B7x-/- mice on day 18 after iv injection of 4T1 tumor
cells (FIG. 4C). Recent work has revealed that Treg.sup.11,12 and
MDSC.sup.13,14 are two major cell populations capable of
suppressing immune responses. The present results suggest that host
cell-expressed B7x promotes expansion of immune suppressor cells
within the tumor microenvironment.
Generation of Monoclonal Antibodies Recognizing Both Human and Mice
B7x
[0058] Monoclonal antibodies (mAbs) were generated against both
human and mice B7x from B7x gene knock-out mice. Briefly, B7x
knock-out mice were immunized with B7x-Ig fusion protein and their
spleen cells were used for generation of hybridomas. Thirty-two
independent clones were obtained whose supernatant reacted with
immobilized B7x-Ig, but not control Ig, in a standard ELISA. Eight
of these were selected for further study. The specificity of these
hybridomas for B7x was confirmed by flow cytometry staining cell
lines overexpressing B7x, but not cells expressing other B7 family
members (B7-1, B7-2, B7h, PD-L1, PD-L2, and B7-H3).
Monoclonal Antibodies Inhibit Metastatic Cancer Progression In
Vivo
[0059] Four mAbs [clones: 37G9 (IgG2b), 1H3 (IgG1), 12D11(IgG1),
19D6 (IgG1)] were purified and their therapeutic effects examined
with B7x/CT26 lung metastatic cancer model. Syngeneic BLAB/c mice
iv injected with 1.times.10.sup.5/per-mouse of B7x/CT26 on day 0
and then i.p injected with 200 .mu.g/per-mouse of each mAb on day
1, 3, 7, 11, 14, or PBS as the control. All mice were killed on day
17 and tumor nodules in each lung were examined. Two clones, 12D11
and 1H3, had very good therapeutic effects. Both mAbs were able to
reduce more than 50% tumor nodules in the lung in such a robust
metastatic cancer model (FIG. 5). The other two mAb clones (37G9
and 19D6) did not have significant therapeutic effects in vivo
(data not shown), which is most likely due to the possibility that
37G9 and 19D6 are unable to block the interaction between B7x and
its receptor(s).
Cancer-Expressed Human B7x Promotes Metastatic Cancer Progression
In Vivo
[0060] To facilitate the translation of this research into clinical
trials, another lung metastatic cancer model was developed in which
the expression of human B7x on CT26 cancer cells significantly
increased metastatic cancer progression in mice in vivo. A CT26
cell line, hB7x/CT26, was made that stably express human B7x on
cell surface. Balb/c mice were iv injected with
1.times.10.sup.5/per-mouse of CT26 or hB7x/CT26 and killed on day
17 to determine tumor nodules in the lung. FIG. 6 shows that
hB7x/CT26 resulted in much more tumor nodules in the lung than
CT26, 112.8 vs 23.8, demonstrating human B7x expressed on cancer
cells can promote metastatic cancer progression in vivo. This
system can be used to identify monoclonal antibodies that are
suitable for human clinical trials.
Monoclonal Antibody Inhibits Human B7x-Expressed Metastatic Cancer
Progression In Vivo
[0061] A lung metastatic cancer model was developed in which the
expression of human B7x on CT26 cancer cells (hB7x/CT26)
significantly increased metastatic cancer progression in mice in
vivo (FIG. 6). To further facilitate the translation of this
research into clinical trials, it was determined whether mAbs were
able to inhibit human B7x-expressed metastatic cancer progression
in vivo. Syngeneic BLAB/c mice were iv injected with
1.times.10.sup.5/per-mouse of hB7x/CT26 on day 0 and then i.p
injected with 200 .mu.g/per-mouse of mAb 1H3 on day 1, 2, 3, 5, 7,
9, 11, 13, 15, or PBS as the control. All mice were killed on day
17 and tumor nodules in each lung were examined. Monoclonal
antibody 1H3 had very good therapeutic effects, and reduced more
than 60% of tumor nodules in the lung in such a robust humanized
metastatic cancer model (FIG. 7).
Monoclonal Antibody-Dependent Cell-Mediated Cytotoxicity
[0062] mAbs were tested to determine whether they were able to act
through antibody-dependent cell-mediated cytotoxicity (ADCC).
B7x/CT26 tumor cell as target cells, antibodies (0.2 .mu.g/ml), and
spleen cells from syngeneic BLAB/c mice as effector cells were
incubated together at 37.degree. C. for 4 hours, and then tumor
cells were analyzed by flow cytometry. Compared to the control
normal IgG, three (1H3, 15D2, 12D11) out of four mAbs significantly
killed B7x-expressed CT26 tumor cells (FIG. 8). These results
indicate that mAbs can kill B7x-positive tumor cells through
ADCC.
B7x Protein is not Detected in Antigen-Presenting Cells and T
Cells
[0063] B7x protein is not detected in antigen-presenting cells
(APC) and T cells in both human and mice, as presented in Table
1.
TABLE-US-00002 TABLE 1 B7x protein is not detected in APCs and T
cells before and after stimuli. Immune cells Stimulation and
disease models Human Dendritic cells CD40L, LPS, PMA + ionomycin,
cytokine cocktail Langerhans cells CD40L, cytokine cocktail
Monocytes LPS + IFN-.gamma. B cells LPS, PMA + ionomycin T cells
PHA, PMA + ionomycin Mouse Dendritic cells LPS, IL-4, IFN-.gamma.,
IL-10, TNF-.alpha., 4T1 cancer, S. pneumoniae infection, B. malayi
infection, Macrophages Thioglycolate, IL-4, LPS + IFN-.gamma., IL-6
+ IL-10, TGF-.beta., Treg, 4T1 cancer, S. pneumoniae infection, B.
malayi infection, B cells LPS, anti-IgM F(ab')2, PMA + ionomycin,
4T1 cancer, S. pneumoniae infection T cells ConA, anti-CD3, PMA +
ionomycin, 4T1 cancer, S. pneumoniae infection Th1, Th2, Treg B7x
protein was examined by flow cytometry with specific monoclonal or
polyclonal Abs.
Mechanisms of treatment for cancers
[0064] The present results show that 1) cancer-expressed mice B7x
promotes metastatic cancer progression in two mice models and that
monoclonal antibodies can inhibit metastatic cancer progression in
vivo, demonstrating that blockage of cancer-associated B7x can be
used as a novel treatment for metastatic cancers; and 2) human B7x
expressed on cancer cells can also promote metastatic cancer
progression in vivo and that a monoclonal antibody can inhibit
human B7x-expressing metastatic cancer progression in vivo,
demonstrating that blockage of cancer-associated human B7x can be
used as a novel treatment for metastatic cancers. There are three
possible mechanisms by which this treatment can inhibit metastatic
cancer progression in vivo (see FIG. 9): (A) antibodies block the
interaction between cancer-expressed B7x and activated T cells,
therefore increasing T cell-mediated immunity against cancer; (B)
antibodies bind cancer-expressed B7x and kill cancer cells; and (C)
antibodies block very low level of B7x expressed by some tissue
cells, therefore inhibiting the expansion of regulatory T cells
(Treg) and myeloid-derived suppressor cells (MDSC) within the tumor
microenvironment.
Discussion
[0065] The present technology has enormous market and clinical
potential for at least three reasons. 1) B7x is expressed in many
human cancers, so it is an excellent therapeutic target for human
cancers such as, for example, cancers of the lung, pancreas,
kidney, brain, gut, prostate, breast, esophagus, skin, thyroid,
stomach and ovary. 2) More than 90% of cancer patients die from
metastasis; therefore, therapies for metastasis are desperately
needed. 3) The antibodies describe herein recognize both human and
mice B7x, so these antibodies can be used in clinical trials.
[0066] Like B7x, CTLA-4 and PD-1 are two members of the B7/CD28
family. An antibody against CTLA-4 (YERVOY.RTM. from Bristol-Myers
Squibb Company) was approved by the FDA in March 2011 as a new drug
for metastatic melanoma. Antibodies against PD-1 (MDX-1106) are in
phase I clinical trials. These existing technologies work by
blockade of the B7/CD28 family members CTLA-4 and PD-1 on activated
T cells, so they increase immunity against cancer but also induce
autoimmune diseases. In contrast, the present technology works by
blockade of B7x on cancer cells, so as to increase specific
immunity against cancer without inducing autoimmune diseases.
Example II
Interaction of mAbs with B7x IgV Domain
[0067] Binding rate constants were estimated and corresponding
equilibrium affinity constants (K.sub.Ds) derived for the
interactions of mAbs with recombinant murine B7x ectodomain, as
well as murine and human B7x-IgV through surface plasmon resonance
(SPR). Antibodies 1H3, 12D11 and 15D12 strongly interacted with all
of these proteins, with dissociation constants summarized in Table
2.
Anti-B7x mAb Therapy in Mouse B7x-Expressing Tumor Models
[0068] To develop a functional screening system for immunotherapy,
tumor cell lines expressing cell surface B7x were established,
since most tumor lines were B7x protein negative. Mouse colon
carcinoma CT26 (which were B7x negative) and B7x/CT26 (which
expressed mouse B7x on the surface) were intravenously (iv)
injected into syngeneic BALB/c mice to induce experimental lung
metastasis. By day 17 after injection, the average number of lung
tumor nodules in the B7x/CT26 group was .about.3.5 fold higher than
that in the CT26 group, suggesting that the expression of B7x on
CT26 tumor cells significantly promotes tumor progression in
vivo.
[0069] The in vivo therapeutic effects of B7x-specific mAbs were
screened in the B7x/CT26-induced pulmonary metastasis model.
B7x/CT26 cells were iv injected into BALB/c mice followed by
intraperitoneal (ip) injection of anti-B7x mAbs. By day 17, lung
tumor nodules were examined. Two mAbs, 1H3 and 12D11, significantly
reduced .about.60% of tumor nodules in lungs. The 4T1 mammary
carcinoma cell line that spontaneously metastasizes to the
lung.sup.20 was tried next. 1H3 treatment significantly reduced
primary tumor-induced metastatic tumor nodules in the lung (FIG.
10B).
Anti-B7x mAb Therapy in a Human B7x-Expressing Tumor Model
[0070] Since both 1H3 and 12D11 recognize human B7x (Table 2), the
therapeutic effects of these two mAbs were tested in a human
B7x-expressing tumor model in vivo using hB7x/CT26, which expressed
human B7x on mouse CT26. Like mouse B7x, the expression of human
B7x on CT26 markedly increased tumor nodules in the lung. Mice were
iv injected with hB7x/CT26 and then treated with 1H3 or 12D11. On
day 17, the average numbers of lung tumor nodules in 1H3-treated
and 12D11-treated groups were 97 and 236, respectively, whereas the
number in control group was 251 (FIG. 10C). Furthermore, 1H3 could
recognize B7x expression on human colon and ovary cancers through
immunohistochemistry. These results suggest that human B7x promoted
tumor growth in vivo and that mAb 1H3 recognized human B7x and
inhibited human B7x expressing tumor progression in vivo. Since 1H3
inhibited both human and mouse B7x-mediated tumor progression, it
was used for the subsequent experiments.
1H3 mAb-Treated Mice Generate Memory Response and Survive B7x/CT26
Rechallenge
[0071] The effect of 1H3 on the survival of mice bearing B7x/CT26
tumors was investigated. In agreement with the lung tumor nodule
results, 1H3 treated mice had a significantly lower mortality than
control IgG-treated mice. By day 60 post-injection of tumor, 100%
of IgG-treated mice were dead, whereas half of 1H3-treated mice
remained alive (FIG. 10D). Then, it was examined whether the
surviving mice had generated a memory response to the tumor. These
mice were rechallenged with the same number of B7x/CT26 cells, and
all of them remained alive for the following 60 days. On day 120,
mice were sacrificed and hematoxylin and eosin (HE) staining of
lung sections from these mice showed they were free of tumor. These
results suggest that the 1H3 treatment induced a memory response
against the tumor.
Anti-B7x Therapy Increases Infiltrating T and NK Cells and
Decreases Infiltrating MDSCs within Tumors
[0072] To dissect the therapeutic mechanisms of 1H3 treatment,
single-cell suspensions were prepared from tumor-bearing lungs and
immune cells were analyzed by flow cytometry. 1H3-treated mice had
significantly higher percentages of CD45+ immune cell infiltrate
than control IgG-treated mice (FIG. 11A). Among these CD45+ cells,
the 1H3 treatment strongly increased infiltration of tumor by CD8 T
cells and NK cells (FIG. 11B), two major types of anti-tumor immune
cells. SPSYVYHQF/H(SEQ ID NO:5)-2L.sup.d tetramer was used to
detect CD8 T cells specific for CT26 tumor antigen epitope AH1
(amino acids 423-431 SPSYVYHQF (SEQ ID NO:5)).sup.21,22. In
agreement with increased total CD8 T cells, 1H3 treatment increased
the percentage of AH1-specific CD8 T cells (FIG. 11C). Recent
studies identified the co-expression of Tim-3 and PD-1
(Tim-3+PD-1+) cells as a unique phenotype of exhausted T cells in
melanoma and leukemia.sup.23,24. Therefore, the effect of 1H3 was
examined on these two inhibitory receptors on CD4 T cells.
1H3-treated mice had significantly fewer CD4 T cells that were
Tim-3+PD-1+, Tim-3+ alone and PD-1+ alone (FIG. 11D), suggesting
1H3 treatment reduce the conversion of CD4 T cells from activated
to exhausted state. Along with these finding, the 1H3 treatment
enhanced CD4 T cells to produce IFN-.gamma. (FIG. 11E), a critical
cytokine for anti-tumor immunity.
[0073] In the tumor microenvironment, suppression of effector T
cell function is often driven by immunosuppressive cells.
Therefore, the effect of 1H3 treatment on immunosuppressive cell
infiltrates was investigated in tumor-bearing lungs. The treatment
did not change the percentage of Foxp3+CD4+ regulatory T cells
(Tregs), but reduced CD11b+Ly6C+ monocytic myeloid-derived
suppressor cells (MDSCs) infiltrating tumor (FIG. 11F). The
combined increase of CD8 T cell, NK and IFN-.gamma.-producing CD4 T
cells and reduction of MDSCs in the 1H3 treatment afforded a
significantly lower ratio of the suppressive MDSCs and Tregs to
effector anti-tumor immune cells (FIG. 11G).
Anti-B7x Therapy Decreases VEGF and TGF-.beta. in the Tumor
Microenvironment
[0074] VEGF stimulates angiogenesis in the tumor microenvironment
and facilitates tumor growth and metastasis.sup.25-28. VEGF
concentration in tumor-bearing lungs from 1H3 treated mice was
significantly lower than that of control mice (FIG. 12A).
Correspondingly, CD31 expression pattern in tumor vasculature
revealed that the 1H3 treatment inhibited intratumor vasculature
(FIG. 12B). The 1H3 treatment also lowered the concentration of
TGF-.beta. in tumor-bearing lungs (FIG. 12C), one of the key
cytokines responsible for suppressing anti-tumor
responses.sup.29,30.
1H3 Kills Tumor Cells Through ADCC but not CDC
[0075] One way in which antibodies can eliminate virus-infected
cells or tumor cells is via antibody-dependent cellular
cytotoxicity (ADCC).sup.31-33. It was examined whether 1H3 kills
tumor cells through ADCC. 1H3 induced death of 50% more target
cells compared to control IgG (FIG. 13A). In agreement with the in
vitro ADCC result, significantly increased numbers of apoptotic
cells were present in tumors from 1H3 treated mice (FIG. 13B).
Antibodies can also eliminate tumor cells via complement-dependent
cytotoxicity (CDC).sup.34; however, 1H3 specific CDC activity could
not be detected (data not shown).
1H3 Blocks B7x-Mediated T Cell Coinhibition
[0076] T cells proliferated vigorously when incubated with anti-CD3
and control Ig with more than 73% of T cells dividing. When T cells
were incubated with anti-CD3 and B7x-Ig, significantly fewer T
cells proliferated, with about 41% dividing. The presence of 1H3 in
the system significantly neutralized B7x-mediated T cell
coinhibition, as 1H3 increased T cell proliferation to >61%.
Furthermore, Fab fragment of 1H3 had a similar neutralizing effect
on B7x-induced T cell coinhibition. These results reveal that 11-13
can partially block B7x-mediated T cell coinhibition. To assess
whether 1H3 therapy depends on ADCC and/or functional
neutralization in vivo, therapeutic efficacies between 1H3 and its
Fab (which cannot cause ADCC) were compared. Mice treated with the
Fab had significantly fewer lung tumor nodules than did mice
treated with control IgG, but had significantly more lung tumor
nodules than did mice treated with 1H3 (FIG. 13C). Taken together,
these results suggest that 1H3 inhibits tumor growth through the
combination of ADCC and functional neutralization.
TABLE-US-00003 TABLE 2 Surface plasmon resonance measurements. mAb
k.sub.on (M.sup.-1 s.sup.-1) k.sub.off (s.sup.-1) K.sub.D (nM)
murine B7x (IgV domain) 1H3 2.455(4).sup.a .times. 10.sup.6
0.001248(7) 0.508(3) 12D11 2.140(4) .times. 10.sup.6 0.001341(8)
0.627(4) 15D12 1.790(3) .times. 10.sup.6 0.001135(7) 0.634(4)
murine B7x 1H3 6.71(3) .times. 10.sup.5 0.001298(7) 1.94(1) 12D11
6.62(4) .times. 10.sup.5 0.001177(7) 1.78(1) 15D12 4.40(3) .times.
10.sup.5 0.001095(6) 2.49(2) human B7x (IgV domain) 1H3 2.53(2)
.times. 10.sup.5 0.00917(4) 36.2(3) 12D11 2.13(1) .times. 10.sup.5
0.00928(3) 43.5(3) 15D12 1.388(7) .times. 10.sup.5 0.01039(2)
74.9(4) .sup.aThe value in parentheses denotes the standard error
in the last digit
Example III
Anti-B7x Antibody Therapy is Better than Anti-PD-1 Antibody Therapy
in Two Lung Metastasis of Cancer Models
[0077] Anti-B7x antibody therapy was compared to anti-programmed
cell death protein 1 (PD-1) antibody therapy in two lung metastasis
of cancer models. Anti-PD-1 therapy is currently in phase 1
clinical trials in cancer patients.
[0078] In one model, BALB/c mice were iv injected with B7x/CT26
tumor on day 0 and then ip injected with normal IgG (control),
anti-B7x mAb 1H3, or anti-PD-1 mAb RMP1-14 on day 1, 3, 7, 11, 14.
On day 17, mice were sacrificed and numbers of lung tumor nodules
were evaluated. Anti-B7x treatment reduced more than 58% of lung
tumor nodules, ***P<0.001; whereas anti-PD-1 treatment reduced
only 34% of lung tumor nodules and did not reach statistical
significance (FIG. 14A).
[0079] In a second model, BALB/c females mice were injected with
B7x/4T1 tumor into the mammary fatpad on day 0 and then ip injected
with normal IgG (control), anti-B7x mAb 1H3, or anti-PD-1 mAb
RMP1-14 on day 8, 11, 13, 15, 18. On day 20, mice were sacrificed
and numbers of lung tumor nodules were evaluated. Anti-B7x
treatment reduced more than 58% of lung tumor nodules, *P<0.05;
whereas anti-PD-1 did not have an effect (FIG. 14B).
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Sequence CWU 1
1
51282PRTHomo sapiens 1Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser
Ile Ile Ser Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala Leu
Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Arg His Ser Ile Thr Val
Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly Ile
Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Ser Asp Ile
Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val 65 70 75 80 His
Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90
95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn
100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly
Thr Tyr 115 120 125 Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn
Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu
Val Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Thr Leu Arg
Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Val Trp
Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val Ser
Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205 Lys
Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215
220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val
225 230 235 240 Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu
Leu Asn Ser 245 250 255 Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala
Ile Ser Trp Ala Leu 260 265 270 Leu Pro Leu Ser Pro Tyr Leu Met Leu
Lys 275 280 2283PRTMouse 2Met Ala Ser Leu Gly Gln Ile Ile Phe Trp
Ser Ile Ile Asn Ile Ile 1 5 10 15 Ile Ile Leu Ala Gly Ala Ile Ala
Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30 Gly Lys His Phe Ile Thr
Val Thr Thr Phe Thr Ser Ala Gly Asn Ile 35 40 45 Gly Glu Asp Gly
Thr Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60 Asn Gly
Ile Val Ile Gln Trp Leu Lys Glu Gly Ile Lys Gly Leu Val 65 70 75 80
His Glu Phe Lys Glu Gly Lys Asp Asp Leu Ser Gln Gln His Glu Met 85
90 95 Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Val Val Gly
Asn 100 105 110 Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala
Gly Thr Tyr 115 120 125 Thr Cys Tyr Ile Arg Thr Ser Lys Gly Lys Gly
Asn Ala Asn Leu Glu 130 135 140 Tyr Lys Thr Gly Ala Phe Ser Met Pro
Glu Ile Asn Val Asp Tyr Asn 145 150 155 160 Ala Ser Ser Glu Ser Leu
Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln 165 170 175 Pro Thr Val Ala
Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190 Glu Val
Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205
Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210
215 220 Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys
Val 225 230 235 240 Thr Asp Ser Glu Val Lys Arg Arg Ser Gln Leu Gln
Leu Leu Asn Ser 245 250 255 Gly Pro Ser Pro Cys Val Phe Ser Ser Ala
Phe Val Ala Gly Trp Ala 260 265 270 Leu Leu Ser Leu Ser Cys Cys Leu
Met Leu Arg 275 280 3849DNAHomo sapiens 3atggcttccc tggggcagat
cctcttctgg agcataatta gcatcatcat tattctggct 60ggagcaattg cactcatcat
tggctttggt atttcaggga gacactccat cacagtcact 120actgtcgcct
cagctgggaa cattggggag gatggaatcc tgagctgcac ttttgaacct
180gacatcaaac tttctgatat cgtgatacaa tggctgaagg aaggtgtttt
aggcttggtc 240catgagttca aagaaggcaa agatgagctg tcggagcagg
atgaaatgtt cagaggccgg 300acagcagtgt ttgctgatca agtgatagtt
ggcaatgcct ctttgcggct gaaaaacgtg 360caactcacag atgctggcac
ctacaaatgt tatatcatca cttctaaagg caaggggaat 420gctaaccttg
agtataaaac tggagccttc agcatgccgg aagtgaatgt ggactataat
480gccagctcag agaccttgcg gtgtgaggct ccccgatggt tcccccagcc
cacagtggtc 540tgggcatccc aagttgacca gggagccaac ttctcggaag
tctccaatac cagctttgag 600ctgaactctg agaatgtgac catgaaggtt
gtgtctgtgc tctacaatgt tacgatcaac 660aacacatact cctgtatgat
tgaaaatgac attgccaaag caacagggga tatcaaagtg 720acagaatcgg
agatcaaaag gcggagtcac ctacagctgc taaactcaaa ggcttctctg
780tgtgtctctt ctttctttgc catcagctgg gcacttctgc ctctcagccc
ttacctgatg 840ctaaaataa 8494852DNAMouse 4atggcttcct tggggcagat
catcttttgg agtattatta acatcatcat catcctggct 60ggggccatcg cactcatcat
tggctttggc atttcaggca agcacttcat cacggtcacg 120accttcacct
cagctggaaa cattggagag gacgggaccc tgagctgcac ttttgaacct
180gacatcaaac tcaacggcat cgtcatccag tggctgaaag aaggcatcaa
aggtttggtc 240cacgagttca aagaaggcaa agacgacctc tcacagcagc
atgagatgtt cagaggccgc 300acagcagtgt ttgctgatca ggtggtagtt
ggcaatgctt ccctgagact gaaaaacgtg 360cagctcacgg atgctggcac
ctacacatgt tacatccgca cctcaaaagg caaagggaat 420gcaaacctag
agtataagac cggagccttc agtatgccag agataaatgt ggactataat
480gccagttcag agagtttacg ctgcgaggct cctcggtggt tcccccagcc
cacagtggcc 540tgggcatctc aagtcgacca aggagccaac ttctcagaag
tctcgaacac cagctttgag 600ttgaactctg agaatgtgac catgaaggtc
gtatctgtgc tctacaatgt cacaatcaac 660aacacatact cctgtatgat
tgaaaatgac attgccaaag ccactgggga catcaaagtg 720acagattcag
aggtcaaaag gcggagtcag ctgcagctgc tcaactccgg gccttccccg
780tgtgtttttt cttctgcctt tgcggctggc tgggcgctcc tatctctctc
ctgttgcctg 840atgctaagat ga 85259PRTArtificial Sequencetumor
antigen 5Ser Pro Ser Tyr Val Tyr His Gln Xaa 1 5
* * * * *