U.S. patent application number 17/367094 was filed with the patent office on 2021-11-04 for methods of treating cancer using pd-1 axis binding antagonists and tigit inhibitors.
The applicant listed for this patent is Genentech, Inc.. Invention is credited to Kristin Bowles, Laetitia Comps-Agrar, Dan Eaton, Jane Grogan, Jason Hackney, Bryan Irving, Robert J. Johnston, Xin Yu.
Application Number | 20210340253 17/367094 |
Document ID | / |
Family ID | 1000005697424 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210340253 |
Kind Code |
A1 |
Grogan; Jane ; et
al. |
November 4, 2021 |
METHODS OF TREATING CANCER USING PD-1 AXIS BINDING ANTAGONISTS AND
TIGIT INHIBITORS
Abstract
The present invention describes combination treatment comprising
a PD-1 axis binding antagonist and an agent that decreases or
inhibits TIGIT expression and/or activity and methods for use
thereof, including methods of treating conditions where enhanced
immunogenicity is desired such as increasing tumor immunogenicity
for the treatment of cancer or chronic infection.
Inventors: |
Grogan; Jane; (San
Francisco, CA) ; Johnston; Robert J.; (San Francisco,
CA) ; Irving; Bryan; (San Francisco, CA) ;
Hackney; Jason; (San Carlos, CA) ; Yu; Xin;
(South San Francisco, CA) ; Eaton; Dan; (San
Rafael, CA) ; Bowles; Kristin; (South San Francisco,
CA) ; Comps-Agrar; Laetitia; (Foster City,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
1000005697424 |
Appl. No.: |
17/367094 |
Filed: |
July 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16818820 |
Mar 13, 2020 |
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17367094 |
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15239569 |
Aug 17, 2016 |
10611836 |
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16818820 |
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14333375 |
Jul 16, 2014 |
9873740 |
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15239569 |
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61992109 |
May 12, 2014 |
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61985884 |
Apr 29, 2014 |
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61950754 |
Mar 10, 2014 |
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61865582 |
Aug 13, 2013 |
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61846941 |
Jul 16, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/507 20130101;
C07K 16/3023 20130101; C07K 16/2818 20130101; C07K 16/3053
20130101; A01K 2267/0331 20130101; A61K 39/3955 20130101; A01K
2217/075 20130101; Y02A 50/30 20180101; C07K 16/303 20130101; C07K
2319/30 20130101; C07K 14/70596 20130101; A01K 2267/0387 20130101;
A01K 67/0276 20130101; C07K 16/3061 20130101; C07K 16/3046
20130101; C07K 16/2827 20130101; A61K 39/39558 20130101; C07K
16/3015 20130101; C07K 16/3038 20130101; C07K 16/2803 20130101;
A01K 2227/105 20130101; C07K 16/3069 20130101; A61K 45/06
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A01K 67/027 20060101 A01K067/027; C07K 14/705 20060101
C07K014/705; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06; C07K 16/30 20060101 C07K016/30 |
Claims
1.-166. (canceled)
167. A method of treating cancer in a human subject in need
thereof, comprising administering to the human subject an effective
amount of: a. an anti-PD-1 antagonistic antibody; and b. an
anti-TIGIT antagonistic antibody.
168. The method of claim 167, wherein the anti-TIGIT antagonistic
antibody is a human antibody or a humanized antibody.
169. The method of claim 167, wherein the anti-TIGIT antagonistic
antibody is a monoclonal antibody.
170. The method of claim 167, wherein the anti-TIGIT antagonistic
antibody is a monoclonal humanized antibody.
171. The method of claim 167, wherein the anti-TIGIT antagonistic
antibody is a monoclonal human antibody.
172. The method of claim 167, wherein the anti-TIGIT antagonistic
antibody is an IgG antibody.
173. The method of claim 172, wherein the anti-TIGIT antagonistic
antibody is an IgG-1 antibody.
174. The method of claim 173, wherein the anti-TIGIT antagonistic
antibody is a monoclonal human IgG-1 antibody or a monoclonal
humanized IgG-1 antibody.
175. The method of claim 172, wherein the anti-TIGIT antagonistic
antibody is an IgG-4 antibody.
176. The method of claim 175, wherein the anti-TIGIT antagonistic
antibody is a monoclonal human IgG-4 antibody or a monoclonal
humanized IgG-4 antibody.
177. The method of claim 167, wherein the anti-PD-1 antagonistic
antibody is nivolumab (MDX-1106).
178. The method of claim 167, wherein the anti-PD-1 antagonistic
antibody is lambrolizumab (MK-3475).
179. The method of claim 167, wherein the anti-PD-1 antagonistic
antibody is a human antibody or a humanized antibody.
180. The method of claim 167, wherein the anti-PD-1 antagonistic
antibody is a monoclonal antibody.
181. The method of claim 167, wherein the anti-PD-1 antagonistic
antibody is a monoclonal humanized antibody.
182. The method of claim 167, wherein the anti-PD-1 antagonistic
antibody is a monoclonal human antibody.
183. The method of claim 167, wherein the anti-PD-1 antagonistic
antibody is an IgG antibody.
184. The method of claim 183, wherein the anti-PD-1 antagonistic
antibody is an IgG-1 antibody.
185. The method of claim 184, wherein the anti-PD-1 antagonistic
antibody is a monoclonal human IgG-1 antibody or a monoclonal
humanized IgG-1 antibody.
186. The method of claim 183, wherein the anti-PD-1 antagonistic
antibody is an IgG-4 antibody.
187. The method of claim 186, wherein the anti-PD-1 antagonistic
antibody is a monoclonal human IgG-4 antibody or a monoclonal
humanized IgG-4 antibody.
188. The method of claim 167, further comprising administering an
effective amount of at least one chemotherapeutic agent.
189. The method of claim 167, wherein the cancer is selected from
the group consisting of a non-small cell lung cancer, a small cell
lung cancer, a renal cell cancer, a colorectal cancer, an ovarian
cancer, a breast cancer, a pancreatic cancer, a gastric carcinoma,
a bladder cancer, an esophageal cancer, a mesothelioma, a melanoma,
a head and neck cancer, a thyroid cancer, a sarcoma, a prostate
cancer, a glioblastoma, a cervical cancer, a thymic carcinoma, a
leukemia, a lymphoma, a myeloma, a mycosis fungoides, a Merkel cell
cancer, and a hematologic malignancy.
190. The method of claim 167, wherein the cancer is selected from
the group consisting of a non-small cell lung cancer, a small cell
lung cancer, a hepatocellular cancer, a renal cell cancer, a head
and neck cancer, a colorectal cancer, a breast cancer, an
esophageal cancer, a gastric carcinoma, a cervical cancer, an
endometrial cancer, a bladder cancer, a melanoma, a lymphoma, and a
Merkel cell cancer.
191. The method of claim 167, wherein the cancer is melanoma.
192. The method of claim 167, wherein the cancer is non-small cell
lung cancer.
193. The method of claim 167, wherein the cancer is a solid
tumor.
194. The method of claim 167, wherein the cancer is a hematologic
malignancy.
195. The method of claim 167, wherein the anti-TIGIT antagonistic
antibody is administered before or after the anti-PD-1 antagonistic
antibody.
196. The method of claim 167, wherein the anti-TIGIT antagonistic
antibody is administered simultaneously with the anti-PD-1
antagonistic antibody.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 15/239,569, filed on Aug. 17, 2016, which is a divisional of
U.S. application Ser. No. 14/333,375, filed on Jul. 16, 2014, now
U.S. Pat. No. 9,873,740, which claims benefit of U.S. Provisional
Application No. 61/992,109, filed on May 12, 2014; U.S. Provisional
Application No. 61/985,884, filed on Apr. 29, 2014; U.S.
Provisional Application No. 61/950,754, filed on Mar. 10, 2014;
U.S. Provisional Application No. 61/865,582, filed on Aug. 13,
2013; and U.S. Provisional Application No. 61/846,941, filed on
Jul. 16, 2013.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 20, 2020, is named 50474-109009_Sequence
Listing_02.20.2020_ST25, and is 26,165 bytes in size.
BACKGROUND OF THE INVENTION
[0003] The provision of two distinct signals to T-cells is a widely
accepted model for lymphocyte activation of resting T lymphocytes
by antigen-presenting cells (APCs). Lafferty et al, Aust. J. Exp.
Biol. Med. ScL 53: 27-42 (1975). This model further provides for
the discrimination of self from non-self and immune tolerance.
Bretscher et al, Science 169: 1042-1049 (1970); Bretscher, P. A.,
P.N.A.S. USA 96: 185-190 (1999); Jenkins et al, J. Exp. Med. 165:
302-319 (1987). The primary signal, or antigen specific signal, is
transduced through the T-cell receptor (TCR) following recognition
of foreign antigen peptide presented in the context of the major
histocompatibility-complex (MHC). The second or co-stimulatory
signal is delivered to T-cells by co-stimulatory molecules
expressed on antigen-presenting cells (APCs), and induce T-cells to
promote clonal expansion, cytokine secretion and effector function.
Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). In the absence
of co-stimulation, T-cells can become refractory to antigen
stimulation, which results in a tolerogenic response to either
foreign or endogenous antigens.
[0004] In the two-signal model, T-cells receive both positive
co-stimulatory and negative co-inhibitory signals. The regulation
of such positive and negative signals is critical to maximize the
host's protective immune responses, while maintaining immune
tolerance and preventing autoimmunity. Negative signals seem
necessary for induction of T-cell tolerance, while positive signals
promote T-cell activation.
[0005] Both co-stimulatory and co-inhibitory signals are provided
to antigen-exposed T cells, and the interplay between
co-stimulatory and co-inhibitory signals is essential to
controlling the magnitude of an immune response. Further, the
signals provided to the T cells change as an infection or immune
provocation is cleared, worsens, or persists, and these changes
powerfully affect the responding T cells and re-shape the immune
response.
[0006] The mechanism of co-stimulation is of therapeutic interest
because the manipulation of co-stimulatory signals has shown to
provide a means to either enhance or terminate cell-based immune
response. Recently, it has been discovered that T cell dysfunction
or anergy occurs concurrently with an induced and sustained
expression of the inhibitory receptor, programmed death 1
polypeptide (PD-1). As a result, therapeutic targeting of PD-1 and
other molecules which signal through interactions with PD-1, such
as programmed death ligand 1 (PD-L1) and programmed death ligand 2
(PD-L2) are an area of intense interest.
[0007] PD-L1 is overexpressed in many cancers and is often
associated with poor prognosis (Okazaki T et al., Intern. Immun.
2007 19(7):813) (Thompson R H et al., Cancer Res 2006, 66(7):3381).
Interestingly, the majority of tumor infiltrating T lymphocytes
predominantly express PD-1, in contrast to T lymphocytes in normal
tissues and peripheral blood T lymphocytes indicating that
up-regulation of PD-1 on tumor-reactive T cells can contribute to
impaired antitumor immune responses (Blood 2009 114(8):1537). This
may be due to exploitation of PD-L1 signaling mediated by PD-L1
expressing tumor cells interacting with PD-1 expressing T cells to
result in attenuation of T cell activation and evasion of immune
surveillance (Sharpe et al., Nat Rev 2002) (Keir M E et al., 2008
Annu. Rev. Immunol. 26:677). Therefore, inhibition of the
PD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing of
tumors.
[0008] The inhibition of PD-1 axis signaling through its direct
ligands (e.g., PD-L1, PD-L2) has been proposed as a means to
enhance T cell immunity for the treatment of cancer (e.g., tumor
immunity). Moreover, similar enhancements to T cell immunity have
been observed by inhibiting the binding of PD-L1 to the binding
partner B7-1. Furthermore, combining inhibition of PD-1 signaling
with other signaling pathways that are deregulated in tumor cells
may further enhance treatment efficacy. There remains a need for
such an optimal therapy for treating, stabilizing, preventing,
and/or delaying development of various cancers.
[0009] All references, publications, and patent applications
disclosed herein are hereby incorporated by reference in their
entirety.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention describes a combination treatment
comprising a PD-1 axis binding antagonist and an agent that
decreases or inhibits TIGIT expression and/or activity.
[0011] Provided herein are methods for treating or delaying
progression of cancer in an individual comprising administering to
the individual an effective amount of a PD-1 axis binding
antagonist and an agent that decreases or inhibits TIGIT expression
and/or activity.
[0012] Provided herein are also methods for reducing or inhibiting
cancer relapse or cancer progression in an individual comprising
administering to the individual an effective amount of a PD-1 axis
binding antagonist and an agent that decreases or inhibits TIGIT
expression and/or activity.
[0013] Provided herein are also methods for treating or delaying
progression of an immune related disease in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and an agent that decreases or
inhibits TIGIT expression and/or activity.
[0014] Provided herein are also methods for reducing or inhibiting
progression of an immune related disease in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and an agent that decreases or
inhibits TIGIT expression and/or activity.
[0015] In some embodiments, the immune related disease is
associated with a T cell dysfunctional disorder. In some
embodiments, the T cell dysfunctional disorder is characterized by
decreased responsiveness to antigenic stimulation. In some
embodiments, the T cell dysfunctional disorder is characterized by
T cell anergy or decreased ability to secrete cytokines,
proliferate or execute cytolytic activity. In some embodiments, the
T cell dysfunctional disorder is characterized by T cell
exhaustion. In some embodiments, the T cells are CD4+ and CD8+ T
cells. In some embodiments, the immune related disease is selected
from the group consisting of unresolved acute infection, chronic
infection and tumor immunity.
[0016] Provided herein are also methods of increasing, enhancing or
stimulating an immune response or function in an individual by
administering to the individual an effective amount of a PD-1 axis
binding antagonist and an agent that decreases or inhibits TIGIT
expression and/or activity.
[0017] Provided herein are also methods of treating or delaying
progression of cancer in an individual comprising administering to
the individual an effective amount of a PD-1 axis binding
antagonist and an agent that modulates the CD226 expression and/or
activity.
[0018] Provided herein are also methods for reducing or inhibiting
cancer relapse or cancer progression in an individual comprising
administering to the individual an effective amount of a PD-1 axis
binding antagonist and an agent that modulates the CD226 expression
and/or activity.
[0019] Provided herein are also methods for treating or delaying
progression of an immune related disease in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and an agent that modulates the CD226
expression and/or activity.
[0020] Provided herein are also methods for reducing or inhibiting
progression of an immune related disease in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and agent that modulates the CD226
expression and/or activity.
[0021] In some embodiments, the immune related disease is
associated with a T cell dysfunctional disorder. In some
embodiments, the T cell dysfunctional disorder is characterized by
decreased responsiveness to antigenic stimulation. In some
embodiments, the T cell dysfunctional disorder is characterized by
T cell anergy, or decreased ability to secrete cytokines,
proliferate or execute cytolytic activity. In some embodiments, the
T cell dysfunctional disorder is characterized by T cell
exhaustion. In some embodiments, the T cells are CD4+ and CD8+ T
cells. In some embodiments, the immune related disease is selected
from the group consisting of unresolved acute infection, chronic
infection and tumor immunity.
[0022] Provided herein are also methods of increasing, enhancing or
stimulating an immune response or function in an individual by
administering to the individual an effective amount of a PD-1 axis
binding antagonist and an agent that modulates the CD226 expression
and/or activity.
[0023] In some embodiments, the agent that modulates the CD226
expression and/or activity is capable of increasing and/or
stimulating CD226 expression and/or activity.
[0024] In some embodiments, the agent that modulates the CD226
expression and/or activity is selected from an agent that inhibits
and/or blocks the interaction of CD226 with TIGIT, an antagonist of
TIGIT expression and/or activity, an antagonist of PVR expression
and/or activity, an agent that inhibits and/or blocks the
interaction of TIGIT with PVR, an agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVR.
[0025] In some embodiments, the agent that inhibits and/or blocks
the interaction of CD226 with TIGIT is a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In some embodiments, the agent that inhibits and/or blocks the
interaction of CD226 with TIGIT is an anti-TIGIT antibody or
antigen-binding fragment thereof.
[0026] In some embodiments, the antagonist of TIGIT expression
and/or activity is a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the antagonist of TIGIT expression and/or activity is
an anti-TIGIT antibody or antigen-binding fragment thereof.
[0027] In some embodiments, the antagonist of PVR expression and/or
activity is a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide.
[0028] In some embodiments, the agent that inhibits and/or blocks
the interaction of TIGIT with PVR is a small molecule inhibitor, an
inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0029] In some embodiments, the agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVR is a
small molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide.
[0030] The present invention also describes a combination treatment
comprising an agent that decreases or inhibits TIGIT expression
and/or activity and an agent that decreases or inhibits one or more
additional immune co-inhibitory receptors.
[0031] Provided herein are methods of increasing, enhancing or
stimulating an immune response or function in an individual by
administering to the individual an effective amount of an agent
that decreases or inhibits TIGIT expression and/or activity and an
agent that decreases or inhibits one or more additional immune
co-inhibitory receptors.
[0032] In some embodiments, the one or more additional immune
co-inhibitory receptor is selected from the group consisting of
PD-1, CTLA-4, LAG3, TIM3, BTLA and VISTA. In some embodiments, the
one or more additional immune co-inhibitory receptor is selected
from the group of PD-1, CTLA-4, LAG3 and TIM3.
[0033] The present invention also describes a combination treatment
comprising an agent that decreases or inhibits TIGIT expression
and/or activity and an agent that increases or activates one or
more additional immune co-stimulatory receptor.
[0034] Provided herein are methods of increasing, enhancing or
stimulating an immune response or function in an individual by
administering to the individual an effective amount of an agent
that decreases or inhibits TIGIT expression and/or activity and an
agent that increases or activates one or more additional immune
co-stimulatory receptor.
[0035] In some embodiments, the one or more additional immune
co-stimulatory receptor is selected from the group consisting of
CD226, OX-40, CD28, CD27, CD137, HVEM, and GITR. In some
embodiments, the one or more additional immune co-stimulatory
receptor is selected from the group of CD226, OX-40, CD27, CD137,
HVEM and GITR. In some embodiments, the one or more additional
immune co-stimulatory receptor is selected from the group
consisting of OX-40 and CD27.
[0036] In some embodiments, any of the above methods further
comprise administering at least one chemotherapeutic agent.
[0037] In some embodiments, the individual in any of the above
methods has cancer. In some embodiments, the individual in any of
the above methods is a human.
[0038] In some embodiments, the CD4 and/or CD8 T cells in the
individual have increased or enhanced priming, activation,
proliferation, cytokine release and/or cytolytic activity relative
to prior to the administration of the combination.
[0039] In some embodiments, the number of CD4 and/or CD8 T cells is
elevated relative to prior to administration of the combination. In
some embodiments, the number of activated CD4 and/or CD8 T cells is
elevated relative to prior to administration of the combination. In
some embodiments, the activated CD4 and/or CD8 T cells is
characterized by .gamma.-IFN.sup.+ producing CD4 and/or CD8 T cells
and/or enhanced cytolytic activity relative to prior to the
administration of the combination. In some embodiments, the CD4
and/or CD8 T cells exhibit increased release of cytokines selected
from the group consisting of IFN-.gamma., TNF-.alpha., and
interleukins.
[0040] In some embodiments, the CD4 and/or CD8 T cell is an
effector memory T cell. In some embodiments, the CD4 and/or CD8
effector memory T cell is characterized by .gamma.-IFN.sup.+
producing CD4 and/or CD8 T cells and/or enhanced cytolytic
activity. In some embodiments, the CD4 and/or CD8 effector memory T
cell is characterized by having the expression of CD44.sup.high
CD62L.sup.low.
[0041] In some embodiments, the cancer in any of the above methods
has elevated levels of T cell infiltration.
[0042] In some embodiments, the agent that decreases or inhibits
TIGIT expression and/or activity is selected from the group
consisting of an antagonist of TIGIT expression and/or activity, an
antagonist of PVR expression and/or activity, and an agent that
inhibits the interaction and/or the intracellular signaling
mediated by TIGIT binding to PVR.
[0043] In some embodiments, the antagonist of TIGIT expression
and/or activity is selected from the group consisting of a small
molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide.
[0044] In some embodiments, the antagonist of PVR expression and/or
activity is selected from the group consisting of a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0045] In some embodiments, the agent that inhibits the
intracellular signaling mediated by TIGIT binding to PVR is
selected from the group consisting of a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0046] In some embodiments, the antagonist of TIGIT expression
and/or activity is an anti-TIGIT antibody or antigen-binding
fragment thereof.
[0047] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof comprises at least one HVR
comprising an amino acid sequence selected from the amino acid
sequences KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ ID NO:2),
QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),
FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6) or
RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT
(SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID
NO:11), and GLRGFYAMDY (SEQ ID NO:12).
[0048] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof comprises a light chain comprising
the amino acid sequence set forth in
TABLE-US-00001 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSP
KLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINN PLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQ
LLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQP
PTFGPGTKLEVK.
[0049] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof comprises a heavy chain comprising
the amino acid sequence set forth in
TABLE-US-00002 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMEIWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARR
PLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGL
IIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGL
RGFYAMDYWGQGTSVTVSS.
[0050] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof comprises a light chain comprising
the amino acid sequence set forth in
TABLE-US-00003 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSP
KLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINN PLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQ
LLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQP PTFGPGTKLEVK
and the antibody heavy chain comprises the amino acid sequence set
forth in
TABLE-US-00004 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMEIWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARR
PLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGL
IIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGL
RGFYAMDYWGQGTSVTVSS.
[0051] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof, wherein the antibody is selected
from a humanized antibody, a chimeric antibody, a bispecific
antibody, a heteroconjugate antibody, and an immunotoxin.
[0052] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof comprises at least one HVR is at
least 90% identical to an HVR set forth in any of
TABLE-US-00005 (SEQ ID NO: 1) KSSQSLYYSGVKENLLA, (SEQ ID NO: 2)
ASIRFT, (SEQ ID NO: 3) QQGINNPLT, (SEQ ID NO: 4) GFTFSSFTMH, (SEQ
ID NO: 5) FIRSGSGIVFYADAVRG, and (SEQ ID NO: 6) RPLGHNTFDS or (SEQ
ID NO: 7) RSSQSLVNSYGNTFLS, (SEQ ID NO: 8) GISNRFS, (SEQ ID NO: 9)
LQGTHQPPT, (SEQ ID NO: 10) GYSFTGHLMN, (SEQ ID NO: 11)
LIIPYNGGTSYNQKFKG, and (SEQ ID NO: 12) GLRGFYAMDY..
[0053] In some embodiments, the anti-TIGIT antibody or fragment
thereof comprises the light chain and/or heavy chain comprising
amino acid sequences at least 90% identical to the amino acid
sequences set forth in
TABLE-US-00006 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSP
KLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINN PLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQ
LLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQP PTFGPGTKLEVK, or
(SEQ ID NO: 15) EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMIHWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARR
PLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGL
IIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGL
RGFYAMDYWGQGTSVTVSS,
respectively.
[0054] In some embodiments, the PD-1 axis binding antagonist is
selected from the group consisting of a PD-1 binding antagonist, a
PD-L1 binding antagonist and a PD-L2 binding antagonist.
[0055] In some embodiments, the PD-1 axis binding antagonist is a
PD-1 binding antagonist. In some embodiments, the PD-1 binding
antagonist inhibits the binding of PD-1 to its ligand binding
partners. In some embodiments, the PD-1 binding antagonist inhibits
the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding
antagonist inhibits the binding of PD-1 to PD-L2. In some
embodiments, the PD-1 binding antagonist inhibits the binding of
PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding
antagonist is an antibody. In some embodiments, the PD-1 binding
antagonist is MDX-1106 (nivolumab). In some embodiments, the PD-1
binding antagonist is Merck 3475 (lambrolizumab). In some
embodiments, the PD-1 binding antagonist is CT-011 (pidilizumab).
In some embodiments, the PD-1 binding antagonist is AMP-224.
[0056] In some embodiments, the PD-1 axis binding antagonist is a
PD-L1 binding antagonist. In some embodiments, the PD-L1 binding
antagonist inhibits the binding of PD-L1 to PD-1. In some
embodiments, the PD-L1 binding antagonist inhibits the binding of
PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist
inhibits the binding of PD-L1 to both PD-1 and B7-1. In some
embodiments, the PD-L1 binding antagonist is an antibody.
[0057] In some embodiments, the PD-L1 binding antagonist is
selected from the group consisting of: YW243.55.S70, MPDL3280A,
MDX-1105 and MEDI 4736.
[0058] In some embodiments, the anti-PD-L1 antibody comprises a
heavy chain comprising HVR-H1 sequence of GFTFSDSWIH (SEQ ID
NO:17), HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:18), and
HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:19); and a light chain
comprising HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO:20), HVR-L2
sequence of SASFLYS (SEQ ID NO:21), and HVR-L3 sequence of
QQYLYHPAT (SEQ ID NO:22).
[0059] In some embodiments, the anti-PD-L1 antibody comprises a
heavy chain variable region comprising the amino acid sequence
of
TABLE-US-00007 (SEQ ID NO: 23)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSA
and a light chain variable region comprising the amino acid
sequence of
TABLE-US-00008 (SEQ ID NO: 24)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.
[0060] In some embodiments, the PD-1 axis binding antagonist is a
PD-L2 binding antagonist. In some embodiments, the PD-L2 binding
antagonist is an antibody. In some embodiments, the PD-L2 binding
antagonist is an immunoadhesin.
[0061] In some embodiments, the cancer being treated is selected
from the group consisting of non-small cell lung cancer, small cell
lung cancer, renal cell cancer, colorectal cancer, ovarian cancer,
breast cancer, pancreatic cancer, gastric carcinoma, bladder
cancer, esophageal cancer, mesothelioma, melanoma, head and neck
cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma,
cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas,
mycoses fungoids, merkel cell cancer, and other hematologic
malignancies.
[0062] In some embodiments, the agent that decreases or inhibits
TIGIT expression and/or activity is administered continuously. In
some embodiments, the agent that decreases or inhibits TIGIT
expression and/or activity is administered intermittently. In some
embodiments, the agent that decreases or inhibits TIGIT expression
and/or activity is administered before the PD-1 axis binding
antagonist. In some embodiments, the agent that decreases or
inhibits TIGIT expression and/or activity is administered
simultaneous with the PD-1 axis binding antagonist. In some
embodiments, the agent that decreases or inhibits TIGIT expression
and/or activity is administered after the PD-1 axis binding
antagonist.
[0063] Also provided herein are kits comprising a PD-1 axis binding
antagonist and a package insert comprising instructions for using
the PD-1 axis binding antagonist in combination with an agent that
decreases or inhibits TIGIT expression and/or activity to treat or
delay progression of cancer in an individual.
[0064] Also provided herein are kits comprising a PD-1 axis binding
antagonist and an agent that decreases or inhibits TIGIT expression
and/or activity, and a package insert comprising instructions for
using the PD-1 axis binding antagonist and the agent that decreases
or inhibits TIGIT expression and/or activity to treat or delay
progression of cancer in an individual.
[0065] Also provided herein are kits comprising an agent that
decreases or inhibits TIGIT expression and/or activity and a
package insert comprising instructions for using the agent that
decreases or inhibits TIGIT expression and/or activity in
combination with a PD-1 axis binding antagonist to treat or delay
progression of cancer in an individual.
[0066] Also provided herein are kits comprising a PD-1 axis binding
antagonist and a package insert comprising instructions for using
the PD-1 axis binding antagonist in combination with an agent that
decreases or inhibits TIGIT expression and/or activity to enhance
immune function of an individual having cancer.
[0067] Also provided herein are kits comprising a PD-1 axis binding
antagonist and an agent that decreases or inhibits TIGIT expression
and/or activity, and a package insert comprising instructions for
using the PD-1 axis binding antagonist and the agent that decreases
or inhibits TIGIT expression and/or activity to enhance immune
function of an individual having cancer.
[0068] Also provided herein are kits comprising an agent that
decreases or inhibits TIGIT expression and/or activity and a
package insert comprising instructions for using the agent that
decreases or inhibits TIGIT expression and/or activity in
combination with a PD-1 axis binding antagonist to enhance immune
function of an individual having cancer.
[0069] Also provided herein are kits comprising a PD-1 axis binding
antagonist and a package insert comprising instructions for using
the PD-1 axis binding antagonist in combination with an agent that
modulates the CD226 expression and/or activity to treat or delay
progression of cancer in an individual.
[0070] Also provided herein are kits comprising a PD-1 axis binding
antagonist and an agent that modulates the CD226 expression and/or
activity, and a package insert comprising instructions for using
the PD-1 axis binding antagonist and the agent that modulates the
CD226 expression and/or activity to treat or delay progression of
cancer in an individual.
[0071] Also provided herein are kits comprising an agent that
modulates the CD226 expression and/or activity and a package insert
comprising instructions for using the agent modulates the CD226
expression and/or activity in combination with a PD-1 axis binding
antagonist to treat or delay progression of cancer in an
individual.
[0072] Also provided herein are kits comprising a PD-1 axis binding
antagonist and a package insert comprising instructions for using
the PD-1 axis binding antagonist in combination with an agent that
modulates the CD226 expression and/or activity to enhance immune
function of an individual having cancer.
[0073] Also provided herein are kits comprising a PD-1 axis binding
antagonist and an agent that modulates the CD226 expression and/or
activity, and a package insert comprising instructions for using
the PD-1 axis binding antagonist and the agent that modulates the
CD226 expression and/or activity to enhance immune function of an
individual having cancer.
[0074] Also provided herein are kits comprising an agent modulates
the CD226 expression and/or activity and a package insert
comprising instructions for using the agent that modulates the
CD226 expression and/or activity in combination with a PD-1 axis
binding antagonist to enhance immune function of an individual
having cancer.
[0075] In some embodiments, the kits comprising the PD-1 axis
binding antagonist is an anti-PD-L1 antibody. In some embodiments,
the kits comprising the PD-1 axis binding antagonist is an
anti-PD-1 antibody. In some embodiments, the kits comprising the
agent that decreases or inhibits TIGIT expression and/or activity
is selected from the group consisting of an antagonist of TIGIT
expression and/or activity, an antagonist of PVR expression and/or
activity, and an agent that inhibits the interaction and/or the
intracellular signaling mediated by TIGIT binding to PVR. In some
embodiments, the kits comprising the antagonist of TIGIT expression
and/or activity is an anti-TIGIT antibody or antigen-binding
fragment thereof.
[0076] In some embodiments, the kits comprises an agent that
modulates the CD226 expression and/or activity which is capable of
increasing and/or stimulating CD226 expression and/or activity. In
some embodiments, the kits comprising the agent that modulates the
CD226 expression and/or activity is selected from an agent that
inhibits and/or blocks the interaction of CD226 with TIGIT, an
antagonist of TIGIT expression and/or activity, an antagonist of
PVR expression and/or activity, an agent that inhibits and/or
blocks the interaction of TIGIT with PVR, an agent that inhibits
and/or blocks the intracellular signaling mediated by TIGIT binding
to PVR. In some embodiments, the kits comprising the agent that
inhibits and/or blocks the interaction of CD226 with TIGIT and/or
the antagonist of TIGIT expression and/or activity is an anti-TIGIT
antibody or antigen-binding fragment thereof.
[0077] In certain aspects, the present disclosure provides a method
for treating or delaying progression of cancer in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and an agent that decreases or
inhibits TIGIT expression and/or activity. In other aspects, the
present disclosure provides use of an effective amount of a PD-1
axis binding antagonist in the manufacture of a medicament for
treating or delaying progression of cancer in an individual,
wherein the PD-1 axis binding agent is used in combination with an
agent that decreases or inhibits TIGIT expression and/or activity.
In other aspects, the present disclosure provides use of an
effective amount of an agent that decreases or inhibits TIGIT
expression and/or activity in the manufacture of a medicament for
treating or delaying progression of cancer in an individual,
wherein the an agent that decreases or inhibits TIGIT expression
and/or activity is used in combination with a PD-1 axis binding
antagonist. In other aspects, the present disclosure provides a
pharmaceutical composition comprising a PD-1 axis binding
antagonist for use in treating or delaying progression of cancer in
combination with an agent that decreases or inhibits TIGIT
expression and/or activity. In other aspects, the present
disclosure provides a pharmaceutical composition comprising an
agent that decreases or inhibits TIGIT expression and/or activity
for use in treating or delaying progression of cancer in
combination with a PD-1 axis binding antagonist.
[0078] In other aspects, the present disclosure provides a method
for reducing or inhibiting cancer relapse or cancer progression in
an individual comprising administering to the individual an
effective amount of a PD-1 axis binding antagonist and an agent
that decreases or inhibits TIGIT expression and/or activity. In
other aspects, the present disclosure provides use of an effective
amount of a PD-1 axis binding antagonist in the manufacture of a
medicament for reducing or inhibiting cancer relapse or cancer
progression in an individual, wherein the PD-1 axis binding agent
is used in combination with an agent that decreases or inhibits
TIGIT expression and/or activity. In other aspects, the present
disclosure provides use of an effective amount of an agent that
decreases or inhibits TIGIT expression and/or activity in the
manufacture of a medicament for reducing or inhibiting cancer
relapse or cancer progression in an individual, wherein the agent
that decreases or inhibits TIGIT expression and/or activity is used
in combination with a PD-1 axis binding antagonist. In other
aspects, the present disclosure provides a pharmaceutical
composition comprising a PD-1 axis binding antagonist for use in
reducing or inhibiting cancer relapse or cancer progression in
combination with an agent that decreases or inhibits TIGIT
expression and/or activity. In other aspects, the present
disclosure provides a pharmaceutical composition comprising an
agent that decreases or inhibits TIGIT expression and/or activity
for use in reducing or inhibiting cancer relapse or cancer
progression in combination with a PD-1 axis binding antagonist.
[0079] In other aspects, the present disclosure provides a method
for treating or delaying progression of an immune related disease
in an individual comprising administering to the individual an
effective amount of a PD-1 axis binding antagonist and an agent
that decreases or inhibits TIGIT expression and/or activity. In
other aspects, the present disclosure provides use of an effective
amount of a PD-1 axis binding antagonist in the manufacture of a
medicament for treating or delaying progression of an immune
related disease in an individual, wherein the PD-1 axis binding
agent is used in combination with an agent that decreases or
inhibits TIGIT expression and/or activity. In other aspects, the
present disclosure provides use of an effective amount of an agent
that decreases or inhibits TIGIT expression and/or activity in the
manufacture of a medicament for treating or delaying progression of
an immune related disease in an individual, wherein the agent that
decreases or inhibits TIGIT expression and/or activity is used in
combination with a PD-1 axis binding antagonist. In other aspects,
the present disclosure provides a pharmaceutical composition
comprising a PD-1 axis binding antagonist for use in treating or
delaying progression of an immune related disease in combination
with an agent that decreases or inhibits TIGIT expression and/or
activity. In other aspects, the present disclosure provides a
pharmaceutical composition comprising an agent that decreases or
inhibits TIGIT expression and/or activity for use in treating or
delaying progression of an immune related disease in combination
with a PD-1 axis binding antagonist.
[0080] In other aspects, the present disclosure provides a
combination comprising an effective amount of a PD-1 axis binding
antagonist and an agent that decreases or inhibits TIGIT expression
and/or activity.
[0081] In other aspects, the present disclosure provides a method
for reducing or inhibiting progression of an immune related disease
in an individual comprising administering to the individual an
effective amount of a PD-1 axis binding antagonist and an agent
that decreases or inhibits TIGIT expression and/or activity. In
other aspects, the present disclosure provides use of an effective
amount of a PD-1 axis binding antagonist in the manufacture of a
medicament for reducing or inhibiting progression of an immune
related disease in an individual, wherein the PD-1 axis binding
agent is used in combination with an agent that decreases or
inhibits TIGIT expression and/or activity. In other aspects, the
present disclosure provides use of an effective amount of an agent
that decreases or inhibits TIGIT expression and/or activity in the
manufacture of a medicament for reducing or inhibiting progression
of an immune related disease in an individual, wherein the agent
that decreases or inhibits TIGIT expression and/or activity is used
in combination with a PD-1 axis binding antagonist. In other
aspects, the present disclosure provides a pharmaceutical
composition comprising a PD-1 axis binding antagonist for use in
reducing or inhibiting progression of an immune related disease in
combination with an agent that decreases or inhibits TIGIT
expression and/or activity. In other aspects, the present
disclosure provides a pharmaceutical composition comprising an
agent that decreases or inhibits TIGIT expression and/or activity
for use in reducing or inhibiting progression of an immune related
disease in combination with a PD-1 axis binding antagonist.
[0082] In certain embodiments that may be combined with any of the
preceding embodiments, the immune related disease is associated
with a T cell dysfunctional disorder. In certain embodiments that
may be combined with any of the preceding embodiments, the immune
related disease is a viral infection. In certain embodiments that
may be combined with any of the preceding embodiments, the viral
infection is a chronic viral infection. In certain embodiments that
may be combined with any of the preceding embodiments, the T cell
dysfunctional disorder is characterized by decreased responsiveness
to antigenic stimulation. In certain embodiments that may be
combined with any of the preceding embodiments, the T cell
dysfunctional disorder is characterized by T cell anergy or
decreased ability to secrete cytokines, proliferate or execute
cytolytic activity. In certain embodiments that may be combined
with any of the preceding embodiments, the T cell dysfunctional
disorder is characterized by T cell exhaustion. In certain
embodiments that may be combined with any of the preceding
embodiments, the T cells are CD4+ and CD8+ T cells. In certain
embodiments that may be combined with any of the preceding
embodiments, the immune related disease is selected from the group
consisting of unresolved acute infection, chronic infection, and
tumor immunity.
[0083] In other aspects, the present disclosure provides a method
of increasing, enhancing or stimulating an immune response or
function in an individual comprising administering to the
individual an effective amount of a PD-1 axis binding antagonist
and an agent that decreases or inhibits TIGIT expression and/or
activity. In other aspects, the present disclosure provides use of
an effective amount of a PD-1 axis binding antagonist in the
manufacture of a medicament for enhancing or stimulating an immune
response or function in an individual, wherein the PD-1 axis
binding agent is used in combination with an agent that decreases
or inhibits TIGIT expression and/or activity. In other aspects, the
present disclosure provides use of an effective amount of an agent
that decreases or inhibits TIGIT expression and/or activity in the
manufacture of a medicament for enhancing or stimulating an immune
response or function in an individual, wherein the agent that
decreases or inhibits TIGIT expression and/or activity is used in
combination with a PD-1 axis binding antagonist. In other aspects,
the present disclosure provides a pharmaceutical composition
comprising a PD-1 axis binding antagonist for use in enhancing or
stimulating an immune response or function in combination with an
agent that decreases or inhibits TIGIT expression and/or activity.
In other aspects, the present disclosure provides a pharmaceutical
composition comprising an agent that decreases or inhibits TIGIT
expression and/or activity for use in enhancing or stimulating an
immune response or function in combination with a PD-1 axis binding
antagonist. In other aspects, the present disclosure provides a
combination comprising an effective amount of a PD-1 axis binding
antagonist and an agent that decreases or inhibits TIGIT expression
and/or activity.
[0084] In other aspects, the present disclosure provides a method
of treating or delaying progression of cancer in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and an agent that modulates CD226
expression and/or activity. In other aspects, the present
disclosure provides use of an effective amount of a PD-1 axis
binding antagonist in the manufacture of a medicament for treating
or delaying progression of cancer in an individual, wherein the
PD-1 axis binding agent is used in combination with an agent that
modulates CD226 expression and/or activity. In other aspects, the
present disclosure provides use of an effective amount of an agent
that modulates CD226 expression and/or activity in the manufacture
of a medicament for treating or delaying progression of cancer in
an individual, wherein the agent that modulates CD226 expression
and/or activity is used in combination with a PD-1 axis binding
antagonist. In other aspects, the present disclosure provides a
pharmaceutical composition comprising a PD-1 axis binding
antagonist for use in treating or delaying progression of cancer in
combination with an agent that modulates CD226 expression and/or
activity. In other aspects, the present disclosure provides a
pharmaceutical composition comprising an agent that modulates CD226
expression and/or activity for use in treating or delaying
progression of cancer in combination with a PD-1 axis binding
antagonist.
[0085] In other aspects, the present disclosure provides a method
for reducing or inhibiting cancer relapse or cancer progression in
an individual comprising administering to the individual an
effective amount of a PD-1 axis binding antagonist and an agent
that modulates CD226 expression and/or activity. In other aspects,
the present disclosure provides use of an effective amount of a
PD-1 axis binding antagonist in the manufacture of a medicament for
reducing or inhibiting cancer relapse or cancer progression in an
individual, wherein the PD-1 axis binding agent is used in
combination with an agent that modulates CD226 expression and/or
activity. In other aspects, the present disclosure provides use of
an effective amount of an agent that modulates CD226 expression
and/or activity in the manufacture of a medicament for reducing or
inhibiting cancer relapse or cancer progression in an individual,
wherein the agent that modulates CD226 expression and/or activity
is used in combination with a PD-1 axis binding antagonist. In
other aspects, the present disclosure provides a pharmaceutical
composition comprising a PD-1 axis binding antagonist for use in
reducing or inhibiting cancer relapse or cancer progression in
combination with an agent that modulates CD226 expression and/or
activity. In other aspects, the present disclosure provides a
pharmaceutical composition comprising an agent that modulates CD226
expression and/or activity for use in reducing or inhibiting cancer
relapse or cancer progression in combination with a PD-1 axis
binding antagonist.
[0086] In other aspects, the present disclosure provides a method
for treating or delaying progression of an immune related disease
in an individual comprising administering to the individual an
effective amount of a PD-1 axis binding antagonist and an agent
that modulates CD226 expression and/or activity. In other aspects,
the present disclosure provides use of an effective amount of a
PD-1 axis binding antagonist in the manufacture of a medicament for
treating or delaying progression of an immune related disease in an
individual, wherein the PD-1 axis binding agent is used in
combination with an agent that modulates CD226 expression and/or
activity. In other aspects, the present disclosure provides use of
an effective amount of an agent that modulates CD226 expression
and/or activity in the manufacture of a medicament for treating or
delaying progression of an immune related disease in an individual,
wherein the agent that modulates CD226 expression and/or activity
is used in combination with a PD-1 axis binding antagonist. In
other aspects, the present disclosure provides a pharmaceutical
composition comprising a PD-1 axis binding antagonist for use in
treating or delaying progression of an immune related disease in
combination with an agent that modulates CD226 expression and/or
activity. In other aspects, the present disclosure provides a
pharmaceutical composition comprising an agent that modulates CD226
expression and/or activity for use in treating or delaying
progression of an immune related disease in combination with a PD-1
axis binding antagonist.
[0087] In other aspects, the present disclosure provides a
combination comprising an effective amount of a PD-1 axis binding
antagonist and an agent that modulates CD226 expression and/or
activity.
[0088] In other aspects, the present disclosure provides a method
for reducing or inhibiting progression of an immune related disease
in an individual comprising administering to the individual an
effective amount of a PD-1 axis binding antagonist and an agent
that modulates CD226 expression and/or activity. In other aspects,
the present disclosure provides use of an effective amount of a
PD-1 axis binding antagonist in the manufacture of a medicament for
reducing or inhibiting progression of an immune related disease in
an individual, wherein the PD-1 axis binding agent is used in
combination with an agent that modulates CD226 expression and/or
activity. In other aspects, the present disclosure provides use of
an effective amount of an agent that modulates CD226 expression
and/or activity in the manufacture of a medicament for reducing or
inhibiting progression of an immune related disease in an
individual, wherein the agent that modulates CD226 expression
and/or activity is used in combination with a PD-1 axis binding
antagonist. In other aspects, the present disclosure provides a
pharmaceutical composition comprising a PD-1 axis binding
antagonist for use in reducing or inhibiting progression of an
immune related disease in combination with an agent that modulates
CD226 expression and/or activity. In other aspects, the present
disclosure provides a pharmaceutical composition comprising an
agent that modulates CD226 expression and/or activity for use in
reducing or inhibiting progression of an immune related disease in
combination with a PD-1 axis binding antagonist.
[0089] In certain embodiments that may be combined with any of the
preceding embodiments, the immune related disease is associated
with a T cell dysfunctional disorder. In certain embodiments that
may be combined with any of the preceding embodiments, the immune
related disease is a viral infection. In certain embodiments that
may be combined with any of the preceding embodiments, the viral
infection is a chronic viral infection. In certain embodiments that
may be combined with any of the preceding embodiments, the T cell
dysfunctional disorder is characterized by decreased responsiveness
to antigenic stimulation. In certain embodiments that may be
combined with any of the preceding embodiments, the T cell
dysfunctional disorder is characterized by T cell anergy, or
decreased ability to secrete cytokines, proliferate or execute
cytolytic activity. In certain embodiments that may be combined
with any of the preceding embodiments, the T cell dysfunctional
disorder is characterized by T cell exhaustion. In certain
embodiments that may be combined with any of the preceding
embodiments, the T cells are CD4+ and CD8+ T cells. In certain
embodiments that may be combined with any of the preceding
embodiments, the immune related disease is selected from the group
consisting of unresolved acute infection, chronic infection and
tumor immunity.
[0090] In other aspects, the present disclosure provides a method
of increasing, enhancing, or stimulating an immune response or
function in an individual comprising administering to the
individual an effective amount of a PD-1 axis binding antagonist
and an agent that modulates CD226 expression and/or activity. In
other aspects, the present disclosure provides use of an effective
amount of a PD-1 axis binding antagonist in the manufacture of a
medicament for enhancing or stimulating an immune response or
function in an individual, wherein the PD-1 axis binding agent is
used in combination with an agent that modulates CD226 expression
and/or activity. In other aspects, the present disclosure provides
use of an effective amount of an agent that modulates CD226
expression and/or activity in the manufacture of a medicament for
enhancing or stimulating an immune response or function in an
individual, wherein the an agent that modulates CD226 expression
and/or activity is used in combination with a PD-1 axis binding
antagonist. In other aspects, the present disclosure provides a
pharmaceutical composition comprising a PD-1 axis binding
antagonist for use in enhancing or stimulating an immune response
or function in combination with an agent that modulates CD226
expression and/or activity. In other aspects, the present
disclosure provides a pharmaceutical composition comprising an
agent that modulates CD226 expression and/or activity for use in
enhancing or stimulating an immune response or function in
combination with a PD-1 axis binding antagonist. In other aspects,
the present disclosure provides a combination comprising an
effective amount of a PD-1 axis binding antagonist and an agent
that modulates CD226 expression and/or activity.
[0091] In certain embodiments that may be combined with any of the
preceding embodiments, the agent that modulates CD226 expression
and/or activity is an agent that increases and/or stimulates CD226
expression and/or activity. In certain embodiments that may be
combined with any of the preceding embodiments, the agent that
modulates CD226 expression and/or activity is an agent that
increases and/or stimulates the interaction of CD226 with PVR. In
certain embodiments that may be combined with any of the preceding
embodiments, the agent that modulates CD226 expression and/or
activity is an agent that increases and/or stimulates the
intracellular signaling mediated by CD226 binding to PVR. In
certain embodiments that may be combined with any of the preceding
embodiments, the agent that modulates CD226 expression and/or
activity is selected from the group consisting of an agent that
inhibits and/or blocks the interaction of CD226 with TIGIT, an
antagonist of TIGIT expression and/or activity, an antagonist of
PVR expression and/or activity, an agent that inhibits and/or
blocks the interaction of TIGIT with PVR, an agent that inhibits
and/or blocks the interaction of TIGIT with PVRL2, an agent that
inhibits and/or blocks the interaction of TIGIT with PVRL3, an
agent that inhibits and/or blocks the intracellular signaling
mediated by TIGIT binding to PVR, an agent that inhibits and/or
blocks the intracellular signaling mediated by TIGIT binding to
PVRL2, an agent that inhibits and/or blocks the intracellular
signaling mediated by TIGIT binding to PVRL3, and combinations
thereof. In certain embodiments that may be combined with any of
the preceding embodiments, the agent that modulates CD226
expression and/or activity is an agent that inhibits and/or blocks
the interaction of CD226 with TIGIT. In certain embodiments that
may be combined with any of the preceding embodiments, the agent
that inhibits and/or blocks the interaction of CD226 with TIGIT is
a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, or an inhibitory polypeptide. In certain embodiments that may
be combined with any of the preceding embodiments, the agent that
inhibits and/or blocks the interaction of CD226 with TIGIT is an
anti-TIGIT antibody or antigen-binding fragment thereof. In certain
embodiments that may be combined with any of the preceding
embodiments, the agent that inhibits and/or blocks the interaction
of CD226 with TIGIT is an inhibitory nucleic acid selected from the
group consisting of an antisense polynucleotide, an interfering
RNA, a catalytic RNA, and an RNA-DNA chimera. In certain
embodiments that may be combined with any of the preceding
embodiments, the antisense polynucleotide targets TIGIT. In certain
embodiments that may be combined with any of the preceding
embodiments, the interfering RNA targets TIGIT. In certain
embodiments that may be combined with any of the preceding
embodiments, the catalytic RNA targets TIGIT. In certain
embodiments that may be combined with any of the preceding
embodiments, the RNA-DNA chimera targets TIGIT. In certain
embodiments that may be combined with any of the preceding
embodiments, the agent that modulates CD226 expression and/or
activity is an antagonist of TIGIT expression and/or activity. In
certain embodiments that may be combined with any of the preceding
embodiments, the antagonist of TIGIT expression and/or activity is
a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide. In certain embodiments that
may be combined with any of the preceding embodiments, the
antagonist of TIGIT expression and/or activity is an anti-TIGIT
antibody or antigen-binding fragment thereof. In certain
embodiments that may be combined with any of the preceding
embodiments, the antagonist of TIGIT expression and/or activity is
an inhibitory nucleic acid selected from the group consisting of an
antisense polynucleotide, an interfering RNA, a catalytic RNA, and
an RNA-DNA chimera. In certain embodiments that may be combined
with any of the preceding embodiments, the antagonist of PVR
expression and/or activity is selected from the group consisting of
a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide. In certain embodiments that
may be combined with any of the preceding embodiments, the agent
that inhibits and/or blocks the interaction of TIGIT with PVR is
selected from the group consisting of a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In certain embodiments that may be combined with any of the
preceding embodiments, the agent that inhibits and/or blocks the
interaction of TIGIT with PVRL2 is selected from the group
consisting of a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide. In certain embodiments that
may be combined with any of the preceding embodiments, the agent
that inhibits and/or blocks the interaction of TIGIT with PVRL3 is
selected from the group consisting of a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In certain embodiments that may be combined with any of the
preceding embodiments, the agent that inhibits and/or blocks the
intracellular signaling mediated by TIGIT binding to PVR is
selected from the group consisting of a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In certain embodiments that may be combined with any of the
preceding embodiments, the agent that inhibits and/or blocks the
interaction of TIGIT with PVRL2 is selected from the group
consisting of a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide. In certain embodiments that
may be combined with any of the preceding embodiments, the agent
that inhibits and/or blocks the interaction of TIGIT with PVRL3 is
selected from the group consisting of a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0092] In other aspects, the present disclosure provides a method
of increasing, enhancing, or stimulating an immune response or
function in an individual comprising administering to the
individual an effective amount of an agent that decreases or
inhibits TIGIT expression and/or activity and an agent that
decreases or inhibits one or more additional immune co-inhibitory
receptors. In other aspects, the present disclosure provides use of
an effective amount of an agent that decreases or inhibits TIGIT
expression and/or activity in the manufacture of a medicament for
enhancing or stimulating an immune response or function in an
individual, wherein the agent that decreases or inhibits TIGIT
expression and/or activity is used in combination with an agent
that decreases or inhibits one or more additional immune
co-inhibitory receptors. In other aspects, the present disclosure
provides use of an effective amount of an agent that decreases or
inhibits one or more additional immune co-inhibitory receptors in
the manufacture of a medicament for enhancing or stimulating an
immune response or function in an individual, wherein the agent
that decreases or inhibits one or more additional immune
co-inhibitory receptors is used in combination with an agent that
decreases or inhibits TIGIT expression and/or activity. In other
aspects, the present disclosure provides a pharmaceutical
composition comprising an agent that decreases or inhibits TIGIT
expression and/or activity for use in enhancing or stimulating an
immune response or function in combination with an agent that
decreases or inhibits one or more additional immune co-inhibitory
receptors. In other aspects, the present disclosure provides a
pharmaceutical composition comprising an agent that decreases or
inhibits one or more additional immune co-inhibitory receptors for
use in enhancing or stimulating an immune response or function in
combination with an agent that decreases or inhibits TIGIT
expression and/or activity. In other aspects, the present
disclosure provides a combination comprising an effective amount of
an agent that decreases or inhibits TIGIT expression and/or
activity and an agent that decreases or inhibits one or more
additional immune co-inhibitory receptors. In certain embodiments
that may be combined with any of the preceding embodiments, the one
or more additional immune co-inhibitory receptor is selected from
the group consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA,
B7H4, and CD96. In certain embodiments that may be combined with
any of the preceding embodiments, the one or more additional immune
co-inhibitory receptor is selected from the group consisting of
PD-1, CTLA-4, LAG3 and TIM3.
[0093] In other aspects, the present disclosure provides a method
of increasing, enhancing, or stimulating an immune response or
function in an individual comprising administering to the
individual an effective amount of an agent that decreases or
inhibits TIGIT expression and/or activity and an agent that
increases or activates one or more additional immune co-stimulatory
receptors. In other aspects, the present disclosure provides use of
an effective amount of an agent that decreases or inhibits TIGIT
expression and/or activity in the manufacture of a medicament for
enhancing or stimulating an immune response or function in an
individual, wherein the agent that decreases or inhibits TIGIT
expression and/or activity is used in combination with an agent
that increases or activates one or more additional immune
co-stimulatory receptors. In other aspects, the present disclosure
provides use of an effective amount of an a agent that increases or
activates one or more additional immune co-stimulatory receptors in
the manufacture of a medicament for enhancing or stimulating an
immune response or function in an individual, wherein the a agent
that increases or activates one or more additional immune
co-stimulatory receptors is used in combination with an agent that
decreases or inhibits TIGIT expression and/or activity. In other
aspects, the present disclosure provides a pharmaceutical
composition comprising an agent that decreases or inhibits TIGIT
expression and/or activity for use in enhancing or stimulating an
immune response or function in combination with an agent that
increases or activates one or more additional immune co-stimulatory
receptors. In other aspects, the present disclosure provides a
pharmaceutical composition comprising an agent that increases or
activates one or more additional immune co-stimulatory receptors
for use in enhancing or stimulating an immune response or function
in combination with an agent that decreases or inhibits TIGIT
expression and/or activity. In other aspects, the present
disclosure provides a combination comprising an effective amount of
an agent that decreases or inhibits TIGIT expression and/or
activity and an agent that increases or activates one or more
additional immune co-stimulatory receptors. In certain embodiments
that may be combined with any of the preceding embodiments, the one
or more additional immune co-stimulatory receptors is selected from
the group consisting of CD226, OX-40, CD28, CD27, CD137, HVEM,
GITR, MICA, ICOS, NKG2D, and 2B4. In certain embodiments that may
be combined with any of the preceding embodiments, the one or more
additional immune co-stimulatory receptors is selected from the
group consisting of CD226, OX-40, CD27, CD137, HVEM and GITR. In
certain embodiments that may be combined with any of the preceding
embodiments, the one or more additional immune co-stimulatory
receptors is selected from the group consisting of OX-40 and
CD27.
[0094] In certain embodiments that may be combined with any of the
preceding embodiments, the method further comprises administering
at least one chemotherapeutic agent. In certain embodiments that
may be combined with any of the preceding embodiments, the
individual has cancer. In certain embodiments that may be combined
with any of the preceding embodiments, the individual is a human.
In certain embodiments that may be combined with any of the
preceding embodiments, CD4 and/or CD8 T cells in the individual
have increased or enhanced priming, activation, proliferation,
cytokine release and/or cytolytic activity relative to prior to the
administration of the combination. In certain embodiments that may
be combined with any of the preceding embodiments, the number of
CD4 and/or CD8 T cells is elevated relative to prior to
administration of the combination. In certain embodiments that may
be combined with any of the preceding embodiments, the number of
activated CD4 and/or CD8 T cells is elevated relative to prior to
administration of the combination. In certain embodiments that may
be combined with any of the preceding embodiments, activated CD4
and/or CD8 T cells are characterized by .gamma.-IFN.sup.+ producing
CD4 and/or CD8 T cells and/or enhanced cytolytic activity relative
to prior to the administration of the combination. In certain
embodiments that may be combined with any of the preceding
embodiments, the CD4 and/or CD8 T cells exhibit increased release
of cytokines selected from the group consisting of IFN-.gamma.,
TNF-.alpha. and interleukins. In certain embodiments that may be
combined with any of the preceding embodiments, the CD4 and/or CD8
T cells are effector memory T cells. In certain embodiments that
may be combined with any of the preceding embodiments, the CD4
and/or CD8 effector memory T cells are characterized by
.gamma.-IFN.sup.+ producing CD4 and/or CD8 T cells and/or enhanced
cytolytic activity. In certain embodiments that may be combined
with any of the preceding embodiments, the CD4 and/or CD8 effector
memory T cells are characterized by having the expression of
CD44.sup.high CD62L.sup.low. In certain embodiments that may be
combined with any of the preceding embodiments, the cancer has
elevated levels of T cell infiltration. In certain embodiments that
may be combined with any of the preceding embodiments, the agent
that decreases or inhibits TIGIT expression and/or activity is
selected from the group consisting of an antagonist of TIGIT
expression and/or activity, an antagonist of PVR expression and/or
activity, an agent that inhibits and/or blocks the interaction of
TIGIT with PVR, an agent that inhibits and/or blocks the
interaction of TIGIT with PVRL2, an agent that inhibits and/or
blocks the interaction of TIGIT with PVRL3, an agent that inhibits
and/or blocks the intracellular signaling mediated by TIGIT binding
to PVR, an agent that inhibits and/or blocks the intracellular
signaling mediated by TIGIT binding to PVRL2, an agent that
inhibits and/or blocks the intracellular signaling mediated by
TIGIT binding to PVRL3, and combinations thereof. In certain
embodiments that may be combined with any of the preceding
embodiments, the antagonist of TIGIT expression and/or activity is
selected from the group consisting of a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In certain embodiments that may be combined with any of the
preceding embodiments, the antagonist of PVR expression and/or
activity is selected from the group consisting of a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide. In certain embodiments that may be combined with any
of the preceding embodiments, the agent that inhibits and/or blocks
the interaction of TIGIT with PVR is selected from the group
consisting of a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide. In certain embodiments that
may be combined with any of the preceding embodiments, the agent
that inhibits and/or blocks the interaction of TIGIT with PVRL2 is
selected from the group consisting of a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In certain embodiments that may be combined with any of the
preceding embodiments, the agent that inhibits and/or blocks the
interaction of TIGIT with PVRL3 is selected from the group
consisting of a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide. In certain embodiments that
may be combined with any of the preceding embodiments, the agent
that inhibits and/or blocks the intracellular signaling mediated by
TIGIT binding to PVR is selected from the group consisting of a
small molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide. In certain embodiments that may be combined
with any of the preceding embodiments, the agent that inhibits
and/or blocks the intracellular signaling mediated by TIGIT binding
to PVRL2 is selected from the group consisting of a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide. In certain embodiments that may be combined with any
of the preceding embodiments, the agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVRL3 is
selected from the group consisting of a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In certain embodiments that may be combined with any of the
preceding embodiments, the antagonist of TIGIT expression and/or
activity is an inhibitory nucleic acid selected from the group
consisting of an antisense polynucleotide, an interfering RNA, a
catalytic RNA, and an RNA-DNA chimera. In certain embodiments that
may be combined with any of the preceding embodiments, the
antisense polynucleotide targets TIGIT.
[0095] In certain embodiments that may be combined with any of the
preceding embodiments, the interfering RNA targets TIGIT. In
certain embodiments that may be combined with any of the preceding
embodiments, the catalytic RNA targets TIGIT. In certain
embodiments that may be combined with any of the preceding
embodiments, the RNA-DNA chimera targets TIGIT. In certain
embodiments that may be combined with any of the preceding
embodiments, the antagonist of TIGIT expression and/or activity is
an anti-TIGIT antibody or antigen-binding fragment thereof. In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-TIGIT antibody or antigen-binding fragment
thereof comprises at least one HVR comprising an amino acid
sequence selected from the amino acid sequences (1)
TABLE-US-00009 (SEQ ID NO: 1) KSSQSLYYSGVKENLLA, (SEQ ID NO: 2)
ASIRFT, (SEQ ID NO: 3) QQGINNPLT, (SEQ ID NO: 4) GFTFSSFTMH, (SEQ
ID NO: 5) FIRSGSGIVFYADAVRG, and (SEQ ID NO: 6) RPLGHNTFDS; or (SEQ
ID NO: 7) (2)RSSQSLVNSYGNTFLS, (SEQ ID NO: 8) GISNRFS, (SEQ ID NO:
9) LQGTHQPPT, (SEQ ID NO: 10) GYSFTGHLMN, (SEQ ID NO: 11)
LIIPYNGGTSYNQKFKG, and (SEQ ID NO: 12) GLRGFYAMDY.
In certain embodiments that may be combined with any of the
preceding embodiments, the anti-TIGIT antibody or antigen-binding
fragment thereof, wherein the antibody light chain comprises the
amino acid sequence set forth in
TABLE-US-00010 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSP
KLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINN PLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQ
LLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQP
PTFGPGTKLEVK.
In certain embodiments that may be combined with any of the
preceding embodiments, the anti-TIGIT antibody or antigen-binding
fragment thereof, wherein the antibody heavy chain comprises the
amino acid sequence set forth in
TABLE-US-00011 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAF
IRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRP
LGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGL
IIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGL
RGFYAMDYWGQGTSVTVSS.
In certain embodiments that may be combined with any of the
preceding embodiments, the anti-TIGIT antibody or antigen-binding
fragment thereof, wherein the antibody light chain comprises the
amino acid sequence set forth in
TABLE-US-00012 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSP
KLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINN PLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQ
LLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQP
PTFGPGTKLEVK,
and the antibody heavy chain comprises the amino acid sequence set
forth in
TABLE-US-00013 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAF
IRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRP
LGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGL
IIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGL
RGFYAMDYWGQGTSVTVSS.
In certain embodiments that may be combined with any of the
preceding embodiments, the anti-TIGIT antibody or antigen-binding
fragment thereof, wherein the antibody is selected from the group
consisting of a humanized antibody, a chimeric antibody, a
bispecific antibody, a heteroconjugate antibody, and an
immunotoxin. In certain embodiments that may be combined with any
of the preceding embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof comprises at least one HVR that is
at least 90% identical to an HVR set forth in any one of (1)
KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ ID NO:2), QQGINNPLT
(SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4), FIRSGSGIVFYADAVRG (SEQ ID
NO:5), and RPLGHNTFDS (SEQ ID NO:6); or (2) RSSQSLVNSYGNTFLS (SEQ
ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ ID NO:9),
GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and
GLRGFYAMDY (SEQ ID NO:12). In certain embodiments that may be
combined with any of the preceding embodiments, the anti-TIGIT
antibody or fragment thereof comprises the light chain comprising
amino acid sequences at least 90% identical to the amino acid
sequences set forth in
TABLE-US-00014 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSP
KLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINN PLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQ
LLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQP
PTFGPGTKLEVK;
and/or the heavy chain comprising amino acid sequences at least 90%
identical to the amino acid sequences set forth in
TABLE-US-00015 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR
RPLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIG
LIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSR
GLRGFYAMDYWGQGTSVTVSS.
In certain embodiments that may be combined with any of the
preceding embodiments, the PD-1 axis binding antagonist is selected
from the group consisting of a PD-1 binding antagonist, a PD-L1
binding antagonist and a PD-L2 binding antagonist. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-1 axis binding antagonist is a PD-1 binding
antagonist. In certain embodiments that may be combined with any of
the preceding embodiments, the PD-1 binding antagonist inhibits the
binding of PD-1 to its ligand binding partners. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-1 binding antagonist inhibits the binding of
PD-1 to PD-L1. In certain embodiments that may be combined with any
of the preceding embodiments, the PD-1 binding antagonist inhibits
the binding of PD-1 to PD-L2. In certain embodiments that may be
combined with any of the preceding embodiments, the PD-1 binding
antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In
certain embodiments that may be combined with any of the preceding
embodiments, the PD-1 binding antagonist is an antibody. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-1 binding antagonist is MDX-1106. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-1 binding antagonist is MK-3475. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-1 binding antagonist is CT-011. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-1 binding antagonist is AMP-224. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-1 axis binding antagonist is a PD-L1 binding
antagonist. In certain embodiments that may be combined with any of
the preceding embodiments, the PD-L1 binding antagonist inhibits
the binding of PD-L1 to PD-1. In certain embodiments that may be
combined with any of the preceding embodiments, the PD-L1 binding
antagonist inhibits the binding of PD-L1 to B7-1. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-L1 binding antagonist inhibits the binding of
PD-L1 to both PD-1 and B7-1. In certain embodiments that may be
combined with any of the preceding embodiments, the PD-L1 binding
antagonist is an anti-PD-L1 antibody. In certain embodiments that
may be combined with any of the preceding embodiments, the PD-L1
binding antagonist is selected from the group consisting of
YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. In certain
embodiments that may be combined with any of the preceding
embodiments, the anti-PD-L1 antibody comprises a heavy chain
comprising HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO:17), HVR-H2
sequence of AWISPYGGSTYYADSVKG (SEQ ID NO:18), and HVR-H3 sequence
of RHWPGGFDY (SEQ ID NO:19); and a light chain comprising HVR-L1
sequence of RASQDVSTAVA (SEQ ID NO:20), HVR-L2 sequence of SASFLYS
(SEQ ID NO:21), and HVR-L3 sequence of QQYLYHPAT (SEQ ID NO:22). In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-PD-L1 antibody comprises a heavy chain
variable region comprising the amino acid sequence of
TABLE-US-00016 (SEQ ID NO: 23)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSA, (SEQ ID NO: 40)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSSASTK, or (SEQ ID NO: 41)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSS, and a light chain variable region comprising
the amino acid sequence of (SEQ ID NO: 24)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY
SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKR.
In certain embodiments that may be combined with any of the
preceding embodiments, the PD-1 axis binding antagonist is a PD-L2
binding antagonist. In certain embodiments that may be combined
with any of the preceding embodiments, the PD-L2 binding antagonist
is an antibody. In certain embodiments that may be combined with
any of the preceding embodiments, the PD-L2 binding antagonist is
an immunoadhesin. In certain embodiments that may be combined with
any of the preceding embodiments, the cancer is selected from the
group consisting of non-small cell lung cancer, small cell lung
cancer, renal cell cancer, colorectal cancer, ovarian cancer,
breast cancer, pancreatic cancer, gastric carcinoma, bladder
cancer, esophageal cancer, mesothelioma, melanoma, head and neck
cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma,
cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas,
mycoses fungoids, merkel cell cancer, and other hematologic
malignancies. In certain embodiments that may be combined with any
of the preceding embodiments, the agent that decreases or inhibits
TIGIT expression and/or activity is administered continuously. In
certain embodiments that may be combined with any of the preceding
embodiments, the agent that decreases or inhibits TIGIT expression
and/or activity is administered intermittently. In certain
embodiments that may be combined with any of the preceding
embodiments, the agent that decreases or inhibits TIGIT expression
and/or activity is administered before the PD-1 axis binding
antagonist. In certain embodiments that may be combined with any of
the preceding embodiments, the agent that decreases or inhibits
TIGIT expression and/or activity is administered simultaneous with
the PD-1 axis binding antagonist. In certain embodiments that may
be combined with any of the preceding embodiments, the agent that
decreases or inhibits TIGIT expression and/or activity is
administered after the PD-1 axis binding antagonist. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-1 axis binding antagonist is administered
before the agent that modulates CD226 expression and/or activity.
In certain embodiments that may be combined with any of the
preceding embodiments, the PD-1 axis binding antagonist is
administered simultaneous with the agent that modulates CD226
expression and/or activity. In certain embodiments that may be
combined with any of the preceding embodiments, the PD-1 axis
binding antagonist is administered after the agent that modulates
CD226 expression and/or activity. In certain embodiments that may
be combined with any of the preceding embodiments, the agent that
decreases or inhibits TIGIT expression and/or activity is
administered before the agent that decreases or inhibits one or
more additional immune co-inhibitory receptors. In certain
embodiments that may be combined with any of the preceding
embodiments, the agent that decreases or inhibits TIGIT expression
and/or activity is administered simultaneous with the agent that
decreases or inhibits one or more additional immune co-inhibitory
receptors. In certain embodiments that may be combined with any of
the preceding embodiments, the agent that decreases or inhibits
TIGIT expression and/or activity is administered after the agent
that decreases or inhibits one or more additional immune
co-inhibitory receptors. In certain embodiments that may be
combined with any of the preceding embodiments, the agent that
decreases or inhibits TIGIT expression and/or activity is
administered before the agent that increases or activates one or
more additional immune co-stimulatory receptors. In certain
embodiments that may be combined with any of the preceding
embodiments, the agent that decreases or inhibits TIGIT expression
and/or activity is administered simultaneous with the agent that
increases or activates one or more additional immune co-stimulatory
receptors. In certain embodiments that may be combined with any of
the preceding embodiments, the agent that decreases or inhibits
TIGIT expression and/or activity is administered after the agent
that increases or activates one or more additional immune
co-stimulatory receptors.
[0096] In other aspects, the present disclosure provides a kit
comprising a PD-1 axis binding antagonist and a package insert
comprising instructions for using the PD-1 axis binding antagonist
in combination with an agent that decreases or inhibits TIGIT
expression and/or activity to treat or delay progression of cancer
in an individual.
[0097] In other aspects, the present disclosure provides a kit
comprising a PD-1 axis binding antagonist and an agent that
decreases or inhibits TIGIT expression and/or activity, and a
package insert comprising instructions for using the PD-1 axis
binding antagonist and the agent that decreases or inhibits TIGIT
expression and/or activity to treat or delay progression of cancer
in an individual.
[0098] In other aspects, the present disclosure provides a kit
comprising an agent that decreases or inhibits TIGIT expression
and/or activity and a package insert comprising instructions for
using the agent that decreases or inhibits TIGIT expression and/or
activity in combination with a PD-1 axis binding antagonist to
treat or delay progression of cancer in an individual.
[0099] In other aspects, the present disclosure provides a kit
comprising a PD-1 axis binding antagonist and a package insert
comprising instructions for using the PD-1 axis binding antagonist
in combination with an agent that decreases or inhibits TIGIT
expression and/or activity to enhance immune function of an
individual having cancer.
[0100] In other aspects, the present disclosure provides a kit
comprising a PD-1 axis binding antagonist and an agent that
decreases or inhibits TIGIT expression and/or activity, and a
package insert comprising instructions for using the PD-1 axis
binding antagonist and the agent that decreases or inhibits TIGIT
expression and/or activity to enhance immune function of an
individual having cancer.
[0101] In other aspects, the present disclosure provides a kit
comprising an agent that decreases or inhibits TIGIT expression
and/or activity and a package insert comprising instructions for
using the agent that decreases or inhibits TIGIT expression and/or
activity in combination with a PD-1 axis binding antagonist to
enhance immune function of an individual having cancer.
[0102] In other aspects, the present disclosure provides a kit
comprising a PD-1 axis binding antagonist and a package insert
comprising instructions for using the PD-1 axis binding antagonist
in combination with an agent that modulates CD226 expression and/or
activity to treat or delay progression of cancer in an
individual.
[0103] In other aspects, the present disclosure provides a kit
comprising a PD-1 axis binding antagonist and an agent that
modulates CD226 expression and/or activity, and a package insert
comprising instructions for using the PD-1 axis binding antagonist
and the agent that modulates CD226 expression and/or activity to
treat or delay progression of cancer in an individual.
[0104] In other aspects, the present disclosure provides a kit
comprising an agent that modulates CD226 expression and/or activity
and a package insert comprising instructions for using the agent
modulates CD226 expression and/or activity in combination with a
PD-1 axis binding antagonist to treat or delay progression of
cancer in an individual.
[0105] In other aspects, the present disclosure provides a kit
comprising a PD-1 axis binding antagonist and a package insert
comprising instructions for using the PD-1 axis binding antagonist
in combination with an agent that modulates CD226 expression and/or
activity to enhance immune function of an individual having
cancer.
[0106] In other aspects, the present disclosure provides a kit
comprising a PD-1 axis binding antagonist and an agent that
modulates CD226 expression and/or activity, and a package insert
comprising instructions for using the PD-1 axis binding antagonist
and the agent that modulates CD226 expression and/or activity to
enhance immune function of an individual having cancer.
[0107] In other aspects, the present disclosure provides a kit
comprising an agent modulates CD226 expression and/or activity and
a package insert comprising instructions for using the agent that
modulates CD226 expression and/or activity in combination with a
PD-1 axis binding antagonist to enhance immune function of an
individual having cancer.
[0108] In certain embodiments that may be combined with any of the
preceding embodiments, the PD-1 axis binding antagonist is an
anti-PD-L1 antibody. In certain embodiments that may be combined
with any of the preceding embodiments, the anti-PD-L1 antibody is
selected from the group consisting of YW243.55.S70, MPDL3280A,
MDX-1105 and MEDI4736. In certain embodiments that may be combined
with any of the preceding embodiments, the anti-PD-L1 antibody
comprises a heavy chain comprising HVR-H1 sequence of GFTFSDSWIH
(SEQ ID NO:17), HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID
NO:18), and HVR-H3 sequence of RHWPGGFDY (SEQ ID NO:19); and a
light chain comprising HVR-L1 sequence of RASQDVSTAVA (SEQ ID
NO:20), HVR-L2 sequence of SASFLYS (SEQ ID NO:21), and HVR-L3
sequence of QQYLYHPAT (SEQ ID NO:22). In certain embodiments that
may be combined with any of the preceding embodiments, the
anti-PD-L1 antibody comprises a heavy chain variable region
comprising the amino acid sequence of
TABLE-US-00017 (SEQ ID NO: 23)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSA, (SEQ ID NO: 40)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSSASTK, or (SEQ ID NO: 41)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSS, and a light chain variable region comprising
the amino acid sequence of (SEQ ID NO: 24)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY
SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKR.
[0109] In certain embodiments that may be combined with any of the
preceding embodiments, the PD-1 axis binding antagonist is an
anti-PD-1 antibody. In certain embodiments that may be combined
with any of the preceding embodiments, the anti-PD-1 antibody is
MDX-1106, MK-3475, or CT-011. In certain embodiments that may be
combined with any of the preceding embodiments, the PD-1 axis
binding antagonist is AMP-224. In certain embodiments that may be
combined with any of the preceding embodiments, the PD-1 axis
binding antagonist is a PD-L2 binding antagonist. In certain
embodiments that may be combined with any of the preceding
embodiments, the PD-L2 binding antagonist is an antibody. In
certain embodiments that may be combined with any of the preceding
embodiments, the PD-L2 binding antagonist is an immunoadhesin.
[0110] In other aspects, the present disclosure provides a kit
comprising an agent that decreases or inhibits TIGIT expression
and/or activity and a package insert comprising instructions for
using the agent that decreases or inhibits TIGIT expression and/or
activity in combination with an agent that decreases or inhibits
one or more additional immune co-inhibitory receptors to treat or
delay progression of cancer in an individual. In other aspects, the
present disclosure provides a kit comprising an agent that
decreases or inhibits TIGIT expression and/or activity and an agent
that decreases or inhibits one or more additional immune
co-inhibitory receptors, and a package insert comprising
instructions for using the agent that decreases or inhibits TIGIT
expression and/or activity and the agent that decreases or inhibits
one or more additional immune co-inhibitory receptors to treat or
delay progression of cancer in an individual. In other aspects, the
present disclosure provides a kit comprising an agent that
decreases or inhibits one or more additional immune co-inhibitory
receptors and a package insert comprising instructions for using
the agent that decreases or inhibits one or more additional immune
co-inhibitory receptors in combination with an agent that decreases
or inhibits TIGIT expression and/or activity to treat or delay
progression of cancer in an individual. In other aspects, the
present disclosure provides a kit comprising an agent that
decreases or inhibits TIGIT expression and/or activity and a
package insert comprising instructions for using the agent that
decreases or inhibits TIGIT expression and/or activity in
combination with an agent that decreases or inhibits one or more
additional immune co-inhibitory receptors to enhance immune
function of an individual having cancer. In other aspects, the
present disclosure provides a kit comprising an agent that
decreases or inhibits TIGIT expression and/or activity and an agent
that decreases or inhibits one or more additional immune
co-inhibitory receptors, and a package insert comprising
instructions for using the agent that decreases or inhibits TIGIT
expression and/or activity and the agent that decreases or inhibits
one or more additional immune co-inhibitory receptors to enhance
immune function of an individual having cancer. In other aspects,
the present disclosure provides a kit comprising an agent that
decreases or inhibits one or more additional immune co-inhibitory
receptors and a package insert comprising instructions for using
the agent that decreases or inhibits one or more additional immune
co-inhibitory receptors in combination with an agent that decreases
or inhibits TIGIT expression and/or activity to enhance immune
function of an individual having cancer. In certain embodiments
that may be combined with any of the preceding embodiments, the one
or more additional immune co-inhibitory receptor is selected from
the group consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA,
B7H4, and CD96. In certain embodiments that may be combined with
any of the preceding embodiments, the one or more additional immune
co-inhibitory receptor is selected from the group consisting of
PD-1, CTLA-4, LAG3 and TIM3.
[0111] In other aspects, the present disclosure provides a kit
comprising an agent that decreases or inhibits TIGIT expression
and/or activity and a package insert comprising instructions for
using the agent that decreases or inhibits TIGIT expression and/or
activity in combination with an agent that increases or activates
one or more additional immune co-stimulatory receptors to treat or
delay progression of cancer in an individual. In other aspects, the
present disclosure provides a kit comprising an agent that
decreases or inhibits TIGIT expression and/or activity and an agent
that increases or activates one or more additional immune
co-stimulatory receptors, and a package insert comprising
instructions for using the agent that decreases or inhibits TIGIT
expression and/or activity and the agent that increases or
activates one or more additional immune co-stimulatory receptors to
treat or delay progression of cancer in an individual. In other
aspects, the present disclosure provides a kit comprising an agent
that increases or activates one or more additional immune
co-stimulatory receptors and a package insert comprising
instructions for using the agent that increases or activates one or
more additional immune co-stimulatory receptors in combination with
an agent that decreases or inhibits TIGIT expression and/or
activity to treat or delay progression of cancer in an individual.
In other aspects, the present disclosure provides a kit comprising
an agent that decreases or inhibits TIGIT expression and/or
activity and a package insert comprising instructions for using the
agent that decreases or inhibits TIGIT expression and/or activity
in combination with an agent that increases or activates one or
more additional immune co-stimulatory receptors to enhance immune
function of an individual having cancer. In other aspects, the
present disclosure provides a kit comprising an agent that
decreases or inhibits TIGIT expression and/or activity and an agent
that increases or activates one or more additional immune
co-stimulatory receptors, and a package insert comprising
instructions for using the agent that decreases or inhibits TIGIT
expression and/or activity and the agent that increases or
activates one or more additional immune co-stimulatory receptors to
enhance immune function of an individual having cancer. In other
aspects, the present disclosure provides a kit comprising an agent
that increases or activates one or more additional immune
co-stimulatory receptors and a package insert comprising
instructions for using the agent that increases or activates one or
more additional immune co-stimulatory receptors in combination with
an agent that decreases or inhibits TIGIT expression and/or
activity to enhance immune function of an individual having cancer.
In certain embodiments that may be combined with any of the
preceding embodiments, the or more additional immune co-stimulatory
receptor is selected from the group consisting of CD226, OX-40,
CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4. In
certain embodiments that may be combined with any of the preceding
embodiments, the one or more additional immune co-stimulatory
receptor is selected from the group consisting of CD226, OX-40,
CD27, CD137, HVEM and GITR. In certain embodiments that may be
combined with any of the preceding embodiments, the one or more
additional immune co-stimulatory receptor is selected from the
group consisting of OX-40 and CD27.
[0112] In certain embodiments that may be combined with any of the
preceding embodiments, the individual is a human. In certain
embodiments that may be combined with any of the preceding
embodiments, the agent that decreases or inhibits TIGIT expression
and/or activity is selected from the group consisting of an
antagonist of TIGIT expression and/or activity, an antagonist of
PVR expression and/or activity, an agent that inhibits and/or
blocks the interaction of TIGIT with PVR, an agent that inhibits
and/or blocks the interaction of TIGIT with PVRL2, an agent that
inhibits and/or blocks the interaction of TIGIT with PVRL3, an
agent that inhibits and/or blocks the intracellular signaling
mediated by TIGIT binding to PVR, an agent that inhibits and/or
blocks the intracellular signaling mediated by TIGIT binding to
PVRL2, and an agent that inhibits and/or blocks the intracellular
signaling mediated by TIGIT binding to PVRL3. In certain
embodiments that may be combined with any of the preceding
embodiments, the antagonist of TIGIT expression and/or activity is
an anti-TIGIT antibody or antigen-binding fragment thereof. In
certain embodiments that may be combined with any of the preceding
embodiments, the agent that modulates CD226 expression and/or
activity is an agent that increases and/or stimulates CD226
expression and/or activity. In certain embodiments that may be
combined with any of the preceding embodiments, the agent that
modulates CD226 expression and/or activity is an agent that
increases and/or stimulates the interaction of CD226 with PVR. In
certain embodiments that may be combined with any of the preceding
embodiments, the agent that modulates CD226 expression and/or
activity is an agent that increases and/or stimulates the
intracellular signaling mediated by CD226 binding to PVR. In
certain embodiments that may be combined with any of the preceding
embodiments, the agent that modulates CD226 expression and/or
activity is selected from the group consisting of an agent that
inhibits and/or blocks the interaction of CD226 with TIGIT, an
antagonist of TIGIT expression and/or activity, an antagonist of
PVR expression and/or activity, an agent that inhibits and/or
blocks the interaction of TIGIT with PVR, an agent that inhibits
and/or blocks the interaction of TIGIT with PVRL2, an agent that
inhibits and/or blocks the interaction of TIGIT with PVRL3, an
agent that inhibits and/or blocks the intracellular signaling
mediated by TIGIT binding to PVR, an agent that inhibits and/or
blocks the intracellular signaling mediated by TIGIT binding to
PVRL2, and an agent that inhibits and/or blocks the intracellular
signaling mediated by TIGIT binding to PVRL3. In certain
embodiments that may be combined with any of the preceding
embodiments, the agent that modulates CD226 expression and/or
activity is an agent that inhibits and/or blocks the interaction of
CD226 with TIGIT. In certain embodiments that may be combined with
any of the preceding embodiments, the agent that inhibits and/or
blocks the interaction of CD226 with TIGIT is a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, or an inhibitory
polypeptide. In certain embodiments that may be combined with any
of the preceding embodiments, the agent that inhibits and/or blocks
the interaction of CD226 with TIGIT is an anti-TIGIT antibody or
antigen-binding fragment thereof. In certain embodiments that may
be combined with any of the preceding embodiments, the anti-TIGIT
antibody or antigen-binding fragment thereof comprises at least one
HVR comprising an amino acid sequence selected from the amino acid
sequences (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ ID
NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),
FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6); or
(2) RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8),
LQGTHQPPT (SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10),
LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and GLRGFYAMDY (SEQ ID NO:12). In
certain embodiments that may be combined with any of the preceding
embodiments, the anti-TIGIT antibody or antigen-binding fragment
thereof, wherein the antibody light chain comprises the amino acid
sequence set forth in
TABLE-US-00018 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQS
PKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGI NNPLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSP
QLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTH
QPPTFGPGTKLEVK.
In certain embodiments that may be combined with any of the
preceding embodiments, the anti-TIGIT antibody or antigen-binding
fragment thereof, wherein the antibody heavy chain comprises the
amino acid sequence set forth in
TABLE-US-00019 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR
RPLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIG
LIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSR
GLRGFYAMDYWGQGTSVTVSS.
In certain embodiments that may be combined with any of the
preceding embodiments, the anti-TIGIT antibody or antigen-binding
fragment thereof, wherein the antibody light chain comprises the
amino acid sequence set forth in
TABLE-US-00020 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQS
PKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGI NNPLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSP
QLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTH
QPPTFGPGTKLEVK,
and the antibody heavy chain comprises the amino acid sequence set
forth in
TABLE-US-00021 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR
RPLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIG
LIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSR
GLRGFYAMDYWGQGTSVTVSS.
BRIEF DESCRIPTION OF THE DRAWINGS
[0113] FIGS. 1A-1D show that TIGIT is highly expressed on exhausted
CD8+ and CD4+ T cells. FIG. 1A depicts MACS-enriched C57BL6/J
splenic CD8+ T cells that were stimulated with plate-bound anti-CD3
and anti-CD28 for 24-48 hours in vitro. Flow cytometry histograms
representative of TIGIT expression (red) relative to isotype
staining (gray). Quantitation of TIGIT MFI is also shown. ***,
P<0.001. Data are representative of 2 independent experiments;
n=3. In FIGS. 1B-1C, C57BL6/J mice were infected with Armstrong
strain LCMV, and splenocytes were analyzed 7 days after infection.
Data are representative of 2 independent experiments; n=5. FIG. 1B
shows flow cytometry histogram representative of TIGIT expression
by naive (CD44.sup.low CD62L.sup.high) and effector memory
(CD44.sup.high CD62L.sup.low) CD4+ and CD8+ T cells. Quantitation
of TIGIT MFI is also shown. ***, P<0.001. FIG. 1C shows flow
cytometry histogram representative of TIGIT expression by
PD-1.sup.high and PD-1.sup.low effector memory CD8+ T cells.
Quantitation of TIGIT MFI is also shown. ***, P<0.001. FIG. 1D
shows that C57BL6/J mice were briefly depleted of CD4+ T cells and
infected with Clone 13 strain LCMV. Splenocytes were analyzed 42
days after infection. Flow cytometry histogram representative of
TIGIT expression by naive (CD44.sup.low CD62L.sup.high), central
memory (CD44.sup.high CD62L.sup.high), and effector memory
(CD44.sup.high CD62L.sup.low) CD8+ T cells. Quantitation of TIGIT
MFI is also shown. ***, P<0.001. Data are representative of 2
independent experiments; n=5. Error bars depict the standard error
of the mean.
[0114] FIG. 2 shows the design of TIGIT.sup.loxP/loxP mice. Exon 1
of TIGIT was flanked by loxP sites using standard techniques.
[0115] FIGS. 3A-3D show that TIGIT-deficient CD8.sup.+ and
CD4.sup.+ T cells respond normally to acute viral infection.
TIGIT.sup.fl/fl CD4.sup.cre (CKO) and TIGIT.sup.fl/fl littermates
(WT) were infected with Armstrong strain LCMV. Splenocytes were
analyzed 7 days after infection. Data are representative of two
independent experiments; n=5. FIG. 3A shows representative FACS
plots gated on CD8.sup.+ T cells, with activated (CD44.sup.high)
cells boxed. Quantitation of activated CD8.sup.+ T cells as a
percentage of total CD8.sup.+ T cells. FIG. 3B shows representative
FACS plots gated on CD8.sup.+ T cells after stimulation in vitro,
with IFN.gamma.-producing cells boxed. Quantitation of
IFNg-producing cells as a percentage of total CD8.sup.+ T cells.
FIG. 3C shows representative FACS plots gated on CD4.sup.+ T cells,
with activated (CD44.sup.high) cells boxed. Quantitation of
activated CD4.sup.+ T cells as a percentage of total CD4.sup.+ T
cells. FIG. 3D shows representative FACS plots gated on CD4.sup.+ T
cells after stimulation in vitro, with IFNg-producing cells boxed.
Quantitation of IFNg-producing cells as a percentage of total
CD4.sup.+ T cells. Error bars depict the standard error of the
mean.
[0116] FIGS. 4A-4H show that TIGIT and PD-1 synergistically
regulate the effector function of exhausted T cells in vivo. In
FIGS. 4A-4E, TIGIT.sup.fl/fl CD4-cre- (WT) and TIGIT.sup.fl/fl
CD4-cre+ (CKO) mice were briefly depleted of CD4+ T cells and
infected with Clone 13 strain LCMV. Splenocytes and liver viral
titers were analyzed 42 days after infection. Data are
representative of 2 independent experiments, and n=6-9 per group.
FIG. 4A depicts representative FACS plots gated on CD8+ T cells,
with activated cells (CD44.sup.high CD62L.sup.low) boxed.
Quantitation of activated cells as a percentage of total CD8+ T
cells. FIG. 4B depicts representative FACS plots gated on CD8+ T
cells after stimulation in vitro, with IFN.gamma.+ cells boxed.
Quantitation of IFN.gamma.-producing cells as a percentage of CD8+
T cells. FIG. 4C depicts representative FACS plots gated on CD4+ T
cells, with activated cells (CD44.sup.high CD62L.sup.low) boxed.
Quantitation of activated cells as a percentage of total CD4+ T
cells. FIG. 4D depicts representative FACS plots gated on CD4+ T
cells after stimulation in vitro, with IFN.gamma.+ cells boxed.
Quantitation of IFN.gamma.-producing cells as a percentage of CD4+
T cells. FIG. 4E depicts quantitation of liver LCMV titers. ***,
P<0.0001. In FIGS. 4F-41I, C57BL6/J mice were briefly depleted
of CD4+ T cells and infected with Clone 13 strain LCMV. Mice were
treated with isotype-matched control, anti-PD-L1, anti-TIGIT, or
anti-PD-L1+ anti-TIGIT antibodies starting 28 days after infection.
Splenocytes and liver viral titers were analyzed 42 days after
infection. Data are representative of 2 independent experiments;
n=10. FIG. 4F depicts representative FACS plots gated on CD8+ T
cells, with activated cells (CD44.sup.high CD62L.sup.low) boxed.
Quantitation of activated cells as a percentage of total CD8+ T
cells. ***, P<0.0001. FIG. 4G depicts representative FACS plots
gated on activated CD8+ T cells after stimulation in vitro, with
IFN.gamma.+ cells boxed. Quantitation of IFN.gamma.-producing cells
as a percentage of activated CD8+ T cells. *. P=0.0352. **,
P=0.0047. FIG. 4H depicts quantitation of liver LCMV titers. *,
P=0.0106. **, P=0.0047. Error bars depict the standard error of the
mean.
[0117] FIGS. 5A-5B show that TIGIT/PD-L1 co-blockade enhances CD4+
T cell effector function during chronic viral infection. C57BL6/J
mice were depleted of CD4+ T cells and infected with Clone 13
strain LCMV. Mice were treated with isotype control, anti-PD-L1,
anti-TIGIT, or anti-PD-L1+ anti-TIGIT antibodies from 28 days after
infection. Splenocytes and liver viral titers were analyzed 42 days
after infection. Data are representative of 2 independent
experiments; n=10. FIG. 5A depicts representative FACS plots gated
on CD4+ T cells, with activated cells (CD44.sup.high CD62L.sup.low)
boxed. Quantitation of activated CD4+ T cells as a percentage of
total CD4+ T cells. FIG. 5B depicts representative FACS plots gated
on CD4+ T cells after stimulation in vitro, with
IFN.gamma.-producing cells boxed. Quantitation of
IFN.gamma.-producing cells as a percentage of total CD4+ T cells.
*, P=0.019. Error bars depict the standard error of the mean.
[0118] FIGS. 6A-6D show that TIGIT expression is elevated in human
breast cancer and correlated with expression of CD8 and inhibitory
co-receptors. Breast cancer gene expression microarray data
generated by the Cancer Gene Atlas Network was analyzed. Gene
expression data is normalized and expressed as relative ratios (log
2). FIG. 6A depicts TIGIT expression in normal and all breast tumor
samples (left) and in breast tumor subtypes (right). ***,
P=6.times.10-12. Box and whisker plots are shown. FIG. 6B depicts
correlation of TIGIT and CD3.epsilon. expression. R2=0.61. FIG. 6C
depicts the correlation of TIGIT and CD8.alpha. (left, R2=0.80) or
CD4 (right, R2=0.42). FIG. 6D depicts the correlation of TIGIT and
PD-1 (left, R.sup.2=0.87), LAG3 (center, R.sup.2=0.80), and CTLA4
(right, R.sup.2=0.76).
[0119] FIGS. 7A-F show that TIGIT and PD-1 inhibit anti-tumor T
cell responses. In FIGS. 7A-7B, BALB/C mice were inoculated with
CT26 colorectal carcinoma cells. Splenocytes and tumor-infiltrating
lymphocytes (TILs) were analyzed 14 days after inoculation, when
tumors had reached approximately 200 mm.sup.3 in size. Data are
representative of one experiment; n=6. FIG. 7A depicts flow
cytometry histogram representative of TIGIT expression by splenic
and tumor-infiltrating CD8+ T cells. Quantitation of TIGIT MFI is
also shown. **, P=0.0023. FIG. 7B depicts flow cytometry histogram
representative of TIGIT expression by splenic and
tumor-infiltrating CD4+ T cells. Quantitation of TIGIT MFI is also
shown. ***, P=0.0002. In FIGS. 7C-7E, BALB/C mice were inoculated
with CT26 colorectal carcinoma cells. When tumors reached
approximately 200 mm3 in size, mice were treated with isotype
control, anti-PD-L1, anti-TIGIT, or anti-PD-L1+ anti-TIGIT
antibodies for three weeks. Data are representative of two
independent experiments; n=10-20 (FIGS. 7C-7D) or 7-10 (FIG. 7E).
FIG. 7C depicts median CT26 tumor volumes over time. FIG. 7D
depicts mouse survival. FIG. 7E shows that approximately 60 days
after initial inoculation, mice in complete remission (CR) that had
received anti-TIGIT+ anti-PD-L1, as well as naive BALB/c mice, were
inoculated with CT26 cells in their left thoracic flanks and
inoculated with EMT6 breast carcinoma cells in their mammary fat
pads. Median (left) and individual (right) tumor volumes for CT26
(squares) and EMT6 (triangles) in CR mice (purple and green) and
naive mice (black and orange) tumors are shown. FIG. 7F shows that
mice were inoculated with CT26 tumors and treated as in FIG. 7C.
Tumor-infiltrating and tumor-draining lymph node resident T cells
were analyzed by flow cytometry. Representative FACS plots of CD8+
TILs after stimulation in vitro, with IFN.gamma.-producing cells
boxed. Quantitation of IFN.gamma.-producing CD8+ TILs as a
percentage of total CD8+ TILs. ***, P=0.0003. Data are
representative of two independent experiments; n=5. Error bars
depict the standard error of the mean.
[0120] FIGS. 8A-8B show that CT26 tumor-infiltrating lymphocyte
TIGIT expression is correlated with Tim-3 expression. BALB/C mice
were inoculated with CT26 colorectal carcinoma cells. Splenocytes
and tumor-infiltrating lymphocytes (TILs) were analyzed
approximately 14 days after inoculation, when tumors had reached
approximately 200 mm.sup.3 in size. Data are representative of one
experiment; n=6. FIG. 8A depicts representative histogram of TIGIT
expression by splenic and tumor-infiltrating CD8.sup.+ T cells.
Quantitation of TIGIT MFI. **, P=0.0026. FIG. 8B depicts
representative histogram of TIGIT expression by splenic and
tumor-infiltrating CD4.sup.+ T cells. Quantitation of TIGIT MFI.
***, P<0.0001. Error bars depict the standard error of the
mean.
[0121] FIGS. 9A-9B show that MC38 tumor-infiltrating lymphocyte
TIGIT expression is correlated with PD-1 and Tim-3 expression.
C57BL6/J mice were inoculated with MC38 colorectal carcinoma cells.
Splenocytes and tumor-infiltrating lymphocytes (TILs) were analyzed
approximately 14 days after inoculation, when tumors had reached
approximately 200 mm.sup.3 in size. Data are representative of one
experiment; n=5. FIG. 9A depicts representative histogram of TIGIT
expression by splenic and tumor-infiltrating CD8.sup.+ T cells.
Quantitation of TIGIT MFI. ***, P<0.0001. FIG. 9B depicts
representative histogram of TIGIT expression by splenic and
tumor-infiltrating CD4.sup.+ T cells. Quantitation of TIGIT MFI. *,
P=0.0136. **, P=0.0029. Error bars depict the standard error of the
mean.
[0122] FIG. 10 shows CT26 tumor growth in mice treated with
anti-PD-L1 and/or anti-TIGIT. Naive BALB/c mice were inoculated
with CT26 tumor cells and treated with anti-PD-L1 and/or anti-TIGIT
or isotype-matched control antibodies, as described in FIGS. 4D-4F.
Tumor volumes over time for individual mice in each treatment group
are shown. Data are representative of two independent
experiments.
[0123] FIGS. 11A-11F show the flow cytometric analysis of CD4.sup.+
TILs and tumor-draining lymph node T cells. BALB/C mice were
inoculated with CT26 colorectal carcinoma cells. When tumors
reached approximately 200 mm.sup.3 in size, mice were treated with
isotype control, anti-PD-L1, anti-TIGIT, or anti-PD-L1+ anti-TIGIT
antibodies for 7 days. Tumors and tumor-draining lymph nodes were
harvested. Data are representative of two independent experiments;
n=5. Representative FACS plots gated on tumor-draining lymph node
CD8.sup.+ T cells after stimulation in vitro, with
IFN.gamma.-producing cells boxed. Quantitation of IFN.gamma..sup.+
cells as a percentage of total CD8.sup.+ T cells. ***, P<0.001.
Quantitation of CD8.sup.+ T cells as a percentage of total TILs.
**, P=0.0065. Quantitation of activated (CD44.sup.high
CD62L.sup.low) CD8.sup.+ T cells as a percentage of total CD8.sup.+
TILs. *, P=0.012. Quantitation of CD8.sup.+ T cells as a percentage
of total tumor-draining lymph node cells. Quantitation of activated
CD8.sup.+ T cells as a percentage of total CD8.sup.+ T cells in the
tumor-draining lymph node. *, P<0.05. FIG. 11C depicts
quantitation of CD4.sup.+ T cells as a percentage of total TILs. *,
P=0.016. FIG. 11D depicts quantitation of activated CD4.sup.+ T
cells as a percentage of total CD4.sup.+ TILs. FIG. 11E depicts
quantitation of CD4.sup.+ T cells as a percentage of total
tumor-draining lymph node cells. FIG. 11F depicts quantitation of
activated CD4.sup.+ T cells as a percentage of total CD4.sup.+ T
cells in the tumor-draining lymph node. FIG. 11A depicts
quantitation of IFN.gamma..sup.+ cells as a percentage of CD4.sup.+
TILs after stimulation in vitro. FIG. 11B depicts quantitation of
IFN.gamma..sup.+ cells as a percentage of CD4.sup.+ T cells in the
tumor-draining lymph node after stimulation in vitro. Error bars
depict the standard error of the mean.
[0124] FIGS. 12A-12C show further flow cytometric analysis of
CD8.sup.+ TILs. BALB/C mice were inoculated with CT26 colorectal
carcinoma cells and treated with isotype control, anti-PD-L1,
anti-TIGIT, or anti-PD-L1+ anti-TIGIT antibodies as described in
FIG. 4. Tumors were harvested after 7 days of treatment and
analyzed by flow cytometry. Data are representative of two
independent experiments; n=5. FIG. 12A depicts quantitation of
TNF.alpha..sup.+ cells as a percentage of total CD8.sup.+ TILs. **,
P<0.01. FIG. 12B depicts quantitation of CD8.sup.+ TILs as a
percentage of total TILs. **, P<0.01. FIG. 12C depicts
quantitation of activated (CD44.sup.high CD62L.sup.low) CD8.sup.+
TILs as a percentage of total CD8.sup.+ TILs. *, P<0.05. Error
bars depict the standard error of the mean.
[0125] FIGS. 13A-13D show the flow cytometric analysis of
tumor-draining lymph node resident CD8.sup.+ T cells. BALB/C mice
were inoculated with CT26 colorectal carcinoma cells and treated
with isotype control, anti-PD-L1, anti-TIGIT, or anti-PD-L1+
anti-TIGIT antibodies as described in FIGS. 4A-4H. Tumor-draining
lymph nodes were harvested after 7 days of treatment and analyzed
by flow cytometry. Data are representative of two independent
experiments; n=5. FIG. 13A depicts representative FACS plots gated
on tumor-draining lymph node resident CD8.sup.+ T cells after
stimulation in vitro, with IFN.gamma.-producing cells boxed.
Quantitation of IFN.gamma..sup.+ cells as a percentage of total
CD8.sup.+ T cells. ***, P<0.001. FIG. 13B depicts quantitation
of CD8.sup.+ T cells as a percentage of total cells in the
tumor-draining lymph node. FIG. 13C depicts quantitation of
activated (CD44.sup.high CD62L.sup.low) CD8.sup.+ T cells as a
percentage of total CD8.sup.+ T cells. *, P<0.05. Error bars
depict the standard error of the mean. FIG. 13D depicts
quantitation of TNF.alpha.-producing cells as a percentage of total
tumor-draining lymph node CD8.sup.+ T cells.
[0126] FIG. 14 shows co-expression of CD226 and TIGIT by
tumor-infiltrating CD8+ T cells. C57BL6/J mice were inoculated with
MC38 colorectal carcinoma cells. Splenocytes and tumor-infiltrating
lymphocytes (TILs) were analyzed approximately 14 days after
inoculation, when tumors had reached approximately 200 mm.sup.3 in
size. Representative histogram of CD226 expression by splenic B
cells (gray), splenic CD8+ T cells (blue), and TIGIT+
tumor-infiltrating CD8+ T cells (red). Data are representative of
two independent experiments; n=5.
[0127] FIG. 15 shows CD226 and TIGIT Co-Immunoprecipate (co-IP) on
transfected cells. COST cells were co-transfected with expression
plasmids containing the cDNA for either TIGIT-HA (5 ng) or
CD226-Flag (10 ng) tagged proteins, or a control plasmid (pRK).
Following transfection, the cells were washed and centrifuged and
cell pellets lysed. The resultant supernatant was pre-cleared and
centrifuged and then equally split into two tubes and
immuno-precipitated with either an anti-HA or an anti-flag using
standard procedures. The immune-precipitated proteins were
subjected to SDS-PAGE and western blotted. Western blots were
probed with either anti-Flag-HRP or anti-HA-HRP.
[0128] FIG. 16 shows TIGIT and CD226 interact in primary CD8+ T
cells. MACS-enriched splenic C57BL6/J CD8+ T cells were stimulated
with plate-bound anti-CD3 and anti-CD28 antibodies and recombinant
IL-2 for 48 hours and lysed. Cell lysates were immunoprecipitated
with anti-TIGIT and probed with anti-CD226. Lanes: molecular weight
ladder (1), input (2), co-immunoprecipitation flow-through (3), and
co-immunoprecipitate. Arrow denotes the expected molecular weight
of CD226.
[0129] FIGS. 17A-17D show the detection of TIGIT/CD226 interaction
by TR-FRET. FIG. 17A depicts the dissociation of Flag-ST-CD226
homodimers by HA-TIGIT. FRET ratio between Flag-ST-CD226 measured
on COS-7 cells expressing a constant amount of Flag-ST-CD226 and
increasing concentrations of HA-TIGIT. FIG. 17B depicts FRET ratio
between Flag-ST-CD226 recorded after a 15-min incubation of either
PBS (white bar) or anti-TIGIT antibody (black bar). FIG. 17C
depicts the association of Flag-ST-CD226 with HA-TIGIT. FRET
intensity between Flag-ST-CD226 and HA-TIGIT over the Flag-ST-CD226
expression as measured by an anti-Flag ELISA on the same batch of
transfected COS-7 cells. FIG. 17D depicts FRET variation between
Flag-ST-CD226 and HA-TIGIT after a 15-min incubation of PBS (white
bar) or anti-TIGIT antibody (black bar). Data in A and C are
representative of 4 independent experiments, each performed in
triplicate. Data in B and D are representative of 2 independent
experiments, each performed in triplicate.
[0130] FIG. 18 shows cell surface expression of Flag-ST-CD226 and
HA-TIGIT. Anti-Flag and anti-HA ELISA on intact COS-7 cells
expressing the indicated tagged-constructs. Data are representative
of 3 independent experiments, each performed in triplicate.
[0131] FIGS. 19A-19D show that CD226 blockade reverses the enhanced
anti-viral T cell response induced by TIGIT/PD-L1 co-blockade. In
FIGS. 19A-19D, C57BL6/J mice were briefly depleted of CD4.sup.+ T
cells and infected with Clone 13 strain LCMV. Mice were treated
with isotype-matched control, anti-CD226, anti-PD-L1+ anti-TIGIT,
or anti-PD-L1+ anti-TIGIT+ anti-CD226 antibodies starting 28 days
after infection. Splenocytes and liver viral titers were analyzed
42 days after infection. FIG. 19A depicts quantitation of CD8.sup.+
T cells as a percentage of splenocytes. FIG. 19B depicts
quantitation of activated CD8.sup.+ T cells as a percentage of
total CD8.sup.+ T cells. ***, P<0.001. FIG. 19C depicts
quantitation of IFNg-producing cells as a percentage of activated
CD8.sup.+ T cells. ***, P<0.001. FIG. 19D depicts quantitation
of liver LCMV titers. ***, P<0.001. Error bars depict the
standard error of the mean.
[0132] FIGS. 20A-20H show that TIGIT expression is elevated in
human cancer and strongly correlated with CD8 and PD-1. Gene
expression analyses of human cancers were performed as described in
Example 11. Scatter plots show per-gene count data, normalized by
library size. Box and whisker plots show the variance stabilized
expression ratio of TIGIT and CD3e. FIG. 20A depicts the
correlation of TIGIT and CD3e RNA expression in LUSC (grey) and
normal lung (black). .rho.=0.86. Quantification of TIGIT/CD3e
expression ratios is also shown. LUSC ratio increase=372%. ***,
P=1.46.times.10.sup.-46. FIG. 20B depicts the correlation of TIGIT
and CD3e RNA expression in COAD (grey) and normal colon (black).
.rho.=0.83. Quantification of TIGIT/CD3e expression ratios is also
shown. COAD ratio increase=116%. ***, P=3.66.times.10.sup.-6. FIG.
20C depicts the correlation of TIGIT and CD3e RNA expression in
UCEC (grey) and normal uterine endrometrium (black). .rho.=0.87.
Quantification of TIGIT/CD3e expression ratios is also shown. UCEC
ratio increase=419%. ***, P=7.41.times.10.sup.-5. FIG. 20D depicts
the correlation of TIGIT and CD3e RNA expression in BRCA (grey) and
normal breast (black). .rho.=0.82. Quantification of TIGIT/CD3e
expression ratios is also shown. BRCA ratio increase=313%. ***,
P=4.6.times.10.sup.-44. FIG. 20E depicts the correlation of TIGIT
and CD3e RNA expression in kidney renal clear cell carcinoma (grey)
and normal kidney (black). .rho.=0.94. Quantification of TIGIT/CD3e
expression ratios is also shown. FIG. 20F depicts the correlation
of TIGIT and CD8A (left) or TIGIT and CD4 (right) in lung squamous
cell carcinoma (grey) and normal lung (black). .rho.=0.77 and 0.48
respectively. FIG. 20G depicts the correlation of TIGIT and PD-1
(Pdcd1) in lung squamous cell carcinoma (grey) and normal lung
(black). .rho.=0.82. FIG. 20H depicts the correlation of TIGIT and
CD226 in lung squamous cell carcinoma (red) and normal lung
(black). .rho.=0.64.
[0133] FIG. 21 shows the analysis of T cell-associated gene
expression in Lung Squamous Cell Carcinoma (LUSC). Gene expression
in LUSC and normal tissue samples was analyzed as described in
Example 11 and a heat map of the genes best correlated with the
gene signature in LUSC samples was generated. Genes and samples
were both clustered using hierarchical clustering using Ward
linkage on the Euclidean distance matrix for the centered and
scaled expression data.
[0134] FIGS. 22A-22G show that TIGIT and PD-1 are coordinately
expressed by human and murine tumor-infiltrating lymphocytes. FIGS.
22A-22C shows analysis of lymphocytes from a freshly resected human
NSCLC tumor, tumor-matched peripheral blood, and normal donor
peripheral blood. Data are representative of two independently
analyzed tumors. FIG. 22A depicts representative FACS plots
representative of TIGIT expression by peripheral and
tumor-infiltrating CD8.sup.+ T cells, with TIGIT.sup.+ cells boxed.
FIG. 22B depicts representative FACS plots representative of TIGIT
expression by peripheral and tumor-infiltrating CD4.sup.+ T cells,
with TIGIT.sup.+ cells boxed. FIG. 22C depicts flow cytometry
histogram representative of TIGIT expression by PD-1.sup.high (red)
and PD-1.sup.low (blue) NSCLC-infiltrating CD8.sup.+ (left) and
CD4.sup.+ (right) T cells. In FIGS. 22D-22G, BALB/C mice were
inoculated with syngeneic CT26 colorectal carcinoma cells.
Splenocytes and tumor-infiltrating lymphocytes (TILs) were analyzed
14 days after inoculation, when tumors had reached approximately
200 mm.sup.3 in size. Data are representative of two independent
experiments; n=5-6. FIG. 22D depicts representative FACS plot of
TIGIT expression by tumor-infiltrating CD8.sup.+ T cells, with
TIGIT cells boxed. FIG. 22E depicts representative FACS plot of
TIGIT expression by tumor-infiltrating CD4.sup.+ T cells, with
TIGIT.sup.+ cells boxed. Quantitation of the frequency of TIGIT T
cells as a percentage of all T cells. *, P=0.0134. ***,
P<0.0001. FIG. 22F depicts flow cytometry histogram
representative of TIGIT expression by PD-1.sup.high and
PD-1.sup.low tumor-infiltrating CD8.sup.+ T cells and by splenic
CD8.sup.+ T cells. Quantitation of TIGIT MFI is also shown. **,
P=0.0023. FIG. 22G depicts flow cytometry histogram representative
of TIGIT expression by PD-1.sup.high and PD-1.sup.low
tumor-infiltrating CD4.sup.+ T cells and by splenic CD4.sup.+ T
cells. Quantitation of TIGIT MFI is also shown. ***, P=0.0002.
Error bars depict the standard error of the mean.
[0135] FIGS. 23A-23D show the characterization of TIGIT expression
by human tumor-infiltrating T cells. FIGS. 23A-23B depict FACS
plots showing TIGIT expression by NSCLC tumor-infiltrating CD8+ and
CD4+ T cells (FIG. 23A) and by donor-matched PBMC CD8+ and CD4+ T
cells (FIG. 23B), with TIGIT+ cells boxed. FIGS. 23C-23D depict
FACS plots showing TIGIT expression by CRC tumor-infiltrating CD8+
and CD4+ T cells (FIG. 23C) and by donor-matched PBMC CD8+ and CD4+
T cells (FIG. 23D), with TIGIT+ cells boxed.
[0136] FIG. 24 shows that the TIGIT:CD226 interaction is not driven
by PVR TIGIT:CD226 and TIGIT Q56R:CD226 interactions were detected
by TR-FRET and the FRET ratio between Flag-ST-CD226 and HA-TIGIT or
HA-TIGIT Q56R shows that WT and Q56R TIGIT bind CD226 with the same
efficacy. Data are representative of three independent experiments
performed in triplicate.
[0137] FIGS. 25A-25C show the efficacy of TIGIT/PD-L1 antibody
co-blockade in mice bearing MC38 tumors. In FIGS. 25A-25C, MC38
tumor-bearing mice were generated as above and treated with
blocking antibodies against PD-L1 (red), TIGIT (blue), TIGIT and
PD-L1 (purple) or isotype-matched control antibodies (black) for
three weeks. N=10 (control, anti-PD-L1 alone, anti-TIGIT alone) or
20 (anti-TIGIT+anti-PD-L1). FIG. 25A depicts median (left) and
individual (right) MC38 tumor volumes over time. FIG. 25B depicts
MC38 tumor volumes after 14 days of antibody treatment. ***,
P=0.0005. **, P=0.0093. *, P=0.0433. FIG. 25C depicts mouse
survival over time. Error bars depict the standard error of the
mean.
[0138] FIGS. 26A-26E show the further characterization of TIGIT
expression by murine tumor-infiltrating T cells. FIG. 26A depicts
that splenic C57BL6/J CD8.sup.+ T cells were enriched by MACS and
cultured with plate-coated anti-CD3 and anti-CD28 agonist
antibodies. Representative histograms of TIGIT (red) and
isotype-matched control (solid gray) staining over time.
Quantitation of TIGIT MFI. ***, P<0.001. Stimulated cells
inducibly expressed PD-1 and constitutively expressed CD226 (data
not shown). Data are representative of two independent experiments;
n=5. In FIGS. 26B-26E, wildtype C57BL6/J mice were subcutaneously
inoculated with syngeneic MC38 colorectal carcinoma cells. Tumors
were allowed to grow without intervention until they reached
150-200 mm.sup.3 in size. Data are representative of two
independent experiments; n=5. FIG. 26B depicts representative FACS
plot of tumor-infiltrating CD8.sup.+ T cells, with TIGIT cells
boxed. Quantitation of the frequency of TIGIT cells as a percentage
of all tumor-infiltrating or splenic CD8.sup.+ T cells. ***,
P<0.0001. FIG. 26C depicts representative FACS plot of
tumor-infiltrating CD4.sup.+ T cells, with TIGIT cells boxed.
Quantitation of the frequency of TIGIT.sup.+ cells as a percentage
of all tumor-infiltrating or splenic CD4.sup.+ T cells. ***,
P<0.0001. FIG. 26D depicts representative histogram of TIGIT
expression by PD-1.sup.high and PD-1.sup.low tumor-infiltrating
CD8.sup.+ T cells (red and blue, respectively) and by splenic
CD8.sup.+ T cells (gray). Quantitation of TIGIT MFI. ***,
P<0.0001. FIG. 26E depicts representative histogram of TIGIT
expression by PD-1.sup.high and PD-1.sup.low tumor-infiltrating
CD4.sup.+ T cells and by splenic CD4.sup.+ T cells. Quantitation of
TIGIT MFI. *, P=0.0136. **, P=0.0029. Error bars depict the
standard error of the mean.
[0139] FIGS. 27A-27B show that tumor-infiltrating CD8.sup.+ and
CD4.sup.+ T cells maintain a high level of CD226 expression.
Wildtype BALB/c mice were inoculated with CT26 tumor cells as
described herein. After tumors have grown to approximately 150-200
mm.sup.3 in size, tumors and spleens were analyzed by flow
cytometry. FIG. 27A depicts quantitation of CD226.sup.+ CD8.sup.+ T
cells, CD4.sup.+ T cells, and non-T cells, as a percentage of all
CD8.sup.+ T cells, CD4.sup.+ T cells, and non-T cells respectively.
FIG. 27B depicts representative histograms of CD226 expression in
tumor and spleen. Data are representative of two independent
experiments; n=5. Error bars depict the standard error of the
mean.
[0140] FIGS. 28A-28F show that TIGIT suppression of CD8.sup.+ T
cell responses is dependent on CD226. BALB/C mice were
subcutaneously inoculated with CT26 colorectal carcinoma cells in
their right thoracic flanks. When tumors reached approximately 200
mm.sup.3 in size, mice were treated with isotype control (black),
anti-CD226 (orange), anti-PD-L1 (red), anti-TIGIT+anti-PD-L1
(purple), or anti-TIGIT+anti-PD-L1+anti-CD226 (green) antibodies
for three weeks. Data are representative of one experiment; n=10
(A-B) or 5 (C-F). FIG. 28A depicts median (left) and individual
(right) CT26 tumor volumes over time. FIG. 28B depicts mouse
survival over time. In FIGS. 28C-28F, after 7 days of treatment,
tumor-infiltrating lymphocytes and tumor-draining lymph
node-resident lymphocytes were assessed by flow cytometry. FIG. 28C
depicts quantitation of IFN.gamma.-producing CD8.sup.+ TILs as a
percentage of total CD8.sup.+ TILs after stimulation in vitro. **,
P<0.01. FIG. 28D depicts quantitation of IFN.gamma.-producing
cells as a percentage of total CD8.sup.+ T cells after stimulation
in vitro. *, P<0.05. FIG. 28E depicts quantitation of CD8.sup.+
TILs as a percentage of total TILs. **, P<0.01. FIG. 28F depicts
quantitation of CD8.sup.+ T cells as a percentage of all
tumor-draining lymph node-resident lymphocytes. Error bars depict
the standard error of the mean.
[0141] FIGS. 29A-29H show that TIGIT impairs CD226 function by
directly disrupting CD226 homodimerization. FIG. 29A depicts that
CD8.sup.+ T cells were MACS-enriched from TIGIT.sup.fl/fl
CD4.sup.cre (CKO) and TIGIT.sup.fl/fl CD4.sup.wt (WT) littermates
and stimulated in the presence of anti-CD226 or isotype-matched
control antibodies as indicated. H.sup.3-thymidine uptake is shown
as a ratio of cells cultured with anti-CD3+PVR-Fc to cells cultured
with anti-CD3 alone. **, P=0.0061. ***, P<0.0001. Data are
representative of two independent experiments; n=5. FIG. 29B
depicts that wildtype C57BL6/J CD8.sup.+ T cells were MACS-enriched
and stimulated in the presence of anti-TIGIT, anti-CD226, and/or
isotype-matched control antibodies as indicated. H.sup.3-thymidine
uptake is shown as a ratio of cells cultured with anti-CD3+PVR-Fc
to cells cultured with anti-CD3 alone. ***, P<0.001 in paired t
tests. FIG. 29C depicts that primary human CD8.sup.+ T cells were
MACS-enriched from blood and stimulated with sub-optimal levels of
plate-bound anti-CD3 in the presence or absence of human
recombinant PVR-Fc. Anti-TIGIT antibodies or isotype-matched
control antibodies were added as indicated. Quantitation of
.sup.3H-thymidine uptake. **, P=0.0071 and 0.0014 respectively.
FIG. 29D depicts that CHO cells were transiently transfected with
increasing concentrations of acceptor and donor FLAG-ST-CD226, as
indicated. Quantification of FRET intensity relative to donor
emission. Data are representative of three independent experiments;
n=3. In FIGS. 29E-29F, CHO cells were transiently transfected with
FLAG-ST-CD226 and with increasing concentrations of HA-TIGIT, as
indicated. Data are representative of two or more independent
experiments; n=4. Data are normalized to the maximal signal. FIG.
29E depicts quantification of the CD226:CD226 FRET ratio (FRET
ratio 1). FIG. 29F depicts quantification of the TIGIT:CD226 FRET
ratio (FRET ratio 2). FIG. 29G depicts anti-FLAG (left) and anti-HA
(right) immunoblots performed on either anti-FLAG or anti-HA
immunoprecipitates prepared from COS-7 cells transfected with
either an empty pRK vector or a combination of Flag-CD226 and
HA-TIGIT. Data are representative of two independent experiments.
FIG. 29H depicts quantification of the TIGIT:CD226 FRET ratio after
incubation with PBS (white) or anti-TIGIT antibodies (red). ***,
P<0.001. Data are representative of 4 independent experiments;
n=3. Error bars depict the standard error of the mean.
[0142] FIG. 30 shows that primary human T cells were MACS-enriched
from blood and stimulated with anti-CD3 and anti-CD28. TIGIT.sup.+
and TIGIT.sup.- cells were sorted, rested, re-stimulated, and
labeled for FRET with the antibodies indicated. Data are
representative of two independent experiments. ***, P<0.001.
Error bars depict the standard error of the mean.
[0143] FIGS. 31A-31C show that TIGIT and PD-1 co-blockade does not
restore the effector function of exhausted CD4.sup.+ T cells during
chronic viral infection. FIG. 31A depicts quantitation of CD8.sup.+
T cells as a percentage of all splenocytes. FIG. 31B depicts
quantitation of GP33 Pentamer.sup.+ cells as a percentage of all
splenic CD8.sup.+ T cells. **, P=0.0040. FIG. 31C depicts
representative FACS plots gated on gp33 pentamer.sup.+ CD8.sup.+ T
cells after stimulation in vitro, with IFN.gamma..sup.+ cells
boxed. Quantitation of IFN.gamma.-producing cells as a percentage
of all gp33 pentamer.sup.+ CD8.sup.+ T cells. *, P=0.0319. **,
P=0.0030. Error bars depict the standard error of the mean.
[0144] FIGS. 32A-32C shows that TIGIT/PD-L1 co-blockade efficacy is
dependent on CD8.sup.+ T cells. In FIGS. 32A-32B, wildtype BALB/c
mice were inoculated with CT26 tumors as described in FIGS. 7A-7F.
When tumors reached 100-150 mm.sup.3 in size, mice were temporarily
depleted of CD8.sup.+ T cells and treated with
anti-TIGIT+anti-PD-L1. Data are representative of one experiment;
n=10/group. FIG. 32A depicts median (left) and individual (right)
CT26 tumor volumes over time. FIG. 32B depicts quantitation of CT26
tumor volumes 17 days after the start of treatment. ***, P=0.0004.
In FIG. 32C, wildtype BALB/c mice were inoculated with CT26 tumors
and treated with anti-TIGIT+anti-PD-L1 and subsequently
re-challenged with CT26 tumors with temporary depletion of
CD8.sup.+ T cells at the time of re-challenge. Data are
representative of two independent experiments; n=5. FIG. 32C
depicts median (left) and individual (right) CT26 tumor volumes
over time. Error bars depict the standard error of the mean.
[0145] FIG. 33 shows that PVR expression on tumor cells is
dispensable for TIGIT/PD-L1 co-blockade efficacy. Wildtype BALB/c
mice were inoculated with wildtype or PVR-deficient (PVR.KO) tumors
as described. When tumors reached 150-200 mm.sup.3 in size, mice
were treated with anti-TIGIT+anti-PD-L1 or isotype-matched control
antibodies. Data are representative of one experiment; n=10/group.
FIG. 33 depicts median (left) and individual (right) CT26 tumor
volumes over time.
[0146] FIG. 34 shows the efficacy of TIGIT/PD-L1 antibody
co-blockade in mice bearing EMT6 tumors. EMT6 tumor-bearing mice
were generated as above and treated with blocking antibodies
against PD-L1 (red), TIGIT (blue), TIGIT and PD-L1 (purple) or
isotype-matched control antibodies (black) for three weeks. N=10
(control, anti-PD-L1 alone, anti-TIGIT alone) or 20
(anti-TIGIT+anti-PD-L1). FIG. 34 depicts median (left) and
individual (right) EMT6 tumor volumes over time.
[0147] FIGS. 35A-35B show that TIGIT regulates tumor-infiltrating
CD8.sup.+ T cell effector function. BALB/C mice were subcutaneously
inoculated with CT26 colorectal carcinoma cells in their right
thoracic flanks and treated with anti-PD-L1, anti-TIGIT, or
anti-PD-L1+ anti-TIGIT, as described in FIGS. 7A-7F. Tumor-draining
lymph node (dLN) resident and tumor-infiltrating T cells were
analyzed by flow cytometry 7 days after the start of treatment.
Data are representative of two independent experiments; n=5. FIG.
35A depicts quantitation of IFN.gamma./TNF.alpha. dual-producing
dLN resident CD8.sup.+ and CD4.sup.+ T cells as percentages of
total dLN resident CD8.sup.+ and CD4.sup.+ T cells respectively.
Dual cytokine production by unstimulated T cells is also shown. **,
P=0.002, 0.003, and 0.001 respectively. FIG. 35B depicts
quantitation of IFN.gamma./TNF.alpha. dual-producing
tumor-infiltrating CD8.sup.+ and CD4.sup.+ T cells as percentages
of total tumor-infiltrating CD8.sup.+ and CD4.sup.+ T cells
respectively. Dual cytokine production by unstimulated T cells is
also shown. ***, P<0.0001. Error bars depict the standard error
of the mean.
[0148] FIGS. 36A-36B show the analysis of lymphocytes from resected
human NSCLC tumors, tumor-matched peripheral blood, and normal
donor peripheral blood. Data are pooled from three independently
acquired sets of samples. FIG. 36A depicts quantitation of
TIGIT.sup.+ cells as a percentage of all CD8.sup.+ T cells. *,
P<0.05. FIG. 36B depicts quantitation of TIGIT.sup.+ cells as a
percentage of all CD4.sup.+ T cells.
[0149] FIGS. 37A-37B show the characterization of TIGIT expression
in human tumors. FIG. 37A depicts representative flow cytometry
histograms of TIGIT expression by NSCLC tumor-resident lymphocytes
(red, CD45.sup.+ FSC.sup.low), myeloid cells (blue, CD45.sup.+
FSC.sup.high), and non-hematopoietic cells (green, CD45.sup.-)
relative to subset-matched isotype staining (gray). FIG. 37B
depicts gating strategy for PD-1.sup.high and PD-1.sup.low NSCLC
tumor-infiltrating CD8.sup.+ and CD4.sup.+ T cells.
DETAILED DESCRIPTION OF THE INVENTION
I. General Techniques
[0150] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Sambrook et
al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current
Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,
(2003)); the series Methods in Enzymology (Academic Press, Inc.):
PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A
Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed.
(1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods
in Molecular Biology, Humana Press; Cell Biology: A Laboratory
Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell
Culture (R.I. Freshney), ed., 1987); Introduction to Cell and
Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;
Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.
Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons;
Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain
Reaction, (Mullis et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: A Practical Approach (D. Catty., ed., IRL Press,
1988-1989); Monoclonal Antibodies: A Practical Approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:
Principles and Practice of Oncology (V. T. DeVita et al., eds., J.
B. Lippincott Company, 1993).
II. Definitions
[0151] The term "PD-1 axis binding antagonist" is a molecule that
inhibits the interaction of a PD-1 axis binding partner with either
one or more of its binding partner, so as to remove T-cell
dysfunction resulting from signaling on the PD-1 signaling
axis--with a result being to restore or enhance T-cell function
(e.g., proliferation, cytokine production, target cell killing). As
used herein, a PD-1 axis binding antagonist includes a PD-1 binding
antagonist, a PD-L1 binding antagonist and a PD-L2 binding
antagonist.
[0152] The term "PD-1 binding antagonists" is a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-1 with one or
more of its binding partners, such as PD-L1, PD-L2. In some
embodiments, the PD-1 binding antagonist is a molecule that
inhibits the binding of PD-1 to its binding partners. In a specific
aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to
PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include
anti-PD-1 antibodies, antigen binding fragments thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules
that decrease, block, inhibit, abrogate or interfere with signal
transduction resulting from the interaction of PD-1 with PD-L1
and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces
the negative co-stimulatory signal mediated by or through cell
surface proteins expressed on T lymphocytes mediated signaling
through PD-1 so as render a dysfunctional T-cell less dysfunctional
(e.g., enhancing effector responses to antigen recognition). In
some embodiments, the PD-1 binding antagonist is an anti-PD-1
antibody. In a specific aspect, a PD-1 binding antagonist is
MDX-1106 described herein. In another specific aspect, a PD-1
binding antagonist is Merck 3745 described herein. In another
specific aspect, a PD-1 binding antagonist is CT-011 described
herein. In another specific aspect, a PD-1 binding antagonist is
AMP-224 described herein.
[0153] The term "PD-L1 binding antagonists" is a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-L1 with either
one or more of its binding partners, such as PD-1, B7-1. In some
embodiments, a PD-L1 binding antagonist is a molecule that inhibits
the binding of PD-L1 to its binding partners. In a specific aspect,
the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1
and/or B7-1. In some embodiments, the PD-L1 binding antagonists
include anti-PD-L1 antibodies, antigen binding fragments thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules
that decrease, block, inhibit, abrogate or interfere with signal
transduction resulting from the interaction of PD-L1 with one or
more of its binding partners, such as PD-1, B7-1. In one
embodiment, a PD-L1 binding antagonist reduces the negative
co-stimulatory signal mediated by or through cell surface proteins
expressed on T lymphocytes mediated signaling through PD-L1 so as
to render a dysfunctional T-cell less dysfunctional (e.g.,
enhancing effector responses to antigen recognition). In some
embodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody.
In a specific aspect, an anti-PD-L1 antibody is YW243.55.870
described herein. In another specific aspect, an anti-PD-L1
antibody is MDX-1105 described herein. In still another specific
aspect, an anti-PD-L1 antibody is MPDL3280A described herein. In
another specific aspect, an anti-PD-L1 antibody is MEDI 4736
described herein.
[0154] The term "PD-L2 binding antagonists" is a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-L2 with either
one or more of its binding partners, such as PD-1. In some
embodiments, a PD-L2 binding antagonist is a molecule that inhibits
the binding of PD-L2 to its binding partners. In a specific aspect,
the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1. In
some embodiments, the PD-L2 antagonists include anti-PD-L2
antibodies, antigen binding fragments thereof, immunoadhesins,
fusion proteins, oligopeptides and other molecules that decrease,
block, inhibit, abrogate or interfere with signal transduction
resulting from the interaction of PD-L2 with either one or more of
its binding partners, such as PD-1. In one embodiment, a PD-L2
binding antagonist reduces the negative co-stimulatory signal
mediated by or through cell surface proteins expressed on T
lymphocytes mediated signaling through PD-L2 so as render a
dysfunctional T-cell less dysfunctional (e.g., enhancing effector
responses to antigen recognition). In some embodiments, a PD-L2
binding antagonist is an immunoadhesin.
[0155] The term "aptamer" refers to a nucleic acid molecule that is
capable of binding to a target molecule, such as a polypeptide. For
example, an aptamer of the invention can specifically bind to a
TIGIT polypeptide, or to a molecule in a signaling pathway that
modulates the expression of TIGIT. The generation and therapeutic
use of aptamers are well established in the art. See, e.g., U.S.
Pat. No. 5,475,096, and the therapeutic efficacy of Macugen.RTM.
(Eyetech, New York) for treating age-related macular
degeneration.
[0156] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native polypeptide disclosed
herein. In a similar manner, the term "agonist" is used in the
broadest sense and includes any molecule that mimics a biological
activity of a native polypeptide disclosed herein. Suitable agonist
or antagonist molecules specifically include agonist or antagonist
antibodies or antibody fragments, fragments or amino acid sequence
variants of native polypeptides, peptides, antisense
oligonucleotides, small organic molecules, etc. Methods for
identifying agonists or antagonists of a polypeptide may comprise
contacting a polypeptide with a candidate agonist or antagonist
molecule and measuring a detectable change in one or more
biological activities normally associated with the polypeptide.
[0157] The terms "TIGIT antagonist" and "antagonist of TIGIT
activity or TIGIT expression" are used interchangeably and refer to
a compound that interferes with the normal functioning of TIGIT,
either by decreasing transcription or translation of TIGIT-encoding
nucleic acid, or by inhibiting or blocking TIGIT polypeptide
activity, or both. Examples of TIGIT antagonists include, but are
not limited to, antisense polynucleotides, interfering RNAs,
catalytic RNAs, RNA-DNA chimeras, TIGIT-specific aptamers,
anti-TIGIT antibodies, TIGIT-binding fragments of anti-TIGIT
antibodies, TIGIT-binding small molecules, TIGIT-binding peptides,
and other polypeptides that specifically bind TIGIT (including, but
not limited to, TIGIT-binding fragments of one or more TIGIT
ligands, optionally fused to one or more additional domains), such
that the interaction between the TIGIT antagonist and TIGIT results
in a reduction or cessation of TIGIT activity or expression. It
will be understood by one of ordinary skill in the art that in some
instances, a TIGIT antagonist may antagonize one TIGIT activity
without affecting another TIGIT activity. For example, a desirable
TIGIT antagonist for use in certain of the methods herein is a
TIGIT antagonist that antagonizes TIGIT activity in response to one
of PVR interaction, PVRL3 interaction, or PVRL2 interaction, e.g.,
without affecting or minimally affecting any of the other TIGIT
interactions.
[0158] The terms "PVR antagonist" and "antagonist of PVR activity
or PVR expression" are used interchangeably and refer to a compound
that interferes with the normal functioning of PVR, either by
decreasing transcription or translation of PVR-encoding nucleic
acid, or by inhibiting or blocking PVR polypeptide activity, or
both. Examples of PVR antagonists include, but are not limited to,
antisense polynucleotides, interfering RNAs, catalytic RNAs,
RNA-DNA chimeras, PVR-specific aptamers, anti-PVR antibodies,
PVR-binding fragments of anti-PVR antibodies, PVR-binding small
molecules, PVR-binding peptides, and other polypeptides that
specifically bind PVR (including, but not limited to, PVR-binding
fragments of one or more PVR ligands, optionally fused to one or
more additional domains), such that the interaction between the PVR
antagonist and PVR results in a reduction or cessation of PVR
activity or expression. It will be understood by one of ordinary
skill in the art that in some instances, a PVR antagonist may
antagonize one PVR activity without affecting another PVR activity.
For example, a desirable PVR antagonist for use in certain of the
methods herein is a PVR antagonist that antagonizes PVR activity in
response to TIGIT interaction without impacting the PVR-CD96 and/or
PVR-CD226 interactions.
[0159] The term "dysfunction" in the context of immune dysfunction,
refers to a state of reduced immune responsiveness to antigenic
stimulation. The term includes the common elements of both
exhaustion and/or anergy in which antigen recognition may occur,
but the ensuing immune response is ineffective to control infection
or tumor growth.
[0160] The term "dysfunctional", as used herein, also includes
refractory or unresponsive to antigen recognition, specifically,
impaired capacity to translate antigen recognition into down-stream
T-cell effector functions, such as proliferation, cytokine
production (e.g., IL-2) and/or target cell killing.
[0161] The term "anergy" refers to the state of unresponsiveness to
antigen stimulation resulting from incomplete or insufficient
signals delivered through the T-cell receptor (e.g. increase in
intracellular Ca' in the absence of ras-activation). T cell anergy
can also result upon stimulation with antigen in the absence of
co-stimulation, resulting in the cell becoming refractory to
subsequent activation by the antigen even in the context of
costimulation. The unresponsive state can often be overridden by
the presence of Interleukin-2. Anergic T-cells do not undergo
clonal expansion and/or acquire effector functions.
[0162] The term "exhaustion" refers to T cell exhaustion as a state
of T cell dysfunction that arises from sustained TCR signaling that
occurs during many chronic infections and cancer. It is
distinguished from anergy in that it arises not through incomplete
or deficient signaling, but from sustained signaling. It is defined
by poor effector function, sustained expression of inhibitory
receptors and a transcriptional state distinct from that of
functional effector or memory T cells. Exhaustion prevents optimal
control of infection and tumors. Exhaustion can result from both
extrinsic negative regulatory pathways (e.g., immunoregulatory
cytokines) as well as cell intrinsic negative regulatory
(costimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).
[0163] "Enhancing T-cell function" means to induce, cause or
stimulate a T-cell to have a sustained or amplified biological
function, or renew or reactivate exhausted or inactive T-cells.
Examples of enhancing T-cell function include: increased secretion
of .gamma.-interferon from CD8.sup.+ T-cells, increased
proliferation, increased antigen responsiveness (e.g., viral,
pathogen, or tumor clearance) relative to such levels before the
intervention. In one embodiment, the level of enhancement is as
least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%,
200%. The manner of measuring this enhancement is known to one of
ordinary skill in the art.
[0164] A "T cell dysfunctional disorder" is a disorder or condition
of T-cells characterized by decreased responsiveness to antigenic
stimulation. In a particular embodiment, a T-cell dysfunctional
disorder is a disorder that is specifically associated with
inappropriate increased signaling through PD-1. In another
embodiment, a T-cell dysfunctional disorder is one in which T-cells
are anergic or have decreased ability to secrete cytokines,
proliferate, or execute cytolytic activity. In a specific aspect,
the decreased responsiveness results in ineffective control of a
pathogen or tumor expressing an immunogen. Examples of T cell
dysfunctional disorders characterized by T-cell dysfunction include
unresolved acute infection, chronic infection and tumor
immunity.
[0165] "Tumor immunity" refers to the process in which tumors evade
immune recognition and clearance. Thus, as a therapeutic concept,
tumor immunity is "treated" when such evasion is attenuated, and
the tumors are recognized and attacked by the immune system.
Examples of tumor recognition include tumor binding, tumor
shrinkage and tumor clearance.
[0166] "Immunogenecity" refers to the ability of a particular
substance to provoke an immune response. Tumors are immunogenic and
enhancing tumor immunogenicity aids in the clearance of the tumor
cells by the immune response. Examples of enhancing tumor
immunogenicity include but not limited to treatment with a PD-1
axis binding antagonist (e.g., anti-PD-L1 antibodies and a TIGIT
inhibitor (e.g., anti-TIGIT antibodies).
[0167] "Sustained response" refers to the sustained effect on
reducing tumor growth after cessation of a treatment. For example,
the tumor size may remain to be the same or smaller as compared to
the size at the beginning of the administration phase. In some
embodiments, the sustained response has a duration at least the
same as the treatment duration, at least 1.5.times., 2.0.times.,
2.5.times., or 3.0.times. length of the treatment duration.
[0168] The term "antibody" includes monoclonal antibodies
(including full length antibodies which have an immunoglobulin Fc
region), antibody compositions with polyepitopic specificity,
multispecific antibodies (e.g., bispecific antibodies, diabodies,
and single-chain molecules, as well as antibody fragments (e.g.,
Fab, F(ab').sub.2, and Fv). The term "immunoglobulin" (Ig) is used
interchangeably with "antibody" herein.
[0169] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains. An IgM antibody consists of 5 of the
basic heterotetramer units along with an additional polypeptide
called a J chain, and contains 10 antigen binding sites, while IgA
antibodies comprise from 2-5 of the basic 4-chain units which can
polymerize to form polyvalent assemblages in combination with the J
chain. In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain at its
other end. The V.sub.L is aligned with the V.sub.H and the CL is
aligned with the first constant domain of the heavy chain
(C.sub.H1). Particular amino acid residues are believed to form an
interface between the light chain and heavy chain variable domains.
The pairing of a V.sub.H and V.sub.L together forms a single
antigen-binding site. For the structure and properties of the
different classes of antibodies, see e.g., Basic and Clinical
Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram
G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page
71 and Chapter 6. The L chain from any vertebrate species can be
assigned to one of two clearly distinct types, called kappa and
lambda, based on the amino acid sequences of their constant
domains. Depending on the amino acid sequence of the constant
domain of their heavy chains (C.sub.H), immunoglobulins can be
assigned to different classes or isotypes. There are five classes
of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains
designated .alpha., .delta., .epsilon., .gamma. and .mu.,
respectively. The .gamma. and .alpha. classes are further divided
into subclasses on the basis of relatively minor differences in the
C.sub.H sequence and function, e.g., humans express the following
subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
[0170] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domains of the heavy chain and light
chain may be referred to as "VH" and "VL", respectively. These
domains are generally the most variable parts of the antibody
(relative to other antibodies of the same class) and contain the
antigen binding sites.
[0171] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and defines the
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
entire span of the variable domains. Instead, it is concentrated in
three segments called hypervariable regions (HVRs) both in the
light-chain and the heavy chain variable domains. The more highly
conserved portions of variable domains are called the framework
regions (FR). The variable domains of native heavy and light chains
each comprise four FR regions, largely adopting a beta-sheet
configuration, connected by three HVRs, which form loops
connecting, and in some cases forming part of, the beta-sheet
structure. The HVRs in each chain are held together in close
proximity by the FR regions and, with the HVRs from the other
chain, contribute to the formation of the antigen binding site of
antibodies (see Kabat et al., Sequences of Immunological Interest,
Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
The constant domains are not involved directly in the binding of
antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody-dependent
cellular toxicity.
[0172] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations and/or post-translation modifications (e.g.,
isomerizations, amidations) that may be present in minor amounts.
Monoclonal antibodies are highly specific, being directed against a
single antigenic site. In contrast to polyclonal antibody
preparations which typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
addition to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method. For example, the monoclonal antibodies to be
used in accordance with the present invention may be made by a
variety of techniques, including, for example, the hybridoma method
(e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et
al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies:
A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2.sup.nd
ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell
Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods
(see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies
(see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et
al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132
(2004), and technologies for producing human or human-like
antibodies in animals that have parts or all of the human
immunoglobulin loci or genes encoding human immunoglobulin
sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735;
WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:
2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);
Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S.
Pat. No. 5,661,016; Marks et al., Bio/Technology 10: 779-783
(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,
Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14:
845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and
Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0173] The term "naked antibody" refers to an antibody that is not
conjugated to a cytotoxic moiety or radiolabel.
[0174] The terms "full-length antibody," "intact antibody" or
"whole antibody" are used interchangeably to refer to an antibody
in its substantially intact form, as opposed to an antibody
fragment. Specifically whole antibodies include those with heavy
and light chains including an Fc region. The constant domains may
be native sequence constant domains (e.g., human native sequence
constant domains) or amino acid sequence variants thereof. In some
cases, the intact antibody may have one or more effector
functions.
[0175] An "antibody fragment" comprises a portion of an intact
antibody, preferably the antigen binding and/or the variable region
of the intact antibody. Examples of antibody fragments include Fab,
Fab', F(ab').sub.2 and Fv fragments; diabodies; linear antibodies
(see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein
Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules and
multispecific antibodies formed from antibody fragments. Papain
digestion of antibodies produced two identical antigen-binding
fragments, called "Fab" fragments, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. The Fab
fragment consists of an entire L chain along with the variable
region domain of the H chain (V.sub.H), and the first constant
domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment which roughly corresponds to two
disulfide linked Fab fragments having different antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having a few additional
residues at the carboxy terminus of the C.sub.H1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s) of
the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0176] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, the
region which is also recognized by Fc receptors (FcR) found on
certain types of cells.
[0177] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three HVRs specific for an antigen) has the ability
to recognize and bind antigen, although at a lower affinity than
the entire binding site.
[0178] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of the sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0179] "Functional fragments" of the antibodies of the invention
comprise a portion of an intact antibody, generally including the
antigen binding or variable region of the intact antibody or the Fc
region of an antibody which retains or has modified FcR binding
capability. Examples of antibody fragments include linear antibody,
single-chain antibody molecules and multispecific antibodies formed
from antibody fragments.
[0180] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10) residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, thereby resulting in a bivalent
fragment, i.e., a fragment having two antigen-binding sites.
Bispecific diabodies are heterodimers of two "crossover" sFv
fragments in which the V.sub.H and V.sub.L domains of the two
antibodies are present on different polypeptide chains. Diabodies
are described in greater detail in, for example, EP 404,097; WO
93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:
6444-6448 (1993).
[0181] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is(are) identical with or
homologous to corresponding sequences in antibodies derived from
another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; Morrison
et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies of interest herein include PRIMATIZED.RTM. antibodies
wherein the antigen-binding region of the antibody is derived from
an antibody produced by, e.g., immunizing macaque monkeys with an
antigen of interest. As used herein, "humanized antibody" is used a
subset of "chimeric antibodies."
[0182] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. In one embodiment, a humanized antibody
is a human immunoglobulin (recipient antibody) in which residues
from an HVR (hereinafter defined) of the recipient are replaced by
residues from an HVR of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
specificity, affinity, and/or capacity. In some instances,
framework ("FR") residues of the human immunoglobulin are replaced
by corresponding non-human residues. Furthermore, humanized
antibodies may comprise residues that are not found in the
recipient antibody or in the donor antibody. These modifications
may be made to further refine antibody performance, such as binding
affinity. In general, a humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the hypervariable
loops correspond to those of a non-human immunoglobulin sequence,
and all or substantially all of the FR regions are those of a human
immunoglobulin sequence, although the FR regions may include one or
more individual FR residue substitutions that improve antibody
performance, such as binding affinity, isomerization,
immunogenicity, etc. The number of these amino acid substitutions
in the FR are typically no more than 6 in the H chain, and in the L
chain, no more than 3. The humanized antibody optionally will also
comprise at least a portion of an immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further
details, see, e.g., Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.
Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);
Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and
Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos.
6,982,321 and 7,087,409.
[0183] A "human antibody" is an antibody that possesses an
amino-acid sequence corresponding to that of an antibody produced
by a human and/or has been made using any of the techniques for
making human antibodies as disclosed herein. This definition of a
human antibody specifically excludes a humanized antibody
comprising non-human antigen-binding residues. Human antibodies can
be produced using various techniques known in the art, including
phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also
available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr.
Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the antigen to a transgenic animal that
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled,
e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and
6,150,584 regarding XENOMOUSE' technology). See also, for example,
Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006)
regarding human antibodies generated via a human B-cell hybridoma
technology.
[0184] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0185] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00022 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0186] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0187] The expression "variable-domain residue-numbering as in
Kabaf" or "amino-acid-position numbering as in Kabat," and
variations thereof, refers to the numbering system used for
heavy-chain variable domains or light-chain variable domains of the
compilation of antibodies in Kabat et al., supra. Using this
numbering system, the actual linear amino acid sequence may contain
fewer or additional amino acids corresponding to a shortening of,
or insertion into, a FR or HVR of the variable domain. For example,
a heavy-chain variable domain may include a single amino acid
insert (residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy-chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0188] "Framework" or "FR" residues are those variable-domain
residues other than the HVR residues as herein defined.
[0189] A "human consensus framework" or "acceptor human framework"
is a framework that represents the most commonly occurring amino
acid residues in a selection of human immunoglobulin VL or VH
framework sequences. Generally, the selection of human
immunoglobulin VL or VH sequences is from a subgroup of variable
domain sequences. Generally, the subgroup of sequences is a
subgroup as in Kabat et al., Sequences of Proteins of Immunological
Interest, 5.sup.th Ed. Public Health Service, National Institutes
of Health, Bethesda, Md. (1991). Examples include for the VL, the
subgroup may be subgroup kappa I, kappa II, kappa III or kappa IV
as in Kabat et al., supra. Additionally, for the VH, the subgroup
may be subgroup I, subgroup II, or subgroup III as in Kabat et al.,
supra. Alternatively, a human consensus framework can be derived
from the above in which particular residues, such as when a human
framework residue is selected based on its homology to the donor
framework by aligning the donor framework sequence with a
collection of various human framework sequences. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain pre-existing amino acid sequence
changes. In some embodiments, the number of pre-existing amino acid
changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less,
5 or less, 4 or less, 3 or less, or 2 or less.
[0190] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al., supra. In one
embodiment, the VH subgroup III consensus framework amino acid
sequence comprises at least a portion or all of each of the
following sequences:
TABLE-US-00023 (HC-FR1) (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS,
(HC-FR2), (SEQ ID NO: 26) WVRQAPGKGLEWV, (HC-FR3, SEQ ID NO: 27)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR, (HC-FR4), (SEQ ID NO: 28)
WGQGTLVTVSA.
[0191] A "VL kappa I consensus framework" comprises the consensus
sequence obtained from the amino acid sequences in variable light
kappa subgroup I of Kabat et al., supra. In one embodiment, the VH
subgroup I consensus framework amino acid sequence comprises at
least a portion or all of each of the following sequences:
TABLE-US-00024 (LC-FR1) (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC,
(LC-FR2) (SEQ ID NO: 30) WYQQKPGKAPKLLIY, (LC-FR3) (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, (LC-FR4) (SEQ ID NO: 32)
FGQGTKVEIKR.
[0192] An "amino-acid modification" at a specified position, e.g.
of the Fc region, refers to the substitution or deletion of the
specified residue, or the insertion of at least one amino acid
residue adjacent the specified residue. Insertion "adjacent" to a
specified residue means insertion within one to two residues
thereof. The insertion may be N-terminal or C-terminal to the
specified residue. The preferred amino acid modification herein is
a substitution.
[0193] An "affinity-matured" antibody is one with one or more
alterations in one or more HVRs thereof that result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody that does not possess those alteration(s). In
one embodiment, an affinity-matured antibody has nanomolar or even
picomolar affinities for the target antigen. Affinity-matured
antibodies are produced by procedures known in the art. For
example, Marks et al., Bio/Technology 10:779-783 (1992) describes
affinity maturation by VH- and VL-domain shuffling. Random
mutagenesis of HVR and/or framework residues is described by, for
example: Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813
(1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J.
Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol.
154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896
(1992).
[0194] As use herein, the term "specifically binds to" or is
"specific for" refers to measurable and reproducible interactions
such as binding between a target and an antibody, which is
determinative of the presence of the target in the presence of a
heterogeneous population of molecules including biological
molecules. For example, an antibody that specifically binds to a
target (which can be an epitope) is an antibody that binds this
target with greater affinity, avidity, more readily, and/or with
greater duration than it binds to other targets. In one embodiment,
the extent of binding of an antibody to an unrelated target is less
than about 10% of the binding of the antibody to the target as
measured, e.g., by a radioimmunoassay (MA). In certain embodiments,
an antibody that specifically binds to a target has a dissociation
constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, or .ltoreq.0.1 nM. In certain embodiments, an
antibody specifically binds to an epitope on a protein that is
conserved among the protein from different species. In another
embodiment, specific binding can include, but does not require
exclusive binding.
[0195] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2 (including IgG2A and IgG2B), IgG-3, or IgG-4 subtypes,
IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. The Ig fusions
preferably include the substitution of a domain of a polypeptide or
antibody described herein in the place of at least one variable
region within an Ig molecule. In a particularly preferred
embodiment, the immunoglobulin fusion includes the hinge, CH2 and
CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule.
For the production of immunoglobulin fusions see also U.S. Pat. No.
5,428,130 issued Jun. 27, 1995. For example, useful immunoadhesins
as second medicaments useful for combination therapy herein include
polypeptides that comprise the extracellular or PD-1 binding
portions of PD-L1 or PD-L2 or the extracellular or PD-L1 or PD-L2
binding portions of PD-1, fused to a constant domain of an
immunoglobulin sequence, such as a PD-L1 ECD-Fc, a PD-L2 ECD-Fc,
and a PD-1 ECD-Fc, respectively. Immunoadhesin combinations of Ig
Fc and ECD of cell surface receptors are sometimes termed soluble
receptors.
[0196] A "fusion protein" and a "fusion polypeptide" refer to a
polypeptide having two portions covalently linked together, where
each of the portions is a polypeptide having a different property.
The property may be a biological property, such as activity in
vitro or in vivo. The property may also be simple chemical or
physical property, such as binding to a target molecule, catalysis
of a reaction, etc. The two portions may be linked directly by a
single peptide bond or through a peptide linker but are in reading
frame with each other.
[0197] A "PD-1 oligopeptide," "PD-L1 oligopeptide," or "PD-L2
oligopeptide" is an oligopeptide that binds, preferably
specifically, to a PD-1, PD-L1 or PD-L2 negative costimulatory
polypeptide, respectively, including a receptor, ligand or
signaling component, respectively, as described herein. Such
oligopeptides may be chemically synthesized using known
oligopeptide synthesis methodology or may be prepared and purified
using recombinant technology. Such oligopeptides are usually at
least about 5 amino acids in length, alternatively at least about
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or
more. Such oligopeptides may be identified using well known
techniques. In this regard, it is noted that techniques for
screening oligopeptide libraries for oligopeptides that are capable
of specifically binding to a polypeptide target are well known in
the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,
4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT
Publication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc.
Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc.
Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in
Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J.
Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,
140:611-616 (1988), Cwirla, S. E. et al. Proc. Natl. Acad. Sci.
USA, 87:6378 (1990); Lowman, H. B. et al. Biochemistry, 30:10832
(1991); Clackson, T. et al. Nature, 352: 624 (1991); Marks, J. D.
et al., J. Mol. Biol., 222:581 (1991); Kang, A. S. et al. Proc.
Natl. Acad. Sci. USA, 88:8363 (1991), and Smith, G. P., Current
Opin. Biotechnol., 2:668 (1991).
[0198] A "blocking" antibody or an "antagonist" antibody is one
that inhibits or reduces a biological activity of the antigen it
binds. In some embodiments, blocking antibodies or antagonist
antibodies substantially or completely inhibit the biological
activity of the antigen. The anti-PD-L1 antibodies of the invention
block the signaling through PD-1 so as to restore a functional
response by T-cells (e.g., proliferation, cytokine production,
target cell killing) from a dysfunctional state to antigen
stimulation.
[0199] An "agonist" or activating antibody is one that enhances or
initiates signaling by the antigen to which it binds. In some
embodiments, agonist antibodies cause or activate signaling without
the presence of the natural ligand.
[0200] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain, including native-sequence
Fc regions and variant Fc regions. Although the boundaries of the
Fc region of an immunoglobulin heavy chain might vary, the human
IgG heavy-chain Fc region is usually defined to stretch from an
amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding
a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues
removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and
without the K447 residue. Suitable native-sequence Fc regions for
use in the antibodies of the invention include human IgG1, IgG2
(IgG2A, IgG2B), IgG3 and IgG4.
[0201] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RT, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors, Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see M. Daeron, Annu. Rev.
Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet,
Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods
4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41
(1995). Other FcRs, including those to be identified in the future,
are encompassed by the term "FcR" herein.
[0202] The term "Fc receptor" or "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus. Guyer et al., J. Immunol. 117: 587 (1976) and
Kim et al., J. Immunol. 24: 249 (1994). Methods of measuring
binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol.
Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology
15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. 279 (8):
6213-6 (2004); WO 2004/92219 (Hinton et al.). Binding to FcRn in
vivo and serum half-life of human FcRn high-affinity binding
polypeptides can be assayed, e.g., in transgenic mice or
transfected human cell lines expressing human FcRn, or in primates
to which the polypeptides having a variant Fc region are
administered. WO 2004/42072 (Presta) describes antibody variants
which improved or diminished binding to FcRs. See also, e.g.,
Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).
[0203] The phrase "substantially reduced," or "substantially
different," as used herein, denotes a sufficiently high degree of
difference between two numeric values (generally one associated
with a molecule and the other associated with a
reference/comparator molecule) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values). The
difference between said two values is, for example, greater than
about 10%, greater than about 20%, greater than about 30%, greater
than about 40%, and/or greater than about 50% as a function of the
value for the reference/comparator molecule.
[0204] The term "substantially similar" or "substantially the
same," as used herein, denotes a sufficiently high degree of
similarity between two numeric values (for example, one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody), such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is, for
example, less than about 50%, less than about 40%, less than about
30%, less than about 20%, and/or less than about 10% as a function
of the reference/comparator value.
[0205] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers that are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
[0206] A "package insert" refers to instructions customarily
included in commercial packages of medicaments that contain
information about the indications customarily included in
commercial packages of medicaments that contain information about
the indications, usage, dosage, administration, contraindications,
other medicaments to be combined with the packaged product, and/or
warnings concerning the use of such medicaments, etc.
[0207] As used herein, the term "treatment" refers to clinical
intervention designed to alter the natural course of the individual
or cell being treated during the course of clinical pathology.
Desirable effects of treatment include decreasing the rate of
disease progression, ameliorating or palliating the disease state,
and remission or improved prognosis. For example, an individual is
successfully "treated" if one or more symptoms associated with
cancer are mitigated or eliminated, including, but are not limited
to, reducing the proliferation of (or destroying) cancerous cells,
decreasing symptoms resulting from the disease, increasing the
quality of life of those suffering from the disease, decreasing the
dose of other medications required to treat the disease, delaying
the progression of the disease, and/or prolonging survival of
individuals.
[0208] As used herein, "delaying progression of a disease" means to
defer, hinder, slow, retard, stabilize, and/or postpone development
of the disease (such as cancer). This delay can be of varying
lengths of time, depending on the history of the disease and/or
individual being treated. As is evident to one skilled in the art,
a sufficient or significant delay can, in effect, encompass
prevention, in that the individual does not develop the disease.
For example, a late stage cancer, such as development of
metastasis, may be delayed.
[0209] As used herein, "reducing or inhibiting cancer relapse"
means to reduce or inhibit tumor or cancer relapse or tumor or
cancer progression.
[0210] As used herein, "cancer" and "cancerous" refer to or
describe the physiological condition in mammals that is typically
characterized by unregulated cell growth. Included in this
definition are benign and malignant cancers as well as dormant
tumors or micrometastatses. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell
cancer, lung cancer (including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, and squamous
carcinoma of the lung), cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer (including gastrointestinal
cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, liver cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma
and various types of head and neck cancer, as well as B-cell
lymphoma (including low grade/follicular non-Hodgkin's lymphoma
(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular
NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL;
high grade lymphoblastic NHL; high grade small non-cleaved cell
NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related
lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic
leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell
leukemia; chronic myeloblastic leukemia; and post-transplant
lymphoproliferative disorder (PTLD), as well as abnormal vascular
proliferation associated with phakomatoses, edema (such as that
associated with brain tumors), and Meigs' syndrome.
[0211] As used herein, "metastasis" is meant the spread of cancer
from its primary site to other places in the body. Cancer cells can
break away from a primary tumor, penetrate into lymphatic and blood
vessels, circulate through the bloodstream, and grow in a distant
focus (metastasize) in normal tissues elsewhere in the body.
Metastasis can be local or distant. Metastasis is a sequential
process, contingent on tumor cells breaking off from the primary
tumor, traveling through the bloodstream, and stopping at a distant
site. At the new site, the cells establish a blood supply and can
grow to form a life-threatening mass. Both stimulatory and
inhibitory molecular pathways within the tumor cell regulate this
behavior, and interactions between the tumor cell and host cells in
the distant site are also significant.
[0212] An "effective amount" is at least the minimum concentration
required to effect a measurable improvement or prevention of a
particular disorder. An effective amount herein may vary according
to factors such as the disease state, age, sex, and weight of the
patient, and the ability of the antibody to elicit a desired
response in the individual. An effective amount is also one in
which any toxic or detrimental effects of the treatment are
outweighed by the therapeutically beneficial effects. For
prophylactic use, beneficial or desired results include results
such as eliminating or reducing the risk, lessening the severity,
or delaying the onset of the disease, including biochemical,
histological and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use, beneficial
or desired results include clinical results such as decreasing one
or more symptoms resulting from the disease, increasing the quality
of life of those suffering from the disease, decreasing the dose of
other medications required to treat the disease, enhancing effect
of another medication such as via targeting, delaying the
progression of the disease, and/or prolonging survival. In the case
of cancer or tumor, an effective amount of the drug may have the
effect in reducing the number of cancer cells; reducing the tumor
size; inhibiting (i.e., slow to some extent or desirably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and desirably stop) tumor metastasis;
inhibiting to some extent tumor growth; and/or relieving to some
extent one or more of the symptoms associated with the disorder. An
effective amount can be administered in one or more
administrations. For purposes of this invention, an effective
amount of drug, compound, or pharmaceutical composition is an
amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective amount of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective amount" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0213] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality. As such, "in conjunction with" refers to
administration of one treatment modality before, during, or after
administration of the other treatment modality to the
individual.
[0214] As used herein, "subject" is meant a mammal, including, but
not limited to, a human or non-human mammal, such as a bovine,
equine, canine, ovine, or feline. Preferably, the subject is a
human. Patients are also subjects herein.
[0215] As used herein, "complete response" or "CR" refers to
disappearance of all target lesions; "partial response" or "PR"
refers to at least a 30% decrease in the sum of the longest
diameters (SLD) of target lesions, taking as reference the baseline
SLD; and "stable disease" or "SD" refers to neither sufficient
shrinkage of target lesions to qualify for PR, nor sufficient
increase to qualify for PD, taking as reference the smallest SLD
since the treatment started.
[0216] As used herein, "progressive disease" or "PD" refers to at
least a 20% increase in the SLD of target lesions, taking as
reference the smallest SLD recorded since the treatment started or
the presence of one or more new lesions.
[0217] As used herein, "progression free survival" (PFS) refers to
the length of time during and after treatment during which the
disease being treated (e.g., cancer) does not get worse.
Progression-free survival may include the amount of time patients
have experienced a complete response or a partial response, as well
as the amount of time patients have experienced stable disease.
[0218] As used herein, "overall response rate" (ORR) refers to the
sum of complete response (CR) rate and partial response (PR)
rate.
[0219] As used herein, "overall survival" refers to the percentage
of individuals in a group who are likely to be alive after a
particular duration of time.
[0220] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclophosphamide
(CYTOXAN.RTM.); alkyl sulfonates such as busulfan, improsulfan, and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; pemetrexed; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
podophyllotoxin; podophyllinic acid; teniposide; cryptophycins
(particularly cryptophycin 1 and cryptophycin 8); dolastatin;
duocarmycin (including the synthetic analogues, KW-2189 and
CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral
alpha-4 integrin inhibitor; a sarcodictyin; spongistatin; nitrogen
mustards such as chlorambucil, chlornaphazine, cholophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;
antibiotics such as the enediyne antibiotics (e.g., calicheamicin,
especially calicheamicin gammalI and calicheamicin omegaI1 (see,
e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186
(1994)); dynemicin, including dynemicin A; an esperamicin; as well
as neocarzinostatin chromophore and related chromoprotein enediyne
antibiotic chromophores), aclacinomysins, actinomycin, authramycin,
azaserine, bleomycins, cactinomycin, carabicin, carminomycin,
carzinophilin, chromomycinis, dactinomycin, daunorubicin,
detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including
ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.) and deoxydoxorubicin),
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such
as mitomycin C, mycophenolic acid, nogalamycin, olivomycins,
peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,
streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin,
zorubicin; anti-metabolites such as methotrexate, gemcitabine
(GEMZAR.RTM.), tegafur (UFTORAL.RTM.), capecitabine (XELODA.RTM.),
an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidine
derivative), as well as other c-Kit inhibitors; anti-adrenals such
as aminoglutethimide, mitotane, trilostane; folic acid replenisher
such as frolinic acid; aceglatone; aldophosphamide glycoside;
aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfornithine; elliptinium acetate; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidainine; maytansinoids such as
maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;
2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide complex
(JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL.RTM.),
albumin-engineered nanoparticle formulation of paclitaxel
(ABRAXANE.TM.), and doxetaxel (TAXOTERE.RTM.); chloranbucil;
6-thioguanine; mercaptopurine; methotrexate; platinum analogs such
as cisplatin and carboplatin; vinblastine (VELBAN.RTM.); platinum;
etoposide (VP-16); ifosfamide; mitoxantrone; vincristine
(ONCOVIN.RTM.); oxaliplatin; leucovovin; vinorelbine
(NAVELBINE.RTM.); novantrone; edatrexate; daunomycin; aminopterin;
ibandronate; topoisomerase inhibitor RFS 2000;
difluorometlhylornithine (DMFO); retinoids such as retinoic acid;
pharmaceutically acceptable salts, acids or derivatives of any of
the above; as well as combinations of two or more of the above such
as CHOP, an abbreviation for a combined therapy of
cyclophosphamide, doxorubicin, vincristine, and prednisolone, and
FOLFOX, an abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0221] Also included in this definition are anti-hormonal agents
that act to regulate, reduce, block, or inhibit the effects of
hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), raloxifene (EVISTA.RTM.),
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and toremifene (FARESTON.RTM.); anti-progesterones;
estrogen receptor down-regulators (ERDs); estrogen receptor
antagonists such as fulvestrant (FASLODEX.RTM.); agents that
function to suppress or shut down the ovaries, for example,
leutinizing hormone-releasing hormone (LHRH) agonists such as
leuprolide acetate (LUPRON.RTM. and ELIGARD.RTM.), goserelin
acetate, buserelin acetate and tripterelin; anti-androgens such as
flutamide, nilutamide and bicalutamide; and aromatase inhibitors
that inhibit the enzyme aromatase, which regulates estrogen
production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, megestrol acetate
(MEGASE.RTM.), exemestane (AROMASIN.RTM.), formestanie, fadrozole,
vorozole (RIVISOR.RTM.), letrozole (FEMARA.RTM.), and anastrozole
(ARIMIDEX.RTM.). In addition, such definition of chemotherapeutic
agents includes bisphosphonates such as clodronate (for example,
BONEFOS.RTM. or OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095,
zoledronic acid/zoledronate (ZOMETA.RTM.), alendronate
(FOSAMAX.RTM.), pamidronate (AREDIA.RTM.), tiludronate
(SKELID.RTM.), or risedronate (ACTONEL.RTM.); as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
anti-sense oligonucleotides, particularly those that inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras,
and epidermal growth factor receptor (EGF-R); vaccines such as
THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN.RTM.); an
anti-estrogen such as fulvestrant; a Kit inhibitor such as imatinib
or EXEL-0862 (a tyrosine kinase inhibitor); EGFR inhibitor such as
erlotinib or cetuximab; an anti-VEGF inhibitor such as bevacizumab;
arinotecan; rmRH (e.g., ABARELIX.RTM.); lapatinib and lapatinib
ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule
inhibitor also known as GW572016); 17AAG (geldanamycin derivative
that is a heat shock protein (Hsp) 90 poison), and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0222] As used herein, the term "cytokine" refers generically to
proteins released by one cell population that act on another cell
as intercellular mediators or have an autocrine effect on the cells
producing the proteins. Examples of such cytokines include
lymphokines, monokines; interleukins ("ILs") such as IL-1,
IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL10,
IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as
IL-23), IL-31, including PROLEUKIN.RTM. rIL-2; a tumor-necrosis
factor such as TNF-.alpha. or TNF-.beta., TGF-.beta.1-3; and other
polypeptide factors including leukemia inhibitory factor ("LIF"),
ciliary neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"),
cardiotrophin ("CT"), and kit ligand ("KL").
[0223] As used herein, the term "chemokine" refers to soluble
factors (e.g., cytokines) that have the ability to selectively
induce chemotaxis and activation of leukocytes. They also trigger
processes of angiogenesis, inflammation, wound healing, and
tumorigenesis. Example chemokines include IL-8, a human homolog of
murine keratinocyte chemoattractant (KC).
[0224] As used herein and in the appended claims, the singular
forms "a," "or," and "the" include plural referents unless the
context clearly dictates otherwise.
[0225] Reference to "about" a value or parameter herein includes
(and describes) variations that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X".
[0226] The phrase "pharmaceutically acceptable salt" as used
herein, refers to pharmaceutically acceptable organic or inorganic
salts of a compound of the invention. Exemplary salts include, but
are not limited, to sulfate, citrate, acetate, oxalate, chloride,
bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate,
tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,
gentisinate, fumarate, gluconate, glucuronate, saccharate, formate,
benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate,
benzenesulfonate, p-toluenesulfonate, pamoate (i.e.,
1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal
(e.g., sodium and potassium) salts, alkaline earth metal (e.g.,
magnesium) salts, and ammonium salts. A pharmaceutically acceptable
salt may involve the inclusion of another molecule such as an
acetate ion, a succinate ion or other counter ion. The counter ion
may be any organic or inorganic moiety that stabilizes the charge
on the parent compound. Furthermore, a pharmaceutically acceptable
salt may have more than one charged atom in its structure.
Instances where multiple charged atoms are part of the
pharmaceutically acceptable salt can have multiple counter ions.
Hence, a pharmaceutically acceptable salt can have one or more
charged atoms and/or one or more counter ion.
[0227] If the compound of the invention is a base, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method available in the art, for example, treatment of the free
base with an inorganic acid, such as hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric
acid and the like, or with an organic acid, such as acetic acid,
maleic acid, succinic acid, mandelic acid, fumaric acid, malonic
acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a
pyranosidyl acid, such as glucuronic acid or galacturonic acid, an
alpha hydroxy acid, such as citric acid or tartaric acid, an amino
acid, such as aspartic acid or glutamic acid, an aromatic acid,
such as benzoic acid or cinnamic acid, a sulfonic acid, such as
p-toluenesulfonic acid or ethanesulfonic acid, or the like.
[0228] If the compound of the invention is an acid, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method, for example, treatment of the free acid with an inorganic
or organic base, such as an amine (primary, secondary or tertiary),
an alkali metal hydroxide or alkaline earth metal hydroxide, or the
like. Illustrative examples of suitable salts include, but are not
limited to, organic salts derived from amino acids, such as glycine
and arginine, ammonia, primary, secondary, and tertiary amines, and
cyclic amines, such as piperidine, morpholine and piperazine, and
inorganic salts derived from sodium, calcium, potassium, magnesium,
manganese, iron, copper, zinc, aluminum and lithium.
[0229] The phrase "pharmaceutically acceptable" indicates that the
substance or composition must be compatible chemically and/or
toxicologically, with the other ingredients comprising a
formulation, and/or the mammal being treated therewith.
[0230] It is understood that aspects and variations of the
invention described herein include "consisting" and/or "consisting
essentially of" aspects and variations.
III. Methods
[0231] In one aspect, provided herein is a method for treating or
delaying progression of cancer in an individual comprising
administering to the individual an effective amount of a PD-1 axis
binding antagonist in combination with an agent that decreases or
inhibits TIGIT expression and/or activity.
[0232] In another aspect, provided herein is a method for reducing
or inhibiting cancer relapse or cancer progression in an individual
comprising administering to the individual an effect amount of a
PD-1 axis binding antagonist in combination with an agent that that
decreases or inhibits TIGIT expression and/or activity. As
disclosed herein, cancer relapse and/or cancer progression include,
without limitation, cancer metastasis.
[0233] In another aspect, provided herein is a method for treating
or delaying progression of an immune related disease in an
individual comprising administering to the individual an effect
amount of a PD-1 axis binding antagonist in combination with an
agent that that decreases or inhibits TIGIT expression and/or
activity.
[0234] In another aspect, provided herein is a method for reducing
or inhibiting progression of an immune related disease in an
individual comprising administering to the individual an effect
amount of a PD-1 axis binding antagonist in combination with an
agent that that decreases or inhibits TIGIT expression and/or
activity.
[0235] In some embodiments, the immune related disease is
associated with T cell dysfunctional disorder. In some embodiments,
the immune related disease is a viral infection. In certain
embodiments, the viral infection is a chronic viral infection. In
some embodiments, T cell dysfunctional disorder is characterized by
decreased responsiveness to antigenic stimulation. In some
embodiments, the T cell dysfunctional disorder is characterized by
T cell anergy or decreased ability to secrete cytokines,
proliferate or execute cytolytic activity. In some embodiments, the
T cell dysfunctional disorder is characterized by T cell
exhaustion. In some embodiments, the T cells are CD4+ and CD8+ T
cells. In some embodiments, the T cell dysfunctional disorder
includes unresolved acute infection, chronic infection and tumor
immunity.
[0236] In another aspect, provided herein is a method for
increasing, enhancing or stimulating an immune response or function
in an individual comprising administering to the individual an
effect amount of a PD-1 axis binding antagonist in combination with
an agent that decreases or inhibits TIGIT expression and/or
activity.
[0237] In another aspect, provided herein is a method of treating
or delaying progression of cancer in an individual comprising
administering to the individual an effective amount of a PD-1 axis
binding antagonist and an agent that modulates the CD226 expression
and/or activity.
[0238] In another aspect, provided herein is a method for reducing
or inhibiting cancer relapse or cancer progression in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and an agent that modulates the CD226
expression and/or activity.
[0239] In another aspect, provided herein is a method for treating
or delaying progression of an immune related disease in an
individual comprising administering to the individual an effective
amount of a PD-1 axis binding antagonist and an agent that
modulates the CD226 expression and/or activity.
[0240] In another aspect, provided herein is a method for reducing
or inhibiting progression of an immune related disease in an
individual comprising administering to the individual an effective
amount of a PD-1 axis binding antagonist and agent that modulates
the CD226 expression and/or activity.
[0241] In some embodiments, the immune related disease is
associated with T cell dysfunctional disorder. In some embodiments,
the immune related disease is a viral infection. In certain
embodiments, the viral infection is a chronic viral infection. In
some embodiments, the T cell dysfunctional disorder is
characterized by decreased responsiveness to antigenic stimulation.
In some embodiments, the T cell dysfunctional disorder is
characterized by T cell anergy, or decreased ability to secrete
cytokines, proliferate or execute cytolytic activity. In some
embodiments, the T cell dysfunctional disorder is characterized by
T cell exhaustion. In some embodiments, the T cells are CD4+ and
CD8+ T cells. In some embodiments, the immune related disease is
selected from the group consisting of unresolved acute infection,
chronic infection and tumor immunity.
[0242] In another aspect, provided herein is a method of
increasing, enhancing or stimulating an immune response or function
in an individual by administering to the individual an effective
amount of a PD-1 axis binding antagonist and an agent that
modulates the CD226 expression and/or activity.
[0243] In some embodiments, the agent that modulates the CD226
expression and/or activity is capable of increasing and/or
stimulating CD226 expression and/or activity; increasing and/or
stimulating the interaction of CD226 with PVR, PVRL2, and/or PVRL3;
and increasing and/or stimulating the intracellular signaling
mediated by CD226 binding to PVR, PVRL2, and/or PVRL3. As used
herein, an agent that is capable of increasing and/or stimulating
CD226 expression and/or activity includes, without limitation,
agents that increase and/or stimulate CD226 expression and/or
activity. As used herein, an agent that is capable of increasing
and/or stimulating the interaction of CD226 with PVR, PVRL2, and/or
PVRL3 includes, without limitation, agents that increase and/or
stimulate the interaction of CD226 with PVR, PVRL2, and/or PVRL3.
As used herein, an agent that is capable of increasing and/or
stimulating the intracellular signaling mediated by CD226 binding
to PVR, PVRL2, and/or PVRL3 includes, without limitation, agents
that increase and/or stimulate the intracellular signaling mediated
by CD226 binding to PVR, PVRL2, and/or PVRL3.
[0244] In some embodiments, the agent that modulates the CD226
expression and/or activity is selected from an agent that inhibits
and/or blocks the interaction of CD226 with TIGIT, an antagonist of
TIGIT expression and/or activity, an antagonist of PVR expression
and/or activity, an agent that inhibits and/or blocks the
interaction of TIGIT with PVR, an agent that inhibits and/or blocks
the interaction of TIGIT with PVRL2, an agent that inhibits and/or
blocks the interaction of TIGIT with PVRL3, an agent that inhibits
and/or blocks the intracellular signaling mediated by TIGIT binding
to PVR, an agent that inhibits and/or blocks the intracellular
signaling mediated by TIGIT binding to PVRL2, an agent that
inhibits and/or blocks the intracellular signaling mediated by
TIGIT binding to PVRL3, and combinations thereof.
[0245] In some embodiments, the agent that inhibits and/or blocks
the interaction of CD226 with TIGIT is a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In some embodiments, the agent that inhibits and/or blocks the
interaction of CD226 with TIGIT is an anti-TIGIT antibody or
antigen-binding fragment thereof. In some embodiments, the agent
that inhibits and/or blocks the interaction of CD226 with TIGIT is
an inhibitory nucleic acid selected from an antisense
polynucleotide, an interfering RNA, a catalytic RNA, and an RNA-DNA
chimera.
[0246] In some embodiments, the antagonist of TIGIT expression
and/or activity is a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the antagonist of TIGIT expression and/or activity is
an anti-TIGIT antibody or antigen-binding fragment thereof. In some
embodiments, the antagonist of TIGIT expression and/or activity is
an inhibitory nucleic acid selected from an antisense
polynucleotide, an interfering RNA, a catalytic RNA, and an RNA-DNA
chimera.
[0247] In some embodiments, the antagonist of PVR expression and/or
activity is a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide. In some embodiments, the
antagonist of PVR expression and/or activity is selected from a
small molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide.
[0248] In some embodiments, the agent that inhibits and/or blocks
the interaction of TIGIT with PVR is a small molecule inhibitor, an
inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In some embodiments, the agent that inhibits and/or blocks the
interaction of TIGIT with PVR is selected from a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0249] In some embodiments, the agent that inhibits and/or blocks
the interaction of TIGIT with PVRL2 is selected from a small
molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide.
[0250] In some embodiments, the agent that inhibits and/or blocks
the interaction of TIGIT with PVRL3 is selected from a small
molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide.
[0251] In some embodiments, the agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVR is a
small molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide. In some embodiments, the agent that
inhibits and/or blocks the intracellular signaling mediated by
TIGIT binding to PVR is selected from a small molecule inhibitor,
an inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0252] In some embodiments, the agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVRL2 is
selected from a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide.
[0253] In some embodiments, the agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVRL3 is
selected from a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide.
[0254] In another aspect, provided herein is a method of
increasing, enhancing or stimulating an immune response or function
in an individual by administering to the individual an effective
amount of an agent that decreases or inhibits TIGIT expression
and/or activity and an agent that decreases or inhibits the
expression and/or activity of one or more additional immune
co-inhibitory receptors. In some embodiments, the one of more
additional immune co-inhibitory receptor is selected from PD-1,
CTLA-4, LAG3, TIM3, BTLA VISTA, B7H4, and CD96. In some
embodiments, one of more additional immune co-inhibitory receptor
is selected from PD-1, CTLA-4, LAG3 and TIM3.
[0255] In another aspect, provided herein is a method of
increasing, enhancing or stimulating an immune response or function
in an individual by administering to the individual an effective
amount of an agent that decreases or inhibits TIGIT expression
and/or activity and an agent that increases or activates the
expression and/or activity of one or more additional immune
co-stimulatory receptors. In some embodiments, the one of more
additional immune co-stimulatory receptor is selected from CD226,
OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4.
In some embodiments, the one or more additional immune
co-stimulatory receptor is selected from CD226, OX-40, CD28, CD27,
CD137, HVEM, and GITR. In some embodiments, the one of more
additional immune co-stimulatory receptor is selected from OX-40
and CD27.
[0256] The methods of this invention may find use in treating
conditions where enhanced immunogenicity is desired such as
increasing tumor immunogenicity for the treatment of cancer or T
cell dysfunctional disorders.
[0257] A variety of cancers may be treated, or their progression
may be delayed.
[0258] In some embodiments, the individual has non-small cell lung
cancer. The non-small cell lung cancer may be at early stage or at
late stage. In some embodiments, the individual has small cell lung
cancer. The small cell lung cancer may be at early stage or at late
stage. In some embodiments, the individual has renal cell cancer.
The renal cell cancer may be at early stage or at late stage. In
some embodiments, the individual has colorectal cancer. The
colorectal cancer may be at early stage or late stage. In some
embodiments, the individual has ovarian cancer. The ovarian cancer
may be at early stage or at late stage. In some embodiments, the
individual has breast cancer. The breast cancer may be at early
stage or at late stage. In some embodiments, the individual has
pancreatic cancer. The pancreatic cancer may be at early stage or
at late stage. In some embodiments, the individual has gastric
carcinoma. The gastric carcinoma may be at early stage or at late
stage. In some embodiments, the individual has bladder cancer. The
bladder cancer may be at early stage or at late stage. In some
embodiments, the individual has esophageal cancer. The esophageal
cancer may be at early stage or at late stage. In some embodiments,
the individual has mesothelioma. The mesothelioma may be at early
stage or at late stage. In some embodiments, the individual has
melanoma. The melanoma may be at early stage or at late stage. In
some embodiments, the individual has head and neck cancer. The head
and neck cancer may be at early stage or at late stage. In some
embodiments, the individual has thyroid cancer. The thyroid cancer
may be at early stage or at late stage. In some embodiments, the
individual has sarcoma. The sarcoma may be at early stage or late
stage. In some embodiments, the individual has prostate cancer. The
prostate cancer may be at early stage or at late stage. In some
embodiments, the individual has glioblastoma. The glioblastoma may
be at early stage or at late stage. In some embodiments, the
individual has cervical cancer. The cervical cancer may be at early
stage or at late stage. In some embodiments, the individual has
thymic carcinoma. The thymic carcinoma may be at early stage or at
late stage. In some embodiments, the individual has leukemia. The
leukemia may be at early stage or at late stage. In some
embodiments, the individual has lymphomas. The lymphoma may be at
early stage or at late stage. In some embodiments, the individual
has myelomas. The myelomas may be at early stage or at late stage.
In some embodiments, the individual has mycoses fungoids. The
mycoses fungoids may be at early stage or at late stage. In some
embodiments, the individual has merkel cell cancer. The merkel cell
cancer may be at early stage or at late stage. In some embodiments,
the individual has hematologic malignancies. The hematological
malignancies may be early stage or late stage. In some embodiments,
the individual is a human.
[0259] In some embodiments of the methods of this invention, the
CD4 and/or CD8 T cells in the individual have increased or enhanced
priming, activation, proliferation, cytokine release and/or
cytolytic activity relative to prior to the administration of the
combination.
[0260] In some embodiments of the methods of this invention, the
number of CD4 and/or CD8 T cells is elevated relative to prior to
administration of the combination. In some embodiments of the
methods of this invention, the number of activated CD4 and/or CD8 T
cells is elevated relative to prior to administration of the
combination.
[0261] In some embodiments of the methods of this invention, the
activated CD4 and/or CD8 T cells is characterized by
.gamma.-IFN.sup.+ producing CD4 and/or CD8 T cells and/or enhanced
cytolytic activity relative to prior to the administration of the
combination.
[0262] In some embodiments of the methods of this invention, the
CD4 and/or CD8 T cells exhibit increased release of cytokines
selected from the group consisting of IFN-.gamma., TNF-.alpha. and
interleukins.
[0263] In some embodiments of the methods of this invention, the
CD4 and/or CD8 T cell is an effector memory T cell. In some
embodiments of the methods of this invention, the CD4 and/or CD8
effector memory T cell is characterized by .gamma.-IFN.sup.+
producing CD4 and/or CD8 T cells and/or enhanced cytolytic
activity. In some embodiments of the methods of this invention, the
CD4 and/or CD8 effector memory T cell is characterized by having
the expression of CD44.sup.high CD62L.sup.low.
[0264] In some embodiments of the methods of this invention, the
cancer has elevated levels of T cell infiltration.
[0265] In some embodiments, the methods of the invention may
further comprise administering an additional therapy. The
additional therapy may be radiation therapy, surgery, chemotherapy,
gene therapy, DNA therapy, viral therapy, RNA therapy,
immunotherapy, bone marrow transplantation, nanotherapy, monoclonal
antibody therapy, or a combination of the foregoing. The additional
therapy may be in the form of an adjuvant or neoadjuvant therapy.
In some embodiments, the additional therapy is the administration
of side-effect limiting agents (e.g., agents intended to lessen the
occurrence and/or severity of side effects of treatment, such as
anti-nausea agents, etc.). In some embodiments, the additional
therapy is radiation therapy. In some embodiments, the additional
therapy is surgery. In some embodiments, the additional therapy may
be one or more of the chemotherapeutic agents described
hereinabove.
[0266] Any of the PD-1 axis binding antagonists and agents that
decreases or inhibits TIGIT expression and/or activity described
below may be used in the methods of the invention.
[0267] In some embodiments, any of the targets described herein
(e.g., PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4,
CD96, B7-1, TIGIT, CD226, OX-40, CD28, CD27, CD137, HVEM, GITR,
MICA, ICOS, NKG2D, 2B4, etc.) is a human protein.
PD-1 Axis Binding Antagonists
[0268] Provided herein is a method for treatment or delaying
progression of cancer in an individual comprising administering to
the individual an effective amount of a PD-1 axis binding
antagonist in combination with an agent that decreases or inhibits
TIGIT expression and/or activity. Provided herein is also a method
for reducing or inhibiting cancer relapse or cancer progression in
an individual comprising administering to the individual an effect
amount of a PD-1 axis binding antagonist in combination with an
agent that that decreases or inhibits TIGIT expression and/or
activity. Provided herein is also a method for treating or delaying
progression of an immune related disease in an individual
comprising administering to the individual an effect amount of a
PD-1 axis binding antagonist in combination with an agent that that
decreases or inhibits TIGIT expression and/or activity. Provided
herein is also a method for reducing or inhibiting progression of
an immune related disease in an individual comprising administering
to the individual an effect amount of a PD-1 axis binding
antagonist in combination with an agent that that decreases or
inhibits TIGIT expression and/or activity. Provided herein is also
a method for increasing, enhancing or stimulating an immune
response or function in an individual comprising administering to
the individual an effect amount of a PD-1 axis binding antagonist
in combination with an agent that decreases or inhibits TIGIT
expression and/or activity.
[0269] For example, a PD-1 axis binding antagonist includes a PD-1
binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding
antagonist.
[0270] In some embodiments, the PD-1 binding antagonist is a
molecule that inhibits the binding of PD-1 to its ligand binding
partners. In a specific aspect the PD-1 ligand binding partners are
PD-L1 and/or PD-L2. In another embodiment, a PD-L1 binding
antagonist is a molecule that inhibits the binding of PD-L1 to its
binding partners. In a specific aspect, PD-L1 binding partners are
PD-1 and/or B7-1. In another embodiment, the PD-L2 binding
antagonist is a molecule that inhibits the binding of PD-L2 to its
binding partners. In a specific aspect, a PD-L2 binding partner is
PD-1. The antagonist may be an antibody, an antigen binding
fragment thereof, an immunoadhesin, a fusion protein, or
oligopeptide.
[0271] In some embodiments, the PD-1 binding antagonist is selected
from MDX-1106 (nivolumab), Merck 3745 (lambrolizumab), CT-011
(pidilizumab), and AMP-224. In some embodiments, the PD-L1 binding
antagonist is selected from YW243.55.S70, MPDL3280A, MDX-1105, and
MEDI 4736. In some embodiments, the PD-L2 binding antagonist is
AMP-224. In some embodiments, the PD-1 binding antagonist is
AMP-224. MDX-1105, also known as BMS-936559, is an anti-PD-L1
antibody described in WO2007/005874. Antibody YW243.55.S70 (SEQ ID
No. 20) is an anti-PD-L1 described in WO 2010/077634 A1 and U.S.
Pat. No. 8,217,149, which are incorporated herein by reference.
MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or
nivolumab, is an anti-PD-1 antibody described in WO2006/121168.
Merck 3745, also known as MK 3475, MK-3475, SCH-900475, or
lambrolizumab, is an anti-PD-1 antibody described in WO2009/114335.
CT-011, also known as hBAT, hBAT-1, or pidilizumab, is an anti-PD-1
antibody described in WO2009/101611. AMP-224, also known as
B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in
WO2010/027827 and WO2011/066342.
[0272] Examples of anti-PD-L1 antibodies useful for the methods of
this invention, and methods for making thereof are described in PCT
patent application WO 2010/077634 A1 and U.S. Pat. No. 8,217,149,
which are incorporated herein by reference.
[0273] In some embodiments, the PD-1 axis binding antagonist is an
anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody
is capable of inhibiting binding between PD-L1 and PD-1 and/or
between PD-L1 and B7-1. In some embodiments, the anti-PD-L1
antibody is a monoclonal antibody. In some embodiments, the
anti-PD-L1 antibody is an antibody fragment selected from the group
consisting of Fab, Fab'-SH, Fv, scFv, and (Fab').sub.2 fragments.
In some embodiments, the anti-PD-L1 antibody is a humanized
antibody. In some embodiments, the anti-PD-L1 antibody is a human
antibody.
[0274] The anti-PD-L1 antibodies useful in this invention,
including compositions containing such antibodies, such as those
described in WO 2010/077634 A1 and U.S. Pat. No. 8,217,149, may be
used in combination with an agent that decreases or inhibits TIGIT
expression and/or activity with or without any additional therapy
(e.g., chemotherapy) to treat cancer or an immune related disease
(e.g., T cell dysfunctional disorder, viral infection, chronic
viral infection, etc.).
[0275] In one embodiment, the anti-PD-L1 antibody contains a heavy
chain variable region polypeptide comprising an HVR-H1, HVR-H2 and
HVR-H3 sequence, wherein:
TABLE-US-00025 (a) the HVR-H1 sequence is (SEQ ID NO: 33)
GFTFSX.sub.1SWIH; (b) the HVR-H2 sequence is (SEQ ID NO: 34)
AWIX.sub.2PYGGSX.sub.3YYADSVKG; (c) the HVR-H3 sequence is (SEQ ID
NO: 19) RHWPGGFDY;
[0276] further wherein: X.sub.1 is D or G; X.sub.2 is S or L;
X.sub.3 is T or S.
[0277] In one specific aspect, X.sub.1 is D; X.sub.2 is S and
X.sub.3 is T. In another aspect, the polypeptide further comprises
variable region heavy chain framework sequences juxtaposed between
the HVRs according to the formula:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the framework
sequences are VH subgroup III consensus framework. In a still
further aspect, at least one of the framework sequences is the
following:
TABLE-US-00026 HC-FR1 is (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 is (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3 is (SEQ ID NO: 27)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 is (SEQ ID NO: 28)
WGQGTLVTVSA.
[0278] In a still further aspect, the heavy chain polypeptide is
further combined with a variable region light chain comprising an
HVR-L1, HVR-L2 and HVR-L3, wherein:
TABLE-US-00027 (a) the HVR-L1 sequence is (SEQ ID NO: 35)
RASQX.sub.4X.sub.5X.sub.6TX.sub.7X.sub.8A; (b) the HVR-L2 sequence
is (SEQ ID NO: 36) SASX.sub.9LX.sub.10S,; (c) the HVR-L3 sequence
is (SEQ ID NO: 37)
QQX.sub.11X.sub.12X.sub.13X.sub.14PX.sub.15T;
further wherein: X.sub.4 is D or V; X.sub.5 is V or I; X.sub.6 is S
or N; X.sub.7 is A or F; X.sub.8 is V or L; X.sub.9 is F or T;
X.sub.10 is Y or A; X.sub.11 is Y, G, F, or S; X.sub.12 is L, Y, F
or W; X.sub.13 is Y, N, A, T, G, F or I; X.sub.14 is H, V, P, T or
I; X.sub.15 is A, W, R, P or T.
[0279] In a still further aspect, X4 is D; X5 is V; X6 is S; X7 is
A; X8 is V; X9 is F; X10 is Y; X11 is Y; X12 is L; X13 is Y; X14 is
H; X15 is A. In a still further aspect, the light chain further
comprises variable region light chain framework sequences
juxtaposed between the HVRs according to the formula:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
a still further aspect, the framework sequences are derived from
human consensus framework sequences. In a still further aspect, the
framework sequences are V.sub.L kappa I consensus framework. In a
still further aspect, at least one of the framework sequence is the
following:
TABLE-US-00028 LC-FR1 is (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 is (SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 is (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 is (SEQ ID NO: 32)
FGQGTKVEIKR.
[0280] In another embodiment, provided is an isolated anti-PD-L1
antibody or antigen binding fragment comprising a heavy chain and a
light chain variable region sequence, wherein:
[0281] (a) the heavy chain comprises and HVR-H1, HVR-H2 and HVR-H3,
wherein further:
TABLE-US-00029 (i) the HVR-H1 sequence is (SEQ ID NO: 33)
GFTFSX.sub.1SWIH; (ii) the HVR-H2 sequence is (SEQ ID NO: 34)
AWIX.sub.2PYGGSX.sub.3YYADSVKG (iii) the HVR-H3 sequence is (SEQ ID
NO: 19) RHWPGGFDY, and
[0282] (b) the light chain comprises and HVR-L1, HVR-L2 and HVR-L3,
wherein further:
TABLE-US-00030 (i) the HVR-L1 sequence is (SEQ ID NO: 35)
RASQX.sub.4X.sub.5X.sub.6TX.sub.7X.sub.8A (ii) the HVR-L2 sequence
is (SEQ ID NO: 36) SASX.sub.9LX.sub.10S; and (iii) the HVR-L3
sequence is (SEQ ID NO: 37)
QQX.sub.11X.sub.12X.sub.13X.sub.14PX.sub.15T;
further wherein: X.sub.1 is D or G; X.sub.2 is S or L; X.sub.3 is T
or S; X.sub.4 is D or V; X.sub.5 is V or I; X.sub.6 is S or N;
X.sub.7 is A or F; X.sub.8 is V or L; X.sub.9 is F or T; X.sub.10
is Y or A; X.sub.11 is Y, G, F, or S; X.sub.12 is L, Y, F or W;
X.sub.13 is Y, N, A, T, G, F or I; X.sub.14 is H, V, P, T or I;
X.sub.15 is A, W, R, P or T.
[0283] In a specific aspect, X.sub.1 is D; X.sub.2 is S and X.sub.3
is T. In another aspect, X.sub.4 is D; X.sub.5 is V; X.sub.6 is S;
X.sub.7 is A; X.sub.8 is V; X.sub.9 is F; X.sub.10 is Y; X.sub.11
is Y; X.sub.12 is L; X.sub.13 is Y; X.sub.14 is H; X.sub.15 is A.
In yet another aspect, X.sub.1 is D; X.sub.2 is S and X.sub.3 is T,
X.sub.4 is D; X.sub.5 is V; X.sub.6 is S; X.sub.7 is A; X.sub.8 is
V; X.sub.9 is F; X.sub.10 is Y; X.sub.11 is Y; X.sub.12 is L;
X.sub.13 is Y; X.sub.14 is H and X.sub.15 is A.
[0284] In a further aspect, the heavy chain variable region
comprises one or more framework sequences juxtaposed between the
HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
a still further aspect, the framework sequences are derived from
human consensus framework sequences. In a still further aspect, the
heavy chain framework sequences are derived from a Kabat subgroup
I, II, or III sequence. In a still further aspect, the heavy chain
framework sequence is a VH subgroup III consensus framework. In a
still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00031 HC-FR1 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 27)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 28)
WGQGTLVTVSA.
[0285] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00032 LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 (SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 (SEQ ID NO: 32)
FGQGTKVEIKR.
[0286] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or
aglycosylation. In still a further embodiment, the effector-less Fc
mutation is an N297A or D265A/N297A substitution in the constant
region.
[0287] In yet another embodiment, provided is an anti-PD-L1
antibody comprising a heavy chain and a light chain variable region
sequence, wherein: [0288] (a) the heavy chain further comprises and
HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence
identity to GFTFSDSWIH (SEQ ID NO:17), AWISPYGGSTYYADSVKG (SEQ ID
NO:18) and RHWPGGFDY (SEQ ID NO:19), respectively, or [0289] (b)
the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3
sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ
ID NO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT (SEQ ID NO:22),
respectively. [0290] (c) In a specific aspect, the sequence
identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100%. In another aspect, the heavy chain variable
region comprises one or more framework sequences juxtaposed between
the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a still further aspect, the heavy
chain framework sequences are derived from a Kabat subgroup I, II,
or III sequence. In a still further aspect, the heavy chain
framework sequence is a V.sub.H subgroup III consensus framework.
In a still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00033 [0290] HC-FR1 (SEQ ID NO: 25)
EVQLVESGGGLVQPGGSLRLSCAAS HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV
HC-FR3 (SEQ ID NO: 27) RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ
ID NO: 28) WGQGTLVTVSA.
[0291] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00034 LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 (SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 (SEQ ID NO: 32)
FGQGTKVEIKR.
[0292] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or
aglycosylation. In still a further embodiment, the effector-less Fc
mutation is an N297A or D265A/N297A substitution in the constant
region.
[0293] In a still further embodiment, provided is an isolated
anti-PD-L1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein:
[0294] (a) the heavy chain sequence has at least 85% sequence
identity to the heavy chain sequence:
TABLE-US-00035 (SEQ ID NO: 23)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSA, (SEQ ID NO: 40)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSSASTK, or (SEQ ID NO: 41)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSS,
or
[0295] (b) the light chain sequences has at least 85% sequence
identity to the light chain sequence:
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID
NO:24).
[0296] In a specific aspect, the sequence identity is 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In another aspect, the heavy chain variable region comprises one or
more framework sequences juxtaposed between the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the heavy chain
framework sequences are derived from a Kabat subgroup I, II, or III
sequence. In a still further aspect, the heavy chain framework
sequence is a VH subgroup III consensus framework. In a still
further aspect, one or more of the heavy chain framework sequences
is the following:
TABLE-US-00036 HC-FR1 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 27)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 28)
WGQGTLVTVSA.
[0297] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00037 LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 (SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 (SEQ ID NO: 32)
FGQGTKVEIKR.
[0298] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect, the minimal effector
function results from production in prokaryotic cells. In a still
further specific aspect the minimal effector function results from
an "effector-less Fc mutation" or aglycosylation. In still a
further embodiment, the effector-less Fc mutation is an N297A or
D265A/N297A substitution in the constant region.
[0299] In a still further embodiment, the invention provides for
compositions comprising any of the above described anti-PD-L1
antibodies in combination with at least one
pharmaceutically-acceptable carrier.
[0300] In a still further embodiment, provided is an isolated
nucleic acid encoding a light chain or a heavy chain variable
region sequence of an anti-PD-L1 antibody, wherein:
[0301] (a) the heavy chain further comprises and HVR-H1, HVR-H2 and
an HVR-H3 sequence having at least 85% sequence identity to
GFTFSDSWIH (SEQ ID NO:17), AWISPYGGSTYYADSVKG (SEQ ID NO:18) and
RHWPGGFDY (SEQ ID NO:19), respectively, and
[0302] (b) the light chain further comprises an HVR-L1, HVR-L2 and
an HVR-L3 sequence having at least 85% sequence identity to
RASQDVSTAVA (SEQ ID NO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT
(SEQ ID NO:22), respectively.
[0303] In a specific aspect, the sequence identity is 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In aspect, the heavy chain variable region comprises one or more
framework sequences juxtaposed between the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the heavy chain
framework sequences are derived from a Kabat subgroup I, II, or III
sequence. In a still further aspect, the heavy chain framework
sequence is a VH subgroup III consensus framework. In a still
further aspect, one or more of the heavy chain framework sequences
is the following:
TABLE-US-00038 HC-FR1 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 27)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 28)
WGQGTLVTVSA.
[0304] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00039 LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 (SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 (SEQ ID NO: 32)
FGQGTKVEIKR.
[0305] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect, the minimal effector
function results from production in prokaryotic cells. In a still
further specific aspect the minimal effector function results from
an "effector-less Fc mutation" or aglycosylation. In still a
further aspect, the effector-less Fc mutation is an N297A or
D265A/N297A substitution in the constant region.
[0306] In another further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein:
[0307] (a) the heavy chain sequence has at least 85% sequence
identity to the heavy chain sequence:
TABLE-US-00040 (SEQ ID NO: 41)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSS,
or
[0308] (b) the light chain sequences has at least 85% sequence
identity to the light chain sequence:
TABLE-US-00041 (SEQ ID NO: 24)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY
SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKR.
[0309] In a specific aspect, the sequence identity is 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In another aspect, the heavy chain variable region comprises one or
more framework sequences juxtaposed between the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the heavy chain
framework sequences are derived from a Kabat subgroup I, II, or III
sequence. In a still further aspect, the heavy chain framework
sequence is a VH subgroup III consensus framework. In a still
further aspect, one or more of the heavy chain framework sequences
is the following:
TABLE-US-00042 HC-FR1 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 27)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 42)
WGQGTLVTVSS.
[0310] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00043 LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 (SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 (SEQ ID NO: 32)
FGQGTKVEIKR.
[0311] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect, the minimal effector
function results from production in prokaryotic cells. In a still
further specific aspect the minimal effector function results from
an "effector-less Fc mutation" or aglycosylation. In still a
further embodiment, the effector-less Fc mutation is an N297A or
D265A/N297A substitution in the constant region.
[0312] In a further aspect, the heavy chain variable region
comprises one or more framework sequences juxtaposed between the
HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
a still further aspect, the framework sequences are derived from
human consensus framework sequences. In a still further aspect, the
heavy chain framework sequences are derived from a Kabat subgroup
I, II, or III sequence. In a still further aspect, the heavy chain
framework sequence is a VH subgroup III consensus framework. In a
still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00044 HC-FR1 (SEQ ID NO: 43)
EVQLVESGGGLVQPGGSLRLSCAASGFTFS HC-FR2 (SEQ ID NO: 44)
WVRQAPGKGLEWVA HC-FR3 (SEQ ID NO: 27)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 45)
WGQGTLVTVSS.
[0313] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00045 LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 (SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 (SEQ ID NO: 46)
FGQGTKVEIK.
[0314] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or
aglycosylation. In still a further embodiment, the effector-less Fc
mutation is an N297A or D265A/N297A substitution in the constant
region.
[0315] In yet another embodiment, provided is an anti-PDL1 antibody
comprising a heavy chain and a light chain variable region
sequence, wherein: [0316] (d) the heavy chain further comprises and
HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence
identity to GFTFSDSWIH (SEQ ID NO:17), AWISPYGGSTYYADSVKG (SEQ ID
NO:18) and RHWPGGFDY (SEQ ID NO:19), respectively, or [0317] (e)
the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3
sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ
ID NO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT (SEQ ID NO:22),
respectively.
[0318] In a specific aspect, the sequence identity is 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In another aspect, the heavy chain variable region comprises one or
more framework sequences juxtaposed between the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a still further aspect, the heavy
chain framework sequences are derived from a Kabat subgroup I, II,
or III sequence. In a still further aspect, the heavy chain
framework sequence is a VH subgroup III consensus framework. In a
still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00046 HC-FR1 (SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 (SEQ ID NO: 26) WVRQAPGKGLEWV HC-FR3 (SEQ ID NO: 27)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 47)
WGQGTLVTVSSASTK.
[0319] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00047 LC-FR1 (SEQ ID NO: 29) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 (SEQ ID NO: 30) WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 31)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 (SEQ ID NO: 32)
FGQGTKVEIKR.
[0320] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or
aglycosylation. In still a further embodiment, the effector-less Fc
mutation is an N297A or D265A/N297A substitution in the constant
region.
[0321] In a still further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein:
[0322] (a) the heavy chain sequence has at least 85% sequence
identity to the heavy chain sequence:
TABLE-US-00048 (SEQ ID NO: 40)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSSASTK,
or
[0323] (b) the light chain sequences has at least 85% sequence
identity to the light chain sequence:
TABLE-US-00049 (SEQ ID NO: 24)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY
SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF GQGTKVEIKR.
[0324] In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain variable region
sequence, wherein the light chain variable region sequence has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity to the amino acid sequence of SEQ ID
NO:24. In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain variable region
sequence, wherein the heavy chain variable region sequence has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity to the amino acid sequence of SEQ ID
NO:40. In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain variable region
sequence, wherein the light chain variable region sequence has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity to the amino acid sequence of SEQ ID
NO:24 and the heavy chain variable region sequence has at least
85%, at least 86%, at least 87%, at least 88%, at least 89%, at
least 90%, at least 91%, at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identity to the amino acid sequence of SEQ ID
NO:40.
[0325] In a still further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
sequence, wherein:
[0326] (a) the heavy chain sequence has at least 85% sequence
identity to the heavy chain sequence:
TABLE-US-00050 (SEQ ID NO: 48)
EVQLVESGGGLVQPGGSLRLSCAASGFTESDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR
RHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP
PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG,
or
[0327] (b) the light chain sequences has at least 85% sequence
identity to the light chain sequence:
TABLE-US-00051 (SEQ ID NO: 49)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY
SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF
GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV
THQGLSSPVTKSFNRGEC.
[0328] In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain sequence,
wherein the light chain sequence has at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% sequence identity
to the amino acid sequence of SEQ ID NO:49. In some embodiments,
provided is an isolated anti-PDL1 antibody comprising a heavy chain
and a light chain sequence, wherein the heavy chain sequence has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity to the amino acid sequence of SEQ ID
NO:48. In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain sequence,
wherein the light chain sequence has at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% sequence identity
to the amino acid sequence of SEQ ID NO:49 and the heavy chain
sequence has at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% sequence identity to the amino acid
sequence of SEQ ID NO:48.
[0329] In a still further aspect, the nucleic acid further
comprises a vector suitable for expression of the nucleic acid
encoding any of the previously described anti-PD-L1 antibodies. In
a still further specific aspect, the vector further comprises a
host cell suitable for expression of the nucleic acid. In a still
further specific aspect, the host cell is a eukaryotic cell or a
prokaryotic cell. In a still further specific aspect, the
eukaryotic cell is a mammalian cell, such as Chinese Hamster Ovary
(CHO).
[0330] The anti-PD-L1 antibody or antigen binding fragment thereof,
may be made using methods known in the art, for example, by a
process comprising culturing a host cell containing nucleic acid
encoding any of the previously described anti-PD-L1 antibodies or
antigen-binding fragment in a form suitable for expression, under
conditions suitable to produce such antibody or fragment, and
recovering the antibody or fragment.
[0331] In a still further embodiment, the invention provides for a
composition comprising an anti-PD-L1 antibody or antigen binding
fragment thereof as provided herein and at least one
pharmaceutically acceptable carrier.
Agents that Decreases or Inhibits TIGIT Expression and/or
Activity
[0332] Provided herein is a method for treatment or delaying
progression of cancer in an individual comprising administering to
the individual an effective amount of a PD-1 axis binding
antagonist in combination with an agent that decreases or inhibits
TIGIT expression and/or activity. Provided herein is also a method
for reducing or inhibiting cancer relapse or cancer progression in
an individual comprising administering to the individual an
effective amount of a PD-1 axis binding antagonist in combination
with an agent that decreases or inhibits TIGIT expression and/or
activity. Provided herein is also a method for treating or delaying
progression of an immune related disease in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist in combination with an agent that
decreases or inhibits TIGIT expression and/or activity. Provided
herein is also a method for reducing or inhibiting progression of
an immune related disease in an individual comprising administering
to the individual an effective amount of a PD-1 axis binding
antagonist in combination with an agent that decreases or inhibits
TIGIT expression and/or activity. Provided herein is also a method
for increasing, enhancing or stimulating an immune response or
function in an individual comprising administering to the
individual an effective amount of a PD-1 axis binding antagonist in
combination with an agent that decreases or inhibits TIGIT
expression and/or activity. Provided herein is also a method for
increasing, enhancing or stimulating an immune response or function
in an individual comprising administering to the individual an
effective amount of an agent that decreases or inhibits TIGIT
expression and/or activity and an agent that decreases or inhibits
one or more additional immune co-inhibitory receptors. Provided
herein is also a method for increasing, enhancing or stimulating an
immune response or function in an individual comprising
administering to the individual an effective amount of an agent
that decreases or inhibits TIGIT expression and/or activity and an
agent that increases or activates one or more additional immune
co-stimulatory receptors. For example, agent that decreases or
inhibits TIGIT expression and/or activity includes an antagonist of
TIGIT expression and/or activity, an antagonist of PVR expression
and/or activity, an agent that inhibits and/or blocks the
interaction of TIGIT with PVR, an agent that inhibits and/or blocks
the interaction of TIGIT with PVRL2, an agent that inhibits and/or
blocks the interaction of TIGIT with PVRL3, an agent that inhibits
and/or blocks the intracellular signaling mediated by TIGIT binding
to PVR, an agent that inhibits and/or blocks the intracellular
signaling mediated by TIGIT binding to PVRL2, an agent that
inhibits and/or blocks the intracellular signaling mediated by
TIGIT binding to PVRL3, and combinations thereof.
[0333] In some embodiments, the antagonist of TIGIT expression
and/or activity includes a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide.
[0334] In some embodiments, the antagonist of PVR expression and/or
activity includes a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide.
[0335] In some embodiments, the agent that inhibits and/or blocks
the interaction of TIGIT with PVR includes a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0336] In some embodiments, the agent that inhibits and/or blocks
the interaction of TIGIT with PVRL2 includes a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0337] In some embodiments, the agent that inhibits and/or blocks
the interaction of TIGIT with PVRL3 includes a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide.
[0338] In some embodiments, the agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVR
includes a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide.
[0339] In some embodiments, the agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVRL2
includes a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide.
[0340] In some embodiments, the agent that inhibits and/or blocks
the intracellular signaling mediated by TIGIT binding to PVRL3
includes a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide.
[0341] In some embodiments, the antagonist of TIGIT expression
and/or activity is an inhibitory nucleic acid selected from an
antisense polynucleotide, an interfering RNA, a catalytic RNA, and
an RNA-DNA chimera.
[0342] In some embodiments, the antagonist of TIGIT expression
and/or activity is an anti-TIGIT antibody or antigen-binding
fragment thereof.
[0343] The anti-TIGIT antibodies useful in this invention,
including compositions containing such antibodies, such as those
described in WO 2009/126688, may be used in combination with PD-1
axis binding antagonists.
Anti-TIGIT Antibodies
[0344] The present invention provides anti-TIGIT antibodies.
Exemplary antibodies include polyclonal, monoclonal, humanized,
bispecific, and heteroconjugate antibodies. It will be understood
by one of ordinary skill in the art that the invention also
provides antibodies against other polypeptides (i.e., anti-PVR
antibodies) and that any of the description herein drawn
specifically to the method of creation, production, varieties, use
or other aspects of anti-TIGIT antibodies will also be applicable
to antibodies specific for other non-TIGIT polypeptides.
Polyclonal Antibodies
[0345] The anti-TIGIT antibodies may comprise polyclonal
antibodies. Methods of preparing polyclonal antibodies are known to
the skilled artisan. Polyclonal antibodies can be raised in a
mammal, for example, by one or more injections of an immunizing
agent and, if desired, an adjuvant. Typically, the immunizing agent
and/or adjuvant will be injected in the mammal by multiple
subcutaneous or intraperitoneal injections. The immunizing agent
may include the TIGIT polypeptide or a fusion protein thereof. It
may be useful to conjugate the immunizing agent to a protein known
to be immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
Monoclonal Antibodies
[0346] The anti-TIGIT antibodies may, alternatively, be monoclonal
antibodies. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature,
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in
vitro.
[0347] The immunizing agent will typically include the TIGIT
polypeptide or a fusion protein thereof. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell [Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells may be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0348] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies [Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
[0349] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against the polypeptide. Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma
cells is determined by immunoprecipitation or by an in vitro
binding assay, such as radioimmunoassay (RIA) or enzyme-linked
immunoabsorbent assay (ELISA). Such techniques and assays are known
in the art. The binding affinity of the monoclonal antibody can,
for example, be determined by the Scatchard analysis of Munson and
Pollard, Anal. Biochem., 107:220 (1980).
[0350] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0351] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0352] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences [U.S.
Pat. No. 4,816,567; Morrison et al., supra] or by covalently
joining to the immunoglobulin coding sequence all or part of the
coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0353] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0354] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
Human and Humanized Antibodies
[0355] The anti-TIGIT antibodies of the invention may further
comprise humanized antibodies or human antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab).sub.2 or other antigen-binding subsequences
of antibodies) which contain minimal sequence derived from
non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0356] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0357] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0358] The antibodies may also be affinity matured using known
selection and/or mutagenesis methods as described above. Preferred
affinity matured antibodies have an affinity which is five times,
more preferably 10 times, even more preferably 20 or 30 times
greater than the starting antibody (generally murine, humanized or
human) from which the matured antibody is prepared.
Bispecific Antibodies
[0359] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for TIGIT, the other one is for any other antigen,
and preferably for a cell-surface protein or receptor or receptor
subunit.
[0360] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities [Milstein and Cuello, Nature, 305:537-539
(1983)]. Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0361] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0362] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0363] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared can be prepared
using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a procedure wherein intact antibodies are proteolytically
cleaved to generate F(ab')2 fragments. These fragments are reduced
in the presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0364] Fab' fragments may be directly recovered from E. coli and
chemically coupled to form bispecific antibodies. Shalaby et al.,
J. Exp. Med. 175:217-225 (1992) describe the production of a fully
humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment
was separately secreted from E. coli and subjected to directed
chemical coupling in vitro to form the bispecific antibody. The
bispecific antibody thus formed was able to bind to cells
overexpressing the ErbB2 receptor and normal human T cells, as well
as trigger the lytic activity of human cytotoxic lymphocytes
against human breast tumor targets.
[0365] Various technique for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
[0366] Antibodies with more than two valencies are contemplated. As
one nonlimiting example, trispecific antibodies can be prepared.
See, e.g., Tutt et al., J. Immunol. 147:60 (1991).
[0367] Exemplary bispecific antibodies may bind to two different
epitopes on a given TIGIT polypeptide herein. Alternatively, an
anti-TIGIT polypeptide arm may be combined with an arm which binds
to a triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the cell expressing the particular TIGIT polypeptide. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express a particular TIGIT polypeptide. These antibodies
possess a TIGIT-binding arm and an arm which binds a cytotoxic
agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or
TETA. Another bispecific antibody of interest binds the TIGIT
polypeptide and further binds tissue factor (TF).
Heteroconjugate Antibodies
[0368] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
Effector Function Engineering
[0369] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) may be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design, 3: 219-230 (1989).
[0370] In some embodiment, anti-TIGIT antibodies were generated
which were hamster-anti-mouse antibodies. Two antibodies, 10A7 and
1F4, also specifically bound to human TIGIT. The amino acid
sequences of the light and heavy chains of the 10A7 antibody were
determined using standard techniques. The light chain sequence of
this antibody is:
TABLE-US-00052 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQS
PKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGI
NNPLTFGDGTKLEIKR
and the heavy chain sequence of this antibody is:
TABLE-US-00053 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR
RPLGHNTFDSWGQGTLVTVSS,
where the complementarity determining regions (CDRs) of each chain
are represented by bold text. Thus, CDR1 of the 10A7 light chain
has the sequence KSSQSLYYSGVKENLLA (SEQ ID NO:1), CDR2 of the 10A7
light chain has the sequence ASIRFT (SEQ ID NO:2), and CDR3 of the
10A7 light chain has the sequence QQGINNPLT (SEQ ID NO:3). CDR1 of
the 10A7 heavy chain has the sequence GFTFSSFTMH (SEQ ID NO:4),
CDR2 of the 10A7 heavy chain has the sequence FIRSGSGIVFYADAVRG
(SEQ ID NO:5), and CDR3 of the 10A7 heavy chain has the sequence
RPLGHNTFDS (SEQ ID NO:6).
[0371] The amino acid sequences of the light and heavy chains of
the 1F4 antibody were also determined. The light chain sequence of
this antibody is:
TABLE-US-00054 (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSP
QLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTH
QPPTFGPGTKLEVK
and the heavy chain sequence of this antibody is:
TABLE-US-00055 (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIG
LIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSR
GLRGFYAMDYWGQGTSVTVSS,
where the complementarity determining regions (CDRs) of each chain
are represented by bold text. Thus, CDR1 of the 1F4 light chain has
the sequence RSSQSLVNSYGNTFLS (SEQ ID NO:7), CDR2 of the 1F4 light
chain has the sequence GISNRFS (SEQ ID NO:8), and CDR3 of the 1F4
light chain has the sequence LQGTHQPPT (SEQ ID NO:9). CDR1 of the
1F4 heavy chain has the sequence GYSFTGHLMN (SEQ ID NO:10), CDR2 of
the 1F4 heavy chain has the sequence LIIPYNGGTSYNQKFKG (SEQ ID
NO:11), and CDR3 of the 1F4 heavy chain has the sequence GLRGFYAMDY
(SEQ ID NO:12).
[0372] The nucleotide sequence encoding the 1F4 light chain was
determined to be
TABLE-US-00056 (SEQ ID NO: 38)
GATGTTGTGTTGACTCAAACTCCACTCTCCCTGTCTGTCAGCTTTGGAG
ATCAAGTTTCTATCTCTTGCAGGTCTAGTCAGAGTCTTGTAAACAGTTA
TGGGAACACCTTTTTGTCTTGGTACCTGCACAAGCCTGGCCAGTCTCCA
CAGCTCCTCATCTTTGGGATTTCCAACAGATTTTCTGGGGTGCCAGACA
GGTTCAGTGGCAGTGGTTCAGGGACAGATTTCACACTCAAGATCAGCAC
AATAAAGCCTGAGGACTTGGGAATGTATTACTGCTTACAAGGTACGCAT
CAGCCTCCCACGTTCGGTCCTGGGACCAAGCTGGAGGTGAAA
and the nucleotide sequence encoding the 1F4 heavy chain was
determined to be
TABLE-US-00057 (SEQ ID NO: 39)
GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAACTT
CAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCCATCT
TATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGA
CTTATTATTCCTTACAATGGTGGTACAAGCTATAACCAGAAGTTCAAGG
GCAAGGCCACATTGACTGTAGACAAGTCATCCAGCACAGCCTACATGGA
GCTCCTCAGTCTGACTTCTGATGACTCTGCAGTCTATTTCTGTTCAAGA
GGCCTTAGGGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAG
TCACCGTCTCCTCA.
[0373] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof comprises at least one HVR
comprising an amino acid sequence selected from the amino acid
sequences set forth in (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT
(SEQ ID NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4),
FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6), or
(2) RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8),
LQGTHQPPT (SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10),
LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and GLRGFYAMDY (SEQ ID
NO:12).
[0374] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof, wherein the antibody light chain
comprises the amino acid sequence set forth in
TABLE-US-00058 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQS
PKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGI NNPLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSP
QLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTH
QPPTFGPGTKLEVK.
[0375] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof, wherein the antibody heavy chain
comprises the amino acid sequence set forth in
TABLE-US-00059 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR
RPLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIG
LIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSR
GLRGFYAMDYWGQGTSVTVSS.
[0376] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof, wherein the antibody light chain
comprises the amino acid sequence set forth in
TABLE-US-00060 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQS
PKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGI NNPLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSP
QLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTH
QPPTFGPGTKLEVK.
and the antibody heavy chain comprises the amino acid sequence set
forth in
TABLE-US-00061 (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR
RPLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIG
LIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSR
GLRGFYAMDYWGQGTSVTVSS.
[0377] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof, wherein the antibody is selected
from a humanized antibody, a chimeric antibody, a bispecific
antibody, a heteroconjugate antibody, and an immunotoxin.
[0378] In some embodiments, the anti-TIGIT antibody or
antigen-binding fragment thereof comprises at least one HVR is at
least 90% identical to an HVR set forth in any of
TABLE-US-00062 (1) (SEQ ID NO: 1) KSSQSLYYSGVKENLLA, (SEQ ID NO: 2)
ASIRFT, (SEQ ID NO: 3) QQGINNPLT, (SEQ ID NO: 4) GFTFSSFTMH, (SEQ
ID NO: 5) FIRSGSGIVFYADAVRG, and (SEQ ID NO: 6) RPLGHNTFDS, or (2)
(SEQ ID NO: 7) RSSQSLVNSYGNTFLS, (SEQ ID NO: 8) GISNRFS, (SEQ ID
NO: 9) LQGTHQPPT, (SEQ ID NO: 10) GYSFTGHLMN, (SEQ ID NO: 11)
LIIPYNGGTSYNQKFKG, and (SEQ ID NO: 12) GLRGFYAMDY.
[0379] In some embodiments, the anti-TIGIT antibody or fragment
thereof comprises the light chain and/or heavy chain comprising
amino acid sequences at least 90% identical to the amino acid
sequences set forth in
TABLE-US-00063 (SEQ ID NO: 13)
DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQS
PKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGI NNPLTFGDGTKLEIKR
or (SEQ ID NO: 14)
DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSP
QLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTH QPPTFGPGTKLEVK,
or (SEQ ID NO: 15)
EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVA
FIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR
RPLGHNTFDSWGQGTLVTVSS or (SEQ ID NO: 16)
EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIG
LIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSR
GLRGFYAMDYWGQGTSVTVSS,
respectively. Agents that Modulate CD226 Expression and/or
Activity
[0380] Provided herein is a method of treating or delaying
progression of cancer in an individual comprising administering to
the individual an effective amount of a PD-1 axis binding
antagonist and an agent that modulates the CD226 expression and/or
activity. Provided herein is also a method for reducing or
inhibiting cancer relapse or cancer progression in an individual
comprising administering to the individual an effective amount of a
PD-1 axis binding antagonist and an agent that modulates the CD226
expression and/or activity. Provided herein is also a method for
treating or delaying progression of an immune related disease in an
individual comprising administering to the individual an effective
amount of a PD-1 axis binding antagonist and an agent that
modulates the CD226 expression and/or activity. Provided herein is
also a method for reducing or inhibiting progression of an immune
related disease in an individual comprising administering to the
individual an effective amount of a PD-1 axis binding antagonist
and agent that modulates the CD226 expression and/or activity.
Provided herein is also a method of increasing, enhancing or
stimulating an immune response or function in an individual by
administering to the individual an effective amount of a PD-1 axis
binding antagonist and an agent that modulates the CD226 expression
and/or activity.
[0381] For example, agents that modulate the CD226 expression
and/or activity are agents capable of increasing and/or stimulating
CD226 expression and/or activity, increasing and/or stimulating the
interaction of CD226 with PVR, PVRL2, and/or PVRL3, and increasing
and/or stimulating the intracellular signaling mediated by CD226
binding to PVR, PVRL2, and/or PVRL3. In some embodiments, agents
capable of increasing and/or stimulating CD226 expression and/or
activity are agents that increase and/or stimulate CD226 expression
and/or activity. In some embodiments, agents capable of increasing
and/or stimulating the interaction of CD226 with PVR, PVRL2, and/or
PVRL3 are agents that increase and/or stimulate the interaction of
CD226 with PVR, PVRL2, and/or PVRL3. In some embodiments, agents
capable of increasing and/or stimulating the intracellular
signaling mediated by CD226 binding to PVR, PVRL2, and/or PVRL3 are
agents that increase and/or stimulate the intracellular signaling
mediated by CD226 binding to PVR, PVRL2, and/or PVRL3.
[0382] In some embodiments, the agent that modulates the CD226
expression and/or activity is selected from an agent that inhibits
and/or blocks the interaction of CD226 with TIGIT, an antagonist of
TIGIT expression and/or activity, an antagonist of PVR expression
and/or activity, an agent that inhibits and/or blocks the
interaction of TIGIT with PVR, an agent that inhibits and/or blocks
the interaction of TIGIT with PVRL2, an agent that inhibits and/or
blocks the interaction of TIGIT with PVRL3, an agent that inhibits
and/or blocks the intracellular signaling mediated by TIGIT binding
to PVR, an agent that inhibits and/or blocks the intracellular
signaling mediated by TIGIT binding to PVRL2, an agent that
inhibits and/or blocks the intracellular signaling mediated by
TIGIT binding to PVRL3, and combinations thereof. In some
embodiments, the agent that inhibits and/or blocks the interaction
of CD226 with TIGIT is selected from a small molecule inhibitor, an
inhibitory antibody or antigen-binding fragment thereof, an
aptamer, an inhibitory nucleic acid, and an inhibitory polypeptide.
In some embodiments, the agent that inhibits and/or blocks the
interaction of CD226 with TIGIT is an anti-TIGIT antibody or
antigen-binding fragment thereof. In some embodiments, the agent
that inhibits and/or blocks the interaction of CD226 with TIGIT is
an inhibitory nucleic acid selected from an antisense
polynucleotide, an interfering RNA, a catalytic RNA, and an RNA-DNA
chimera.
[0383] In some embodiments, the antagonist of TIGIT expression
and/or activity is a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the antagonist of TIGIT expression and/or activity is
an anti-TIGIT antibody or antigen-binding fragment thereof. In some
embodiments, the antagonist of TIGIT expression and/or activity is
an inhibitory nucleic acid selected from an antisense
polynucleotide, an interfering RNA, a catalytic RNA, and an RNA-DNA
chimera. In some embodiments, the antagonist of PVR expression
and/or activity is a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the agent that inhibits and/or blocks the interaction
of TIGIT with PVR is a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the agent that inhibits and/or blocks the interaction
of TIGIT with PVRL2 is a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the agent that inhibits and/or blocks the interaction
of TIGIT with PVRL3 is a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the agent that inhibits and/or blocks the
intracellular signaling mediated by TIGIT binding to PVR is a small
molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide. In some embodiments, the agent that
inhibits and/or blocks the intracellular signaling mediated by
TIGIT binding to PVRL2 is a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the agent that inhibits and/or blocks the
intracellular signaling mediated by TIGIT binding to PVRL3 is a
small molecule inhibitor, an inhibitory antibody or antigen-binding
fragment thereof, an aptamer, an inhibitory nucleic acid, and an
inhibitory polypeptide.
[0384] In some embodiments, the antagonist of TIGIT expression
and/or activity includes a small molecule inhibitor, an inhibitory
antibody or antigen-binding fragment thereof, an aptamer, an
inhibitory nucleic acid, and an inhibitory polypeptide. In some
embodiments, the antagonist of PVR expression and/or activity
includes a small molecule inhibitor, an inhibitory antibody or
antigen-binding fragment thereof, an aptamer, an inhibitory nucleic
acid, and an inhibitory polypeptide. In some embodiments, the agent
that inhibits the intracellular signaling mediated by TIGIT binding
to PVR is selected from the group consisting of a small molecule
inhibitor, an inhibitory antibody or antigen-binding fragment
thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory
polypeptide. In some embodiments, the antagonist of TIGIT
expression and/or activity is an anti-TIGIT antibody or
antigen-binding fragment thereof. In some embodiments, the
antagonist of TIGIT expression and/or activity is an inhibitory
nucleic acid selected from an antisense polynucleotide, an
interfering RNA, a catalytic RNA, and an RNA-DNA chimera.
Combinations of T Cell Targets for Immunoregulatory Antibody
Therapy
[0385] In addition to specific antigen recognition through the TCR,
T-cell activation is regulated through a balance of positive and
negative signals provided by co-stimulatory receptors. These
surface proteins are typically members of either the TNF receptor
or B7 superfamilies. Activating co-stimulatory receptors include
CD226, CD28, OX40, GITR, CD137, CD27, HVEM, MICA, ICOS, NKG2D, and
2B4. Inhibitory co-stimulatory receptors include CTLA-4, PD-1,
TIM-3, BTLA, VISTA, LAG-3, B7H4, and CD96. Agonistic antibodies
directed against activating co-stimulatory molecules and blocking
antibodies against negative co-stimulatory molecules may enhance
T-cell stimulation to promote tumor destruction.
[0386] Provided herein is a method of increasing, enhancing or
stimulating an immune response or function in an individual by
administering to the individual an effective amount of an agent
that decreases or inhibits TIGIT expression and/or activity and an
agent that decreases or inhibits one or more additional immune
co-inhibitory receptors. In some embodiments, the one or more
additional immune co-inhibitory receptor is selected from PD-1,
CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and CD96. In some
embodiments, the one or more additional immune co-inhibitory
receptor is selected from PD-1, CTLA-4, LAG3 and TIM3.
[0387] Provided herein is also a method of increasing, enhancing or
stimulating an immune response or function in an individual by
administering to the individual an effective amount of an agent
that decreases or inhibits TIGIT expression and/or activity and an
agent that increases or activates one or more additional immune
co-stimulatory receptor. In some embodiments, the one or more
additional immune co-stimulatory receptor is selected from CD226,
OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4.
In some embodiments, the one or more additional immune
co-stimulatory receptor is selected from CD226, OX-40, CD27, CD137,
HVEM and GITR. In some embodiments, the one or more additional
immune co-stimulatory receptor is selected from OX-40 and CD27.
IV Kits
[0388] In another aspect, provided is a kit comprising a PD-1 axis
binding antagonist and a package insert comprising instructions for
using the PD-1 axis binding antagonist in combination with an agent
that decreases or inhibits TIGIT expression and/or activity to
treat or delay progression of cancer in an individual or for
enhancing immune function of an individual having cancer. Any of
the PD-1 axis binding antagonists and/or agents that decreases or
inhibits TIGIT expression and/or activity described herein may be
included in the kit.
[0389] In another aspect, provided is a kit comprising a PD-1 axis
binding antagonist and an agent that decreases or inhibits TIGIT
expression and/or activity, and a package insert comprising
instructions for using the PD-1 axis binding antagonist and the
agent that decreases or inhibits TIGIT expression and/or activity
to treat or delay progression of cancer in an individual or for
enhancing immune function of an individual having cancer. Any of
the PD-1 axis binding antagonists and/or agents that decreases or
inhibits TIGIT expression and/or activity described herein may be
included in the kit.
[0390] In another aspect, provided is a kit comprising an agent
that decreases or inhibits TIGIT expression and/or activity and a
package insert comprising instructions for using the agent that
decreases or inhibits TIGIT expression and/or activity in
combination with a PD-1 axis binding antagonist to treat or delay
progression of cancer in an individual or for enhancing immune
function of an individual having cancer. Any of the PD-1 axis
binding antagonists and/or agents that decreases or inhibits TIGIT
expression and/or activity described herein may be included in the
kit.
[0391] In another aspect, provided is a kit comprising a PD-1 axis
binding antagonist and a package insert comprising instructions for
using the PD-1 axis binding antagonist in combination with an agent
that modulates the CD226 expression and/or activity to treat or
delay progression of cancer in an individual. Any of the PD-1 axis
binding antagonists and/or agents that modulate the CD226
expression and/or activity described herein may be included in the
kit.
[0392] In another aspect, provided is a kit comprising a PD-1 axis
binding antagonist and an agent that modulates the CD226 expression
and/or activity, and a package insert comprising instructions for
using the PD-1 axis binding antagonist and the agent that modulates
the CD226 expression and/or activity to treat or delay progression
of cancer in an individual. Any of the PD-1 axis binding
antagonists and/or agents that modulate the CD226 expression and/or
activity described herein may be included in the kit.
[0393] In another aspect, provided is a kit comprising an agent
that modulates the CD226 expression and/or activity and a package
insert comprising instructions for using the agent modulates the
CD226 expression and/or activity in combination with a PD-1 axis
binding antagonist to treat or delay progression of cancer in an
individual. Any of the PD-1 axis binding antagonists and/or agents
that modulate the CD226 expression and/or activity described herein
may be included in the kit.
[0394] In another aspect, provided is a kit comprising a PD-1 axis
binding antagonist and a package insert comprising instructions for
using the PD-1 axis binding antagonist in combination with an agent
that modulates the CD226 expression and/or activity to enhance
immune function of an individual having cancer. Any of the PD-1
axis binding antagonists and/or agents that modulate the CD226
expression and/or activity described herein may be included in the
kit.
[0395] In another aspect, provided is a kit comprising a PD-1 axis
binding antagonist and an agent that modulates the CD226 expression
and/or activity, and a package insert comprising instructions for
using the PD-1 axis binding antagonist and the agent that modulates
the CD226 expression and/or activity to enhance immune function of
an individual having cancer. Any of the PD-1 axis binding
antagonists and/or agents that modulate the CD226 expression and/or
activity described herein may be included in the kit.
[0396] In another aspect, provided is a kit comprising an agent
modulates the CD226 expression and/or activity and a package insert
comprising instructions for using the agent that modulates the
CD226 expression and/or activity in combination with a PD-1 axis
binding antagonist to enhance immune function of an individual
having cancer. Any of the PD-1 axis binding antagonists and/or
agents that modulate the CD226 expression and/or activity described
herein may be included in the kit.
[0397] In another aspect, provided is a kit comprising an agent
that decreases or inhibits TIGIT expression and/or activity and a
package insert comprising instructions for using the agent that
decreases or inhibits TIGIT expression and/or activity in
combination with an agent that decreases or inhibits one or more
additional immune co-inhibitory receptors to treat or delay
progression of cancer in an individual or to enhance immune
function of an individual having cancer. Any of the agents that
decrease or inhibit TIGIT expression and/or activity and/or agents
that decrease or inhibit one or more additional immune
co-inhibitory receptors described herein may be included in the
kit.
[0398] In another aspect, provided is a kit comprising an agent
that decreases or inhibits TIGIT expression and/or activity and an
agent that decreases or inhibits one or more additional immune
co-inhibitory receptors, and a package insert comprising
instructions for using the agent that decreases or inhibits TIGIT
expression and/or activity and the agent that decreases or inhibits
one or more additional immune co-inhibitory receptors to treat or
delay progression of cancer in an individual or to enhance immune
function of an individual having cancer. Any of the agents that
decrease or inhibit TIGIT expression and/or activity and/or agents
that decrease or inhibit one or more additional immune
co-inhibitory receptors described herein may be included in the
kit.
[0399] In another aspect, provided is a kit comprising an agent
that decreases or inhibits one or more additional immune
co-inhibitory receptors and a package insert comprising
instructions for using the agent that decreases or inhibits one or
more additional immune co-inhibitory receptors in combination with
an agent that decreases or inhibits TIGIT expression and/or
activity to treat or delay progression of cancer in an individual
or to enhance immune function of an individual having cancer. Any
of the agents that decrease or inhibit TIGIT expression and/or
activity and/or agents that decrease or inhibit one or more
additional immune co-inhibitory receptors described herein may be
included in the kit.
[0400] In another aspect, provided is a kit comprising an agent
that decreases or inhibits TIGIT expression and/or activity and a
package insert comprising instructions for using the agent that
decreases or inhibits TIGIT expression and/or activity in
combination with an agent that increases or activates one or more
additional immune co-stimulatory receptors to treat or delay
progression of cancer in an individual or to enhance immune
function of an individual having cancer. Any of the agents that
decrease or inhibit TIGIT expression and/or activity and/or agents
that increase or activate one or more additional immune
co-stimulatory receptors described herein may be included in the
kit.
[0401] In another aspect, provided is a kit comprising an agent
that decreases or inhibits TIGIT expression and/or activity and an
agent that increases or activates one or more additional immune
co-stimulatory receptors, and a package insert comprising
instructions for using the agent that decreases or inhibits TIGIT
expression and/or activity and the agent that increases or
activates one or more additional immune co-stimulatory receptors to
treat or delay progression of cancer in an individual or to enhance
immune function of an individual having cancer. Any of the agents
that decrease or inhibit TIGIT expression and/or activity and/or
agents that increase or activate one or more additional immune
co-stimulatory receptors described herein may be included in the
kit.
[0402] In another aspect, provided is a kit comprising an agent
that increases or activates one or more additional immune
co-stimulatory receptors and a package insert comprising
instructions for using the agent that increases or activates one or
more additional immune co-stimulatory receptors in combination with
an agent that decreases or inhibits TIGIT expression and/or
activity to treat or delay progression of cancer in an individual
or to enhance immune function of an individual having cancer. Any
of the agents that decrease or inhibit TIGIT expression and/or
activity and/or agents that increase or activate one or more
additional immune co-stimulatory receptors described herein may be
included in the kit.
[0403] In some embodiments, the kit comprises a container
containing one or more of the PD-1 axis binding antagonists and
agents that decreases or inhibits TIGIT expression and/or activity
described herein. In some embodiments, the kit comprises a
container containing one or more of the PD-1 axis binding
antagonists and agents that modulates CD226 expression and/or
activity described herein. In some embodiments, the kit comprises a
container containing one or more of the agents that decrease or
inhibit TIGIT expression and/or activity and agents that decrease
or inhibit one or more additional immune co-inhibitory receptors
described herein. In some embodiments, the kit comprises a
container containing one or more of the agents that decrease or
inhibit TIGIT expression and/or activity and agents that increase
or activate one or more additional immune co-stimulatory receptors
described herein. Suitable containers include, for example,
bottles, vials (e.g., dual chamber vials), syringes (such as single
or dual chamber syringes) and test tubes. The container may be
formed from a variety of materials such as glass or plastic. In
some embodiments, the kit may comprise a label (e.g., on or
associated with the container) or a package insert. The label or
the package insert may indicate that the compound contained therein
may be useful or intended for treating or delaying progression of
cancer in an individual or for enhancing immune function of an
individual having cancer. The kit may further comprise other
materials desirable from a commercial and user standpoint,
including other buffers, diluents, filters, needles, and
syringes.
EXAMPLES
[0404] The invention can be further understood by reference to the
following examples, which are provided by way of illustration and
are not meant to be limiting.
Example 1: TIGIT is Highly Expressed on Exhausted CD8.sup.+ and
CD4.sup.+ T Cells and Correlated with PD-1 Expression
[0405] To confirm that CD8.sup.+ T cells are competent to express
TIGIT after stimulation in vitro, MACS-enriched C57BL6/J splenic
CD8.sup.+ T cells were stimulated with plate-bound anti-CD3 and
anti-CD28 for 24-48 hours in vitro. Flow cytometry was used to
measure TIGIT expression. In line with TIGIT's expression by
CD4.sup.+ T cells (Yu, X., et al. The surface protein TIGIT
suppresses T cell activation by promoting the generation of mature
immunoregulatory dendritic cells. Nature immunology 10, 48-57
(2009)), murine CD8.sup.+ T cells expressed TIGIT within 48 hours
of stimulation in vitro (FIG. 1A).
[0406] To assess TIGIT expression by activated CD8.sup.+ T cells in
vivo, C57BL6/J mice were infected with Armstrong strain Lymphocytic
Choriomeningitis Virus (LCMV), and splenocytes were analyzed 7 days
after infection. Briefly, for acute infections, mice were
intravenously infected with 2.times.10.sup.6 plaque-forming units
(PFU) Armstrong strain LCMV. Flow cytometry was used to measure
TIGIT expression by naive (CD44.sup.low CD62L.sup.high) and
effector memory (CD44.sup.high CD62L.sup.low) CD8.sup.+ and CD4+ T
cells. At the peak of the LCMV T cell response, a subset of
CD4.sup.+ effector memory T cells (T.sub.EM) and nearly all
CD8.sup.+ T.sub.EM cells strongly expressed TIGIT (FIG. 1B). Flow
cytometry was used to measure TIGIT expression by PD-1.sup.high and
PD-1.sup.low effector memory CD8.sup.+ T cells. Interestingly,
TIGIT expression was near perfectly correlated with PD-1 expression
(FIG. 1C).
[0407] Because PD-1 is associated with T cell exhaustion, TIGIT
expression was examined on chronically stimulated T cells. Briefly,
for chronic infections, C57BL6/J mice were intravenously infected
with 2.times.10.sup.6 PFU Clone 13 strain LCMV and treated with 500
ug and 250 ug of depleting anti-CD4 antibodies (clone GK1.5) 3 days
before and 4 days after infection, respectively. Where indicated,
mice infected with Clone 13 strain LCMV received intraperitoneal
injections of 200 ug of isotype control antibodies, 200 ug of
anti-PD-L1 antibodies, and/or 500 ug of anti-TIGIT antibodies 3
times per week from days 28 to 42 post-infection. Splenocytes were
analyzed 42 days after infection. Flow cytometry was used to
measure TIGIT expression by naive (CD44.sup.low CD62L.sup.high),
central memory (CD44.sup.high CD62L.sup.high), and effector memory
(CD44.sup.high CD62L.sup.low) CD8.sup.+ T cells. Indeed, in mice
chronically infected with Clone 13 strain LCMV, TIGIT was highly
expressed predominantly on PD-1.sup.high T cells but not on naive
cells, PD-1.sup.low T.sub.EM cells, or central memory T cells (FIG.
1D).
Example 2: A Role of TIGIT in T Cell Exhaustion in TIGIT Deficient
Mice
[0408] To characterize the role of TIGIT in T cell exhaustion, mice
in which TIGIT was conditionally deleted in T cells were generated
(TIGIT.sup.fl/fl CD4-cre.sup.+ (CKO), FIG. 2). Briefly, CD4.sup.cre
mice and TIGIT.sup.loxP/loxP mice were generated on a C57BL/6J
background with standard techniques and crossed. The quality-tested
ES cell line (Art B6/3.6 (genetic background: C57BL/6 NTac) was
grown on a mitotically inactivated feeder layer comprised of mouse
embryonic fibroblasts in ES cell culture medium containing Leukemia
inhibitory factor and Fetal Bovine Serum. The cells were
electroporated with the linearized DNA targeting vector according
to Taconic Artemis' Standard Operation Procedures. G418 and
Gancyclovir selection were used as mechanisms for enrichment of
homologously recombined clones. Resistant ES cell colonies (ES
clones) with a distinct morphology were isolated on day 8 after
transfection and analysed by Southern Blotting and/or PCR in a
primary screen. Homologous recombinant ES cell clones were expanded
and frozen in liquid nitrogen after extensive molecular validation.
The neo cassette was removed by flpE recombinase before
microinjection into Bl/6 female albino donors. Chimeric offspring
were produced and tails were screened by PCR for germline
transmission. TIGIT expression was ablated with 96% efficiency from
T cells in TIGIT.sup.loxP/loxP CD4.sup.cre mice.
[0409] Mice whose T cells lacked TIGIT mounted a CD4.sup.+ and
CD8.sup.+ T cell response to acute Armstrong strain LCMV infection
that was similar to wild-type mice (FIGS. 3A-3D).
[0410] To assess the effect in a chronic infection setting,
TIGIT.sup.fl/fl CD4-cre.sup.- (WT) and TIGIT.sup.fl/fl
CD4-cre.sup.+ (CKO) mice were briefly depleted of CD4.sup.+ T cells
and infected with Clone 13 strain LCMV. Splenocytes and liver viral
titers were analyzed 42 days after infection. After chronic
infection with Clone 13 strain LCMV, significantly more CD8.sup.+
and CD4.sup.+ T cells from TIGIT.sup.fl/fl CD4-cre.sup.+ (CKO) mice
were competent to produce interferon gamma (IFN .gamma.) than were
T cells from wildtype littermate mice (TIGIT.sup.fl/fl
CD4-cre.sup.- (WT)) (82-86% increase, P<0.01, FIGS. 4A-4D).
Furthermore, viral loads were significantly reduced in chronically
infected TIGIT.sup.fl/fl CD4-cre.sup.+ (CKO) mice (68% decrease,
P<0.0001, FIG. 4E).
[0411] These results suggest that TIGIT plays an important role in
regulating T cell activity and response during chronic immune
responses such as during a chronic viral infection, and that TIGIT
can regulate the effector function, in particular the competency to
produce effector cytokines, such as IFN.gamma. and TNF.alpha., of
chronically stimulated or exhausted CD8.sup.+ and CD4.sup.+ T
cells.
Example 3: TIGIT and PD-1 Synergistically Regulate the Effector
Function of Exhausted T Cells In Vivo
[0412] Since TIGIT expression was closely correlated with PD-1
expression, especially in CD8.sup.+ T cells during acute and
chronic viral infection (FIGS. 1A-1D), blocking TIGIT and PD-1 in
combination may restore T cell effector function to greater levels
than would be obtained by blocking either co-receptor singly.
[0413] To test this hypothesis, C57BL6/J mice were briefly depleted
of CD4.sup.+ T cells and infected with Clone 13 strain LCMV. For
chronic infections, mice were intravenously infected with
2.times.10.sup.6 PFU Clone 13 strain LCMV and treated with 500 ug
and 250 ug of depleting anti-CD4 antibodies (clone GK1.5) 3 days
before and 4 days after infection, respectively. Where indicated,
mice infected with Clone 13 strain LCMV received intraperitoneal
injections of 200 ug of isotype control antibodies, 200 ug of
anti-PD-L1 antibodies, and/or 500 ug of anti-TIGIT antibodies 3
times per week from days 28 to 42 post-infection. Treatment was
started at 28 days post-infection because the T cell response is
largely exhausted at this time-point in this model of chronic viral
infection (Wherry et al, Molecular Signature of CD8+ T cell
Exhaustion During Chronic Viral Infection, Immunity. 2007 October;
27(4):670-84). Splenocytes and liver viral titers were analyzed 42
days after infection.
[0414] In these mice, anti-PD-L1 treatment induced more robust
CD8.sup.+ T cell activation than did treatment with matched isotype
control antibodies (88% increase, P<0.0001, FIG. 4F), as
previously reported (Barber, D. L., et al. Restoring function in
exhausted CD8 T cells during chronic viral infection. Nature 439,
682-687 (2006)). Anti-TIGIT treatment had no apparent effect on
CD8.sup.+ T cell activation on its own or in combination with
anti-PD-L1 (FIG. 4F). Similarly, blockade of PD-1 alone moderately
increased CD8.sup.+ T cell cytokine competency, whereas blockade of
TIGIT alone had no effect (FIG. 4G). However, the frequency of
IFN.gamma.-producing CD8.sup.+ T cells was increased dramatically
in mice treated with both anti-TIGIT and anti-PD-L1, and to a
significantly greater extent than seen in mice treated with
anti-PD-L1 alone (FIG. 4G 93% increase, P=0.0050). A similar effect
was observed with CD4.sup.+ T cells (FIGS. 5A-5B). As also shown in
FIGS. 31A-31C, TIGIT/PD-L1 co-blockade significantly enhanced
CD8.sup.+ T cell effector function, but not CD4.sup.+ T cell
effector function, in mice compared to mice treated with anti-PD-L1
alone. Similar effects were also observed on T cell expansion and
effector function in LCMV gp33 antigen-specific T cells (FIGS.
31A-31C). These results demonstrate a strong synergy between PD-1
and TIGIT on exhausted CD8.sup.+ T cells, and indicate that TIGIT
specifically regulates CD8.sup.+ T cell cytokine competency and
effector function.
[0415] Consistent with these results, LCMV viral loads were
moderately reduced in mice treated with anti-PD-L1 alone, not
reduced in mice treated with anti-TIGIT alone, and substantially
reduced in mice treated with both anti-TIGIT and anti-PD-L1 (68%
viral titer reduction with anti-PD-L1 treatment, P=0.0004. 92%
viral titer reduction with anti-TIGIT+ anti-PD-L1 treatment,
P<0.0001, FIG. 4H). These data demonstrate a strong synergy
between the inhibitory effects of PD-1 and TIGIT, and suggest that
unlike PD-1, TIGIT is not a broad inhibitor of effector T cell
activation, but rather has a restricted role in limiting T cell
cytokine competency and effector function.
Example 4: TIGIT Expression is Elevated in Human Breast Cancer and
Correlated with Expression of CD8 and Inhibitory Co-Receptors
[0416] T cell exhaustion is also a major immunological feature of
cancer, with many tumor-infiltrating lymphocytes (TILs) expressing
high levels of inhibitory co-receptors and lacking the capacity to
produce effector cytokines (Wherry, E. J. T cell exhaustion. Nature
immunology 12, 492-499 (2011); Rabinovich, G. A., Gabrilovich, D.
& Sotomayor, E. M. Immunosuppressive strategies that are
mediated by tumor cells. Annual review of immunology 25, 267-296
(2007)).
[0417] To determine if TIGIT inhibits TIL effector function, breast
cancer gene expression microarray data generated by the Cancer
Genome Atlas Network (CGAN) was analyzed (Network, C. G. A.
Comprehensive molecular portraits of human breast tumours. Nature
490, 61-70 (2012)).
[0418] TIGIT expression was significantly elevated in breast tumors
overall (135% increase relative to normal samples,
P=6.times.10.sup.-12, FIG. 6A) and across the four major molecular
subtypes of breast cancer (FIG. 6A) (Perou, C. M., et al. Molecular
portraits of human breast tumours. Nature 406, 747-752 (2000);
Sorlie, T., et al. Gene expression patterns of breast carcinomas
distinguish tumor subclasses with clinical implications.
Proceedings of the National Academy of Sciences of the United
States of America 98, 10869-10874 (2001)). Expression of TIGIT was
highly correlated with expression of CD3c, consistent with its
expression by TILs (R.sup.2=0.61, FIG. 6B). Interestingly, TIGIT
expression was highly correlated with CD8a but not with CD4, or
only moderately correlated with CD4, suggesting that TIGIT might
primarily regulate CD8.sup.+ TIL function (CD8.alpha.,
R.sup.2=0.80. CD4, R.sup.2=0.42. FIG. 6C).
[0419] Given the co-expression of TIGIT and PD-1 during chronic
viral infection, we also assessed the correlation of PD-1 and other
inhibitory co-receptors with TIGIT in breast cancer. Correlation
between TIGIT and PD-1, CTLA4, and LAG3 was very strong (PD-1,
R.sup.2=0.87. CTLA4, R.sup.2=0.76. LAG3, R.sup.2=0.80. FIG. 6D).
Collectively, these data suggested that TIGIT was expressed by
TILs, especially CD8.sup.+ T cells, and that it might suppress
their function.
Example 5: TIGIT and PD-1 Inhibit Anti-Tumor T Cell Responses
[0420] To better characterize TIGIT by TILs in mice, BALB/C mice
were inoculated with CT26 colorectal carcinoma cells. Briefly,
BALB/c mice were subcutaneously inoculated with 1.times.10.sup.5
CT26 colon carcinoma cells suspended in matrigel (BD Biosciences)
into the right unilateral thoracic flank. After two weeks, mice
bearing tumors of approximately 200 mm.sup.3 were randomly
recruited into treatment groups receiving 35 mg/kg of isotype
control antibodies, anti-PD-L1 antibodies, and/or anti-TIGIT
antibodies by intraperitoneal injection 3 times per week for 3
weeks. Tumors were measured 2 times per week by caliper. Animals
whose tumors became ulcerated/necrotic or grew larger than 2000
mm.sup.3 were euthanized. Splenocytes and tumor-infiltrating
lymphocytes (TILs) were analyzed 14 days after inoculation, when
tumors had reached approximately 200 mm.sup.3 in size.
[0421] Consistent with TIGIT expression in human tumors (FIGS.
6A-6D), both CD8.sup.+ and CD4.sup.+ CT26 TILs expressed high
levels of TIGIT (FIGS. 7A-7B). Furthermore, in line with the
chronic viral infection studies, TIL TIGIT expression was tightly
correlated with expression of other inhibitory co-receptors
including PD-1 (FIGS. 7A-7B) and Tim-3 (FIGS. 8A-8B). A similar
pattern of TIGIT expression was found in MC38 colon carcinoma
tumors (FIGS. 9A-9B).
[0422] To test the physiological relevance of TIGIT expression in
the context of an anti-tumor immune response, BALB/C mice with
established CT26 tumors (approximately 200 mm.sup.3 in size) were
treated with 200 ug isotype control, 200 ug anti-PD-L1, 500 ug
anti-TIGIT, or 200 ug anti-PD-L1+500 ug anti-TIGIT antibodies for
three weeks.
[0423] CT26 tumor growth was only slightly slowed by treatment with
anti-TIGIT or anti-PD-L1 alone, both of which resulted in a modest
3 day increase in median survival (FIGS. 7C-7D). However,
combination therapy with both anti-PD-L1 and anti-TIGIT
dramatically reduced tumor growth (75% decrease in median tumor
volume by day 16, P<0.0001, FIG. 7C and FIG. 10). Moreover, 70%
of the mice receiving both anti-TIGIT and anti-PD-L1 experienced
complete and durable tumor remission and survived for the duration
of the study, even in the absence of further antibody treatment
(FIGS. 7C-7D). These effects were also observed in tumor-bearing
mice treated with a combination of blocking antibodies against
TIGIT and PD-1.
[0424] To test the immunity of these surviving mice to CT26 tumor
cells, approximately 60 days after initial inoculation, mice in
complete remission (CR) that had received anti-TIGIT+anti-PD-L1, as
well as naive BALB/c mice, were re-inoculated with CT26 cells in
their left (not previously inoculated) unilateral thoracic flanks.
These mice were also inoculated with 1.times.10.sup.5 EMT6 breast
carcinoma cells in matrigel into the fourth mammary fat pad. Tumors
were measured 2 times per week. Animals whose tumors became
ulcerated/necrotic or whose total tumor burden exceeded 2000
m.sup.3 were euthanized.
[0425] As shown in FIG. 7E, both tumors grew readily in naive
control mice, but only EMT6 tumors grew in mice that had previously
cleared a CT26 tumor. These results indicated that co-blockade of
TIGIT and PD-1 during tumorigenesis established a state of specific
immunity to CT26 tumor cells.
[0426] To determine if the efficacy of TIGIT/PD-L1 co-blockade was
mediated by CD8.sup.+ T cells, CT26-tumor bearing mice were
subjected to CD8.sup.+ T cell ablation using depleting antibodies
at the initiation of treatment with anti-TIGIT and anti-PDL1. Mice
treated with anti-TIGIT and anti-PD-L1 antibodies were unable to
reject CT26 tumors when depleted of CD8.sup.+ T cells at the start
of treatment (1532% increase in mean tumor volume after 17 days of
treatment, P=0.0004, FIGS. 32A-32B). Additionally, CD8.sup.+ T cell
depletion impaired the ability of previously treated CR mice to
control re-inoculated CT26 tumors (FIG. 32C). Taken together, these
results demonstrated that anti-TIGIT and anti-PD-L1 acted through
CD8.sup.+ T cells to elicit effective primary and secondary
anti-tumor immune responses.
[0427] To determine if PVR expression of tumor cells is dispensable
for TIGIT/PD-L1 co-blockade efficacy, wildtype BALB/c mice were
inoculated with wildtype CT26 tumors (which express PVR) or
PVR-deficient CT26 tumors. Briefly, wildtype CT26 tumor cells were
transiently transfected with a nucleic acid that reduced expression
of PVR. Approximately two weeks after transfection, CT26 cells were
subcloned on the basis of loss of PVR expression by flow cytometry
and qPCR. When tumors reached 150-200 mm.sup.3 in size, mice were
treated with anti-TIGIT and anti-PD-L1 antibodies, or
isotype-matched control antibodies. Mice treated with anti-TIGIT
and anti-PD-L1 antibodies were able to reject both wildtype and
PVR-deficient tumors, as compared to tumor-inoculated mice treated
with control antibodies (FIG. 33). These results demonstrated that
anti-TIGIT and anti-PD-L1 act independently of tumor-expressed
PVR.
[0428] The efficacy of TIGIT/PD-L1 co-blockade in the MC38 tumor
model was also tested and confirmed. Wildtype C57BL6/J mice were
subcutaneously inoculated with syngeneic MC38 colorectal carcinoma
cells and treated established tumors with a combination of TIGIT
and PD-L1 blocking antibodies, as before. Unlike the CT26 model,
treatment with anti-PD-L1 alone was sufficient to induce a complete
response in some mice (FIGS. 25A-25C). However, as in the CT26
model, treatment of MC38 tumor-bearing mice with both anti-TIGIT
and anti-PD-L1 synergistically reversed tumor growth and induced
tumor clearance in most mice (FIGS. 26A-26E). These effects were
also observed in mice inoculated with syngeneic EMT6 breast
carcinoma cells (FIG. 34).
[0429] These results demonstrated that co-blockade of TIGIT and
PD-1 could elicit a sustained and antigen-specific anti-tumor
immune response. These results also suggested that adaptive
anti-tumor responses were fully functionally and reactivated in
therapeutically treated mice.
[0430] To assess the functional effects of TIGIT and PD-1 blockades
on the tumor-infiltrating lymphocytes themselves, mice were
inoculated with CT26 tumor cells and treated with anti-TIGIT and/or
anti-PD-L1 as before. Seven days after the start of treatment,
tumors and tumor-draining lymph nodes were collected for analysis
by flow cytometry.
[0431] Tumor-infiltrating and tumor-draining lymph node resident
CD4.sup.+ T cells produced little IFN.gamma., and did not produce
more upon TIGIT/PD-1 blockade (FIGS. 11A-11F). However,
tumor-infiltrating CD8.sup.+ T cells from mice treated with both
anti-TIGIT and anti-PD-L1, but not those from mice treated with
anti-TIGIT or anti-PD-L1 alone, were significantly more competent
to produce IFN.gamma. upon stimulation in vitro (174% increase
relative to control, P=0.0001, FIG. 13D). Similar results were
observed for CD8.sup.+ TIL production of TNF.alpha. (FIGS.
12A-12F).
[0432] Interestingly, mice treated with either anti-TIGIT or
anti-PD-L1 alone, or both, all saw increased cytokine competency of
tumor-draining lymph node resident CD8.sup.+ T cells (75-113%
increase, P<0.001, FIGS. 13A-13D), suggesting that lymph
node-resident CD8+ T cells were under lesser degree of suppression
than their tumor-infiltrating counterparts. Accumulation and
phenotypic activation of tumor-infiltrating and tumor-draining
lymph node resident CD8+ T cells and CD4+ T cells were unchanged
and weakly enhanced by single antibody treatment and dual antibody
and dual antibody treatment, respectively (FIGS. 12A-12C and FIGS.
13A-13D). The frequencies of IFN.gamma./TNF.alpha. dual-producing
CD8.sup.+ T cells in tumors and tumor-draining lymph nodes followed
similar patterns (FIGS. 35A-35B).
[0433] Consequently, while blockade of either TIGIT or PD-L1 alone
was sufficient to enhance CD8.sup.+ T cell effector function in
tumor-draining lymph nodes, blockade of both receptors was
necessary to restore the function of exhausted CD8.sup.+ T cells
within the tumor itself, consistent with the notion that tumor
microenvironments are highly immunosuppressive.
Example 6: TIGIT Co-Expression with CD226 on Tumor-Infiltrating
CD8+ T Cells
[0434] TIGIT competes with the co-stimulatory receptor CD226 for
binding to Poliovirus Receptor (PVR) (Yu, X., et al. The surface
protein TIGIT suppresses T cell activation by promoting the
generation of mature immunoregulatory dendritic cells. Nature
immunology 10, 48-57 (2009). Given that CD226 deficiency can
enhance T cell exhaustion during chronic viral infection (Cella,
M., et al. Loss of DNAM-1 contributes to CD8+ T-cell exhaustion in
chronic HIV-1 infection. European Journal of Immunology 40(4),
949-954 (2010); Welch, M., et al. CD8 T cell defect of TNA-a and
IL-2 in DNAM-1 deficient mice delays clearance in vivo of a
persistent virus infection. Virology 429(2) 163-170 (2012)), it is
possible that TIGIT may inhibit T cell responses in part by
interfering with CD226 activity.
[0435] To evaluate whether there is a relationship between CD226
and TIGIT in inhibiting T cell responses, the expression of TIGIT
and CD226 was determined on tumor infiltrating CD8+ T cells.
[0436] As shown in FIG. 14, C57BL6/J mice were inoculated with MC38
colorectal carcinoma cells. Splenocytes and tumor-infiltrating
lymphocytes (TILs) were analyzed by FACs analysis approximately 14
days after inoculation, when tumors had reached approximately 200
mm3 in size. Representative histogram of CD226 expression by
splenic B cells (gray), splenic CD8+ T cells (blue), and
TIGIT+tumor-infiltrating CD8+ T cells (red). Data are
representative of two independent experiments; n=5. FIG. 14
illustrates that splenic CD8+ T cells highly express CD226 and
furthermore, that tumor-infiltrating TIGIT+ CD8+ T cells also
highly expressed CD226. The data demonstrates that TIGIT and CD226
are coordinately expressed on murine tumor-infiltrating CD8.sup.+ T
cells, and may regulate each other's function on CD8.sup.+ T cells.
This observation is similar to that in activated CD4.sup.+ T cells
and NK cells, which also co-express TIGIT and CD226.
Example 7: Co-Immunoprecipitation of TIGIT and CD226 on Transfected
Cells
[0437] To determine whether TIGIT interacts with CD226 at the cell
surface, cells were co-transfected with human-TIGIT and human-CD226
and subjected to immunoprecipiation. Briefly, COS 7 Cells in 15 cm
plates were co-transfected with expression plasmids containing the
cDNA for either TIGIT-HA (5 ng) or CD226-Flag (10 ng) tagged
proteins, or a control plasmid (pRK). 23 hrs after transfection the
cells were washed with PBS and harvested in 4 ml of ice cold PBS
and centrifuged at 300.times.g for 5 min and cell pellets were
re-suspended in 2 ml of Lysis buffer at 4.degree. C. The cells were
lysed over 50 min with vortexing every 15 min and subsequently
centrifuged at 10,00.times.g for 15 min at 4 C. The resultant
supernatant was pre-cleared with 160 .mu.l of CL6B sepahrose slurry
by rotating for 30 min at 4.degree. C., and centrifuged for 2 min
at 3000.times.g. The supernatant was equally split into two tubes
and immuno-precipitated with either an anti-HA or an anti-flag
using standard procedures. The immune-precipitated proteins were
subjected to SDS-PAGE and western blotted. Western blots were
probed with either anti-Flag-HRP or anti-HA-HRP.
[0438] As shown in FIG. 15, anti-TIGIT pulled down CD226 and
anti-CD226 pulled down TIGIT, demonstrating that TIGIT and CD226
are in physical contact at the cell surface.
Example 8: TIGIT and CD226 Interact in Primary CD8+ T Cells
[0439] In addition to demonstrating the ability of CD226 and TIGIT
to interact in transfected cells, the interaction of CD226 and
TIGIT in primary CD8+ T cells was also evaluated. Briefly,
MACS-enriched splenic C57BL6/J CD8+ T cells were stimulated with
plate-bound anti-CD3 and anti-CD28 antibodies and recombinant IL-2
for 48 hours and lysed. Cell lysates were immunoprecipitated with
anti-TIGIT and probed with anti-CD226. FIG. 16 illustrates that
TIGIT and CD226 interact in activated primary CD8+ cells as both
were detectable in the co-immunoprecipitate. This data demonstrates
that CD226 and TIGIT also interact with each other on primary
cells.
Example 9: TIGIT/CD226 Interaction on Transfected Cells Using
TR-FRET (Time Resolved-Fluorescence Resonance Energy Transfer)
[0440] To assess whether there was any molecular interaction
between TIGIT and CD226, TR-FRET methodology was employed. FRET
(Fluorescence Resonance Energy Transfer) is based on the transfer
of energy between two fluorophores, a donor and an acceptor, when
in close proximity. Molecular interactions between biomolecules can
be assessed by coupling each partner with a fluorescent label and
by detecting the level of energy transfer. When two entities come
close enough to each other, excitation of the donor by an energy
source triggers an energy transfer towards the acceptor, which in
turn emits specific fluorescence at a given wavelength. Because of
these spectral properties, a donor-acceptor complex can be detected
without the need for physical separation from the unbound partners.
The combination of time resolved (TR) measurements of FRET allow
the signal to be cleared of background fluorescence. This is
typically done by introducing a time delay between the system
excitation and fluorescence measurement to allow the signal to be
cleared of all non-specific short-lived emissions.
[0441] Using TR-FRET, here we demonstrate that TIGIT and CD226
elicited a FRET when expressed in the same cell, indicating
molecular interaction of these two molecules. Briefly, COS-7 cells
were transfected with SNAP-tagged (ST) CD226 and HA-TIGIT using
Lipofectamine 2000 (Life Technologies) and seeded in a white
96-well plate (Costar) at 100,000 cells per well. 24 hours later,
cells were labeled with 100 nM of donor-conjugated benzyl-guanine
SNAP-Lumi-4Tb (Cisbio) and 1 .mu.M donor-conjugated benzyl-guanine
SNAP-A647 (New England Biolabs) diluted in DMEM 10% FCS for 1 h at
37.degree. C., 5% CO2. After three washes in PBS, the FRET signal
was recorded at 665 nm for 400 .mu.s after a 60 .mu.s delay
following laser excitation at 343 nm using a Safire2 plate reader
(Tecan). When the anti-TIGIT antibody was tested, the FRET signal
was also recorded after a 15 min incubation. The FRET ratio was
then calculated as the FRET intensity divided by the donor emission
at 620 nm. The FRET intensity being: (signal at 665 nm from cells
labeled with SNAP-donor and acceptor)--(signal at 665 nm from the
same batch of transfected cells labeled with SNAP-donor only).
[0442] As shown in FIGS. 17A-17D, TIGIT was able to directly
disrupt and cause dissociation of CD226 homodimers. As shown in
FIG. 17A, the dissociation of Flag-ST-CD226 homodimers was observed
with increasing concentrations of HA-TIGIT as illustrated by the
decreasing FRET ratio between Flag-ST-CD226 measured on COS-7 cells
expressing a constant amount of Flag-ST-CD226 and increasing
concentrations of HA-TIGIT. However, as shown in FIG. 17B, when
anti-TIGIT antibody was added to the cell culture, this blocked the
ability of TIGIT and CD226 to associate. This is illustrated by the
lack of a decrease in the FRET intensity of Flag-ST-CD226
homodimers. This demonstrates that CD226 and TIGIT are associated
as complexes but that anti-TIGIT antibodies can disrupt these
interactions (FIGS. 17A-17B).
[0443] Using TR-FRET, the ability of TIGIT to associate with CD226
was also demonstrated and shown in FIGS. 17C-17D. Briefly, after
SNAP-tag labeling using 1 .mu.M of donor-conjugated benzyl-guanine
SNAP-A647 (see above), cells were washed three times in PBS and
incubated with 2 nM of anti-HA donor-conjugated Lumi-4Tb (Cisbio)
diluted in PBS+0.2% BSA for 2 hours at room temperature. The FRET
signal was then recorded. In that case, the FRET intensity is:
(signal at 665 nm from cells labeled with SNAP-acceptor and anti-HA
donor)--(signal at 665 nm from mock transfected cells labeled with
SNAP-acceptor and anti-HA donor).
[0444] As shown in FIG. 17C, association of Flag-ST-CD226 with
HA-TIGIT was observed as illustrated by the increasing FRET
intensity between Flag-ST-CD226 and HA-TIGIT measured on COS-7
cells expressing a constant amount of Flag-ST-CD226 and increasing
concentrations of HA-TIGIT. When anti-TIGIT antibody was added, the
FRET intensity decreased between Flag-ST-CD226 with HA-TIGIT, as
shown in FIG. 17D, suggesting that the interaction of TIGIT with
CD226 can be blocked by an anti-TIGIT blocking antibody.
[0445] To confirm the cell surface expression of Flag-ST-CD226 and
HA-TIGIT in the FRET experiments, anti-Flag and anti-HA ELISA on
intact COS-7 cells expressing the indicated tagged-constructs was
performed. Briefly, COST cells were fixed with 4% paraformaldehyde,
washed twice, and blocked in phosphate-buffered saline+1% fetal
calf serum (FCS). Cells were then incubated with an anti-HA
monoclonal antibody (clone 3F10, Roche applied science) or
anti-Flag-M2 monoclonal antibody (Sigma), both conjugated with
horseradish peroxidase. After washes, cells were incubated with a
SuperSignal ELISA substrate (Pierce) and chemoluminescence was
detected on a Safire2 plate reader (Tecan). Specific signal was
calculated by subtracting the signal recorded on mock transfected
cells. As illustrated in FIG. 18, cell surface expression of both
CD226 and TIGIT were confirmed in the ELISA assay.
[0446] To confirm that the TIGIT:CD226 interaction is not driven by
PVR binding, Flag-ST-CD226 and HA-TIGIT (WT) or HA-TIGIT Q56R were
generated as described in Stengel et al., (2012) PNAS
109(14):5399-5904 and FRET ratios were determined as described. As
shown in FIG. 24, WT TIGIT and Q56R TIGIT bind CD226 with the same
efficacy.
[0447] This data not only demonstrates the CD226 and TIGIT are
associated as complexes, but that an anti-TIGIT antibody can
disrupt these interactions and that the TIGIT:CD226 interaction is
not driven by PVR binding. The data supports a role for TIGIT in
limiting CD226-mediated activation of T cells and that interference
with CD226 activity may be an important mechanism of action by
which TIGIT inhibits T cell responses and activity.
Example 10: CD226 Blockade Reverses the Effectors of TIGIT/PD-L1
Blockade In Vivo
[0448] To test the physiological relevance of the CD226 and TIGIT
interaction, mice were chronically infected with Clone 13 LCMV and
then treated with anti-TIGIT+anti-PD-L1 in the absence or presence
of anti-CD226 blocking antibodies. Briefly, C57BL6/J mice were
briefly depleted of CD4.sup.+ T cells and infected with Clone 13
strain LCMV. For chronic infections, mice were intravenously
infected with 2.times.10.sup.6 PFU Clone 13 strain LCMV and treated
with 500 ug and 250 ug of depleting anti-CD4 antibodies (clone
GK1.5) 3 days before and 4 days after infection, respectively.
Where indicated, mice infected with Clone 13 strain LCMV received
intraperitoneal injections of 200 ug of isotype control antibodies,
500 ug of anti-CD226 antibodies, 200 ug of anti-PD-L1
antibodies+500 ug of anti-TIGIT antibodies, or 500 ug of anti-CD226
antibodies+200 ug of anti-PD-L1 antibodies+500 ug of anti-TIGIT
antibodies 3 times per week from days 28 to 42 post-infection.
Treatment was started at 28 days post-infection because the T cell
response is largely exhausted at this time-point in this model of
chronic viral infection (Wherry et al, Molecular Signature of CD8+
T cell Exhaustion During Chronic Viral Infection, Immunity. 2007
October; 27(4):670-84). Splenocytes and liver viral titers were
analyzed 42 days after infection.
[0449] In these mice, anti-CD226 treatment alone had limited
effects on CD8.sup.+ T cell frequency, activation, or cytokine
competency (FIGS. 19A-19C). However, anti-CD226 treatment potently
reversed the increases in CD8.sup.+ T cell activation and IFNg
production seen in mice treated with anti-PD-L1+anti-TIGIT (59% and
58% decreases, respectively, P<0.001. FIGS. 19B-19D).
[0450] Consistent with these results, LCMV viral loads were
significantly higher in mice treated with
anti-CD226+anti-PD-L1+anti-TIGIT than in mice treated with
anti-PD-L1+anti-TIGIT alone (272% increase, P<0.001, FIG.
19D).
[0451] This data suggests that a primary mechanism by which TIGIT
limits chronic T cell responses is interference with CD226-mediated
co-stimulation. The data identifies a previously unknown role for
TIGIT in interacting with and disrupting CD226, resulting in the
reduction or loss of a key co-stimulatory signal in CD8.sup.+ T
cells. The data demonstrates that interference with CD226-mediated
T cell costimulation may be a major mechanism by which TIGIT limits
chronic T cell responses such as during cancer or chronic viral
infection. The data also defines an essential parameter for
anti-TIGIT antibodies intended to restore the effector function of
chronically stimulated or exhausted CD8.sup.+ or CD4.sup.+ T cells
by interfering with TIGIT's ability to interact with CD226 and/or
TIGIT's ability to disrupt CD226 dimerization.
Materials and Methods
[0452] Mice. C57BL/6J and BALB/c mice were purchased from the
Jackson Laboratory and Charles River Laboratories. CD4.sup.cre mice
and TIGIT.sup.loxP/loxP mice were generated on a C57BL/6J
background with standard techniques and crossed. TIGIT expression
was ablated with 96% efficiency from T cells in TIGIT.sup.loxP/loxP
CD4.sup.cre mice.
[0453] Flow cytometry. Single cell suspensions of spleen, lymph
node, and tumor were prepared with gentle mechanical disruption.
Surface staining was performed with commercial antibodies against
CD4, CD8, CD44, CD62L, PD-1 (eBiosciences) and CD226 (Biolegend).
TIGIT antibodies were generated at Genentech as previously
described (Yu, X. et al. The surface protein TIGIT suppresses T
cell activation by promoting the generation of mature
immunoregulatory dendritic cells. Nature immunology 10, 48-57
(2009)) and conjugated to Alexa Fluor 647 according to the
manufacturer's directions (Molecular Probes).
[0454] For intracellular cytokine staining (ICS), cells were
stimulated for 4 hours with 20 ng/mL Phorbol 12-myristate
13-acetate (PMA, Sigma) and 1 .mu.M Ionomycin (Sigma) in the
presence of 3 .mu.g/mL Brefeldin A (eBiosciences). After
stimulation, cells were stained for surface markers as described
and fixed and permeabilized with eBioscience's FoxP3 fixation
buffer set according to the manufacturer's directions. Fixed cells
were stained with antibodies against IFN.gamma. and TNF.alpha.
(eBiosciences).
[0455] Blocking antibodies. A blocking anti-TIGIT IgG2a monoclonal
antibody (clone 10A7, reactive against both mouse and human TIGIT)
was generated as previously described and cloned onto a murine
IgG2a backbone. A blocking anti-PD-L1 IgG2a monoclonal antibody
(clone 25A1) was generated by immunizing Pdl1.sup.-/- mice with a
PD-1-Fc fusion protein and cloned onto a murine IgG2a backbone.
Clone 25A1 was modified with previously described mutations
abolishing binding to Fc.gamma. receptors. A blocking anti-CD226
IgG2a monoclonal antibody (clone 37F6) was generated by
immunization of hamsters with recombinant murine CD226 and cloned
onto a murine IgG2a backbone. These antibodies were also used in
tests described in other Examples described herein.
[0456] Viral infections. For acute infections, mice were
intravenously infected with 2.times.10.sup.6 plaque-forming units
(PFU) Armstrong strain LCMV. For chronic infections, mice were
intravenously infected with 2.times.10.sup.6 PFU Clone 13 strain
LCMV and treated with 500 ug and 250 ug of depleting anti-CD4
antibodies (clone GK1.5) 3 days before and 4 days after infection,
respectively. Where indicated, mice infected with Clone 13 strain
LCMV received intraperitoneal injections of 200 ug of isotype
control antibodies, 200 ug of anti-PD-L1 antibodies, and/or 500 ug
of anti-TIGIT antibodies 3 times per week from days 28 to 42
post-infection.
[0457] Viral titer assay. Monolayers of MC57 cells were cultured
with an overlay of 1% methylcellulose and infected with serially
diluted liver homogenates from LCMV-infected mice. 72 hours after
infection, the cells were fixed with 4% paraformaldehyde and
permeabilized with 0.5% Triton-X. Viral plaques were stained with
anti-LCMV NP (clone VL-4) and HRP-conjugated anti-rat IgG and
visualized with 0-phenylenediamine (OPD, Sigma).
[0458] Bioinformatics. Breast cancer gene expression data
microarray data was obtained from the Cancer Gene Atlas Network
(Network, T.C.G.A. Comprehensive genomic characterization of
squamous cell lung cancers. Nature 489, 519-525 (2012)). Processing
and normalization of microarray data were performed using the R
programming language (http://r-project.org) and Bioconductor's
limma package (http://bioconductor.org). Microarray intensity
values from each channel were preprocessed using the
normal+exponential background correction method, as previously
described.sup.22. Corrected intensity values were then normalized
using quantiles normalization, as previously described.sup.23.
Normalized log-ratio data was calculated by subtracting the
reference channel from the test channel for each array. Data were
further filtered using a non-specific filter, as previously
described.sup.24, removing probes that do not map to known genes,
and reducing the dataset to one probe per gene. For differential
expression analysis, moderated t-statistics were calculated with
the limma package, as previously described (Smyth, G. K. Linear
models and empirical bayes methods for assessing differential
expression in microarray experiments. Statistical applications in
genetics and molecular biology 3, Article3 (2004)). To evaluate
correlation, Pearson's correlation coefficients were used.
[0459] CT26 colon carcinoma. BALB/c were subcutaneously inoculated
with 1.times.10.sup.5 CT26 colon carcinoma cells suspended in
matrigel (BD Biosciences) into the right unilateral thoracic flank.
After two weeks, mice bearing tumors of approximately 200 mm.sup.3
were randomly recruited into treatment groups receiving 35 mg/kg of
isotype control antibodies, anti-PD-L1 antibodies, and/or
anti-TIGIT antibodies by intraperitoneal injection 3 times per week
for 3 weeks. Tumors were measured 2 times per week by caliper.
Animals whose tumors became ulcerated/necrotic or grew larger than
2000 mm.sup.3 were euthanized.
[0460] EMT6 breast carcinoma. BALB/c mice were subcutaneously
inoculated in the fourth mammary fat pad with 1.times.10.sup.5
syngeneic EMT6 breast carcinoma cells in matrigel (BD Biosciences).
After two weeks, mice bearing tumors of 150-200 mm.sup.3 were
randomly recruited into treatment groups receiving 35 mg/kg of
isotype control antibodies, anti-PD-L1 antibodies, and/or
anti-TIGIT antibodies by intraperitoneal injection 3 times per week
for 3 weeks. Tumors were measured 2 times per week by caliper, and
tumor volumes were calculated using the modified ellipsoid formula,
1/2.times. (length.times.width). Animals whose tumors shrank to 32
mm.sup.3 or smaller were considered to be in complete response
(CR). Animals whose tumors grew to larger than 2000 mm.sup.3 were
considered to have progressed and were euthanized. Animals whose
tumors became ulcerated prior to progression or complete response
were euthanized and removed from the study.
[0461] CT26 re-challenge. Where indicated, BALB/c mice previously
inoculated with CT26 colon carcinoma cells as described above were
re-inoculated with CT26 cells into the left (not previously
inoculated) unilateral thoracic flank. These mice were also
inoculated with 1.times.10.sup.5 EMT6 breast carcinoma cells in
matrigel into the fourth mammary fat pad. Tumors were measured 2
times per week. Animals whose tumors became ulcerated/necrotic or
whose total tumor burden exceeded 2000 mm.sup.3 were
euthanized.
[0462] Statistics. Statistical tests were conducted using unpaired
(paired where specified) 2-tailed Student's t-tests. Error bars
depict the standard error of the mean.
[0463] Animal Study Oversight. All animal studies were approved by
Genentech's Institutional Animal Care and Use Committee.
Example 11: TIGIT Expression is Elevated in Human Cancer and
Correlated with Expression of CD8 and PD-1 and CD8+ T Cell
Infiltration
[0464] Materials and Methods
[0465] Bioinformatics. Processing and analysis of RNA-sequencing
data was performed using the R programming language
(http://www.r-project.org) along with several packages from the
Bioconductor project (http://www.bioconductor.org). RNA-sequencing
data for cancer and matched normal samples were obtained from the
TCGA for five different indications: breast cancer (Network, C.G.A.
Comprehensive molecular portraits of human breast tumours. Nature
490, 61-70 (2012)), colon adenocarcinoma (Network, T.C.G.A.
Comprehensive molecular characterization of human colon and rectal
cancer. Nature 487, 330-337 (2012)), renal clear cell carcinoma
(Network, C.G.A. Comprehensive molecular characterization of clear
cell renal cell carcinoma. Nature 499, 43-49 (2013)), lung squamous
cell carcinoma (Network, T.C.G.A. Comprehensive genomic
characterization of squamous cell lung cancers. Nature 489, 519-525
(2012)), and endometrial carcinoma (Network, T.C.G.A. Integrated
genomic characterization of endometrial carcinoma. Nature 497,
67-73 (2012)).
[0466] Raw RNA-seq reads were processed using the HTSeqGenie
Bioconductor package. Briefly, reads were aligned to the human
genome (NCBI build 37) using the GSNAP algorithm (Wu, T. D. &
Nacu, S. Fast and SNP-tolerant detection of complex variants and
splicing in short reads. Bioinformatics (Oxford, England) 26,
873-881 (2010)). Uniquely aligned read pairs that fell within exons
were counted to give an estimate of gene expression level for
individual genes. We used the library size estimation from the
edgeR package (Robinson, M.D., McCarthy, D. J. & Smyth, G. K.
edgeR: a Bioconductor package for differential expression analysis
of digital gene expression data. Bioinformatics (Oxford, England)
26, 139-140 (2010)) to normalize across different samples for their
respective sequencing depths.
[0467] To derive a T cell specific gene signature, we manually
curated the T cell genes identified by the IRIS project, removing
genes associated with cell cycle processes, genes highly expressed
in other tissues, and known co-activating and co-inhibitory
receptors. This yields a 15-gene signature that is specific to T
cells. To calculate the T cell gene expression signature score in
the lung squamous cell carcinoma data, we first performed a
variance stabilizing transform on the raw count data using the voom
function from the limma Bioconductor package. We then calculated
the first eigenvector of the centered and scaled
variance-stabilized data from the 15-gene T cell signature. This
approach yields a robust per-sample estimate of relative T cell
abundance. A linear model including the T cell signature score was
then fit for each gene, again using the limma package. We then
ranked the genes by their correlation with the T cell signature in
our linear model, choosing only genes positively correlated with
the T cell signature. For visualizing T cell-associated genes as a
heatmap, we centered and scaled the variance-stabilized data to
unit variance, allowing for comparison of genes with different
average expression levels.
[0468] To determine the correlation between expression of TIGIT and
other genes, we normalized RNA-sequencing count data to account for
differences in library size, using the method from the edgeR
Bioconductor package (Robinson, M. D., McCarthy, D. J. & Smyth,
G. K. edgeR: a Bioconductor package for differential expression
analysis of digital gene expression data. Bioinformatics (Oxford,
England) 26, 139-140 (2010)). We then calculated Spearman's rank
correlation coefficient on the normalized counts. We consider rho
>0.75 to be indicative of strong correlation, rho .ltoreq.0.75
but >0.5 to be indicative of moderate correlation, and
rho.ltoreq.0.5 but >0.25 to be indicative of weak
correlation.
[0469] For calculation of TIGIT/CD3.epsilon. ratios across each
indication, we first calculated the variance-stabilized data for
each RNA-sequencing data set. We then calculated the log 2 ratio of
the variance-stabilized data for TIGIT and CD3.epsilon.. To
calculate the difference between tumor and normal samples, we
performed standard linear model analysis using standard R
functions. We accepted a p-value of <0.01 as evidence of a
significant difference between tumor and normal.
[0470] To identify genes associated with tumor-infiltrating T
cells, we used a gene signature-based approach to interrogate gene
expression data from the Cancer Genome Atlas (TCGA) lung squamous
cell carcinoma (LUSC) collection (Network, T.C.G.A. Comprehensive
genomic characterization of squamous cell lung cancers. Nature 489,
519-525 (2012)). Using immune cell-specific gene sets defined by
the Immune Response In Silico project (Abbas, A. R. et al. Immune
response in silico (IRIS): immune-specific genes identified from a
compendium of microarray expression data. Genes and immunity 6,
319-331 (2005)), and the methods described above, we developed a
highly specific 15 gene signature. Examining the genes most highly
associated with the T cell signature, we identified several
co-inhibitory receptors previously associated with T cell
dysfunction in tumors, particularly PD-1 (FIG. 21). In LUSC,
expression of TIGIT and CDR were highly correlated, with a
Spearman's rank correlation coefficient (.rho. of 0.82 (FIG. 20A).
Indeed, TIGIT and CDR expression were also highly correlated in
many additional TCGA tumor gene expression datasets, including
colon adenocarcinoma (COAD), uterine corpus endrometroid carcinoma
(UCEC), breast carcinoma (BRCA), and kidney renal clear cell
carcinoma (KIRC), with .rho. ranging from 0.83 to 0.94 (FIGS.
20B-20E). Furthermore, expression of TIGIT was elevated relative to
expression of CDR in many tumor samples, with increased TIGIT/CD3c
ratios in LUSC, COAC, UCEC, and BRCA tumor samples compared to
matched normal tissue (116%-419% increase, FIGS. 20A-20D). The
ratio of TIGIT to CDR expression in KIRC samples was unchanged,
though expression of both TIGIT and CDR was much higher in KIRC
samples than in normal tissue samples (FIG. 20E). These data
indicated that TIGIT expression was up-regulated by
tumor-infiltrating lymphocytes (TILs) in a broad range of solid
tumors.
[0471] TIGIT has been previously described as an inhibitor of CD4+
T cell priming, with no known function in CD8+ T cells. However,
TIGIT expression in LUSC samples was highly correlated with CD8A
and only weakly correlated with CD4 (.rho.=0.77 and 0.48
respectively, FIG. 20F). Expression of TIGIT was also correlated
with expression of its complementary co-stimulatory receptor,
CD226, as well as with expression of PD-1, a key mediator of T cell
suppression in tumors and during other chronic immune responses
(.rho.=0.64 and 0.82 respectively, FIGS. 20G-20H). Although some
non-lymphocyte cell sources of these genes exist in tumors, these
data strongly suggested that tumor-infiltrating T cells,
particularly "exhausted" CD8.sup.+ T cells, expressed high levels
of TIGIT.
Example 12: TIGIT and PD-1 are Coordinately Expressed by Human and
Murine Tumor-Infiltrating Lymphocytes
[0472] Materials and Methods
[0473] Human tumor and PBMC samples. Matched whole blood and fresh
surgically resected tumor tissues were obtained from Conversant
Biosciences or Foundation Bio. All specimens were obtained with
written informed consent and collected using a protocol approved by
the Hartford Hospital Institutional Review Board (IRB) (NSCLC
patient 1, depicted in FIGS. 22A-22G) or the Western IRB (NSCLC
patient 2 and CRC patient 1, depicted in FIGS. 23A-23D and FIGS.
37A-37B). Normal adult whole blood was obtained from a healthy
volunteer. PBMCs were purified from whole blood by Ficoll gradient
centrifugation. Tumor tissues were cut into small pieces, and
incubated with collagenase and DNAse (Roche), and disassociated
using a gentleMACS Disassociator (Miltenyi).
[0474] Flow cytometry. Single cell suspensions of mouse spleen,
lymph node, and tumor were prepared with gentle mechanical
disruption. Surface staining was performed with commercial
antibodies against CD4, CD8, CD44, CD62L, PD-1 (eBiosciences) and
CD226 (Biolegend). TIGIT antibodies were generated at Genentech and
conjugated to Alexa Fluor 647 according to the manufacturer's
directions (Molecular Probes).
[0475] For intracellular cytokine staining (ICS), cells were
stimulated for 4 hours with 20 ng/mL Phorbol 12-myristate
13-acetate (PMA, Sigma) and 1 .mu.M Ionomycin (Sigma) in the
presence of 3 .mu.g/mL Brefeldin A (eBiosciences). After
stimulation, cells were stained for surface markers as described
and fixed and permeabilized with eBioscience FoxP3 fixation buffer
set according to the manufacturer's directions. Fixed cells were
stained with antibodies against IFN.gamma. and TNF.alpha.
(eBiosciences).
[0476] Human tumor and PBMC samples were prepared as described
above. Surface staining was performed with a viability dye
(Molecular Probes), commercial antibodies against CD45
(eBiosciences), CD3, CD4, CD8, PD-1 (BD Biosciences), and with
anti-TIGIT antibodies prepared as described above.
[0477] All samples were acquired on LSR-II or LSR-Fortessa
instruments (BD Biosciences) and analyzed using FlowJo software
(Treestar).
[0478] To confirm up-regulation of TIGIT by tumor-infiltrating T
cells, we assessed TIGIT protein expression on human non-small-cell
lung carcinoma tumor-infiltrating T cells, matched peripheral T
cells, and normal donor peripheral T cells. Cell surface TIGIT was
expressed by subsets of NSCLC-infiltrating CD8.sup.+ and CD4.sup.+
T cells (51% and 39% respectively, FIGS. 22A-22B. FIGS. 36A-36B
further demonstrates that cell surface TIGIT was expressed by a
large percentage of NSCLC-infiltrating CD8.sup.+ and CD4.sup.+ T
cells (58% and 28% TIGIT respectively, FIGS. 36A-36B).
Interestingly, peripheral CD8.sup.+ and CD4.sup.+ T cells from the
NSCLC tumor donor also expressed higher levels of TIGIT than did
cells from healthy donors (FIGS. 22A-22B and FIGS. 36A-36B).
Similar results were obtained with a second set of matched NSCLC
and PBMC samples and in a set of matched colorectal carcinoma (CRC)
and PBMC samples (FIGS. 23A-23D and FIGS. 37A-37B). Nearly all
tumor-infiltrating T cells expressing high levels of TIGIT
co-expressed PD-1, consistent with the correlation between TIGIT
and PD-1 expression described in FIG. 22C.
[0479] To extend our human findings into pre-clinical cancer
models, we characterized TIGIT expression by T cells infiltrating
subcutaneous CT26 and MC38 colorectal tumors in wildtype BALB/c
mice and C57BL6/J mice, respectively. Two weeks post-inoculation,
when CT26 and MC38 tumors had become established and grown to
150-200 mm.sup.3 in size, TIGIT was expressed by approximately 50%
of tumor-infiltrating CD8.sup.+ T cells and 25% of
tumor-infiltrating CD4.sup.+ T cells, at levels similar to those of
primary CD8.sup.+ T cells stimulated in vitro (FIGS. 22D-22E and
FIGS. 26A-26E). In both CD8.sup.+ and CD4.sup.+ murine TILs, CD226
was constitutively expressed, and TIGIT and PD-1 expression were
again tightly correlated (FIGS. 22F-22G).
[0480] These results confirmed that TIGIT was highly expressed by
tumor-infiltrating T cells, and that expression of TIGIT occurred
in parallel with expression other co-inhibitory receptors, most
notably PD-1.
Example 13: TIGIT Suppression of CD8+ T Cells Responses is
Dependent on CD226
[0481] Unlike PD-1 or CTLA-4, there is no direct biochemical
evidence of a T cell inhibitory signaling cascade initiated by
TIGIT in cis. However, co-inhibitory receptors can also function by
limiting the activity of a complementary co-stimulatory receptor,
such as with the suppression of CD28 signaling by CTLA-4. Having
established TIGIT as a negative regulator of tumor-infiltrating and
anti-viral CD8.sup.+ T cells, we asked whether TIGIT induced T cell
exhaustion indirectly via suppression of its complementary
co-stimulatory receptor, CD226, which is highly expressed by
peripheral and tumor-infiltrating CD8.sup.+ T cells (FIGS.
27A-27B).
[0482] Wildtype BALB/c mice bearing 150-200 mm.sup.3 CT26 tumors
were treated with a combination of anti-PD-L1 and anti-TIGIT
antibodies in the presence or absence of blocking anti-CD226
antibody, or with anti-CD226 alone. Treatment with anti-CD226 alone
slightly accelerated tumor growth, relative to control mice,
resulting in a decreased median survival of 2 days (anti-CD226
alone vs. control, P=0.0118, FIGS. 28A-28B). Strikingly, the
addition of anti-CD226 blocking antibodies to mice treated with
anti-TIGIT and anti-PD-L1 co-blockade greatly enhanced tumor growth
and fully reversed the efficacy of TIGIT/PD-L1 co-blockade on tumor
regression and survival (FIGS. 28A-28B). A similar effect was
observed on LCMV titers in chronically infected mice treated with
anti-TIGIT, anti-PD-L1, and/or anti-CD226 (FIG. 19D). These data
indicated that CD226 contributed to anti-tumor and other chronic T
cell responses, and that TIGIT suppressed these responses at least
in part by suppression of CD226.
[0483] To more fully understand how TIGIT and CD226 activity
affected anti-tumor T cell responses, we tested how CD226 alone and
in concert with TIGIT influenced T cell activation, tumor
infiltration, and effector function. We analyzed tumors and
tumor-draining lymph nodes from CT26 tumor-bearing mice treated as
above for seven days. As before, co-blockade of PD-L1 and TIGIT
enhanced IFN.gamma. production of both tumor-infiltrating and
tumor-draining lymph node-resident CD8.sup.+ T cells (130% and 99%
increase, respectively, P<0.001, FIGS. 28C-28D). Blockade of
CD226 alone had no effect on IFN.gamma. production by
tumor-infiltrating and tumor-draining lymph node-resident CD8.sup.+
T cells, suggesting that the effects of CD226 co-stimulation were
already limited in exhausted T cells (FIGS. 28C-28E). However,
CD226 blockade did impair both the frequency and effector function
of tumor-infiltrating CD8.sup.+ T cells in mice treated with
combination anti-TIGIT and anti-PD-L1 (57% decrease, P=0.0015, FIG.
28D). Treatment with anti-CD226 had no such effect on CD8.sup.+ T
cells residing in the tumor-draining lymph nodes, whereas
anti-PD-L1 alone enhanced CD8.sup.+ T cell effector function,
suggesting that PD-L1 blockade was sufficient to enhance CD8.sup.+
T cell effector function even in the absence of CD226. CD226
blockade also resulted in a reduced frequency of tumor-infiltrating
CD8.sup.+ T cells (53% reduction, P=0.0044, FIGS. 28E-28F). Taken
together, these data suggested that CD226 functions to support both
the accumulation and effector function of tumor-infiltrating
CD8.sup.+ T cells, and that TIGIT counteracts the latter.
Example 14: TIGIT Impairs CD226 Function by Directly Disrupting
CD226 Homodimerization
[0484] To test if TIGIT may antagonize CD226 activity in cis,
TIGIT's effect on CD226 co-stimulation in vitro was tested.
TIGIT-deficient CD8.sup.+ T cells stimulated with sub-optimal
levels of anti-CD3 responded more robustly to PVR co-stimulation
than did wildtype littermate CD8.sup.+ T cells, and this enhanced
response was dependent on CD226 (46% increase in proliferation,
P=0.0061, FIG. 29A). Consistent with these data, wildtype CD8.sup.+
T cells, stimulated with sub-optimal anti-CD3 and PVR, proliferated
more robustly in the presence of anti-TIGIT antibodies than they
did in the presence of isotype-matched control antibodies, and this
effect was also dependent on CD226 (105% increase in proliferation,
P=0.0010, FIG. 29B).
[0485] To test the relevance of TIGIT to primary human CD8.sup.+ T
cells, we purified CD8.sup.+ T cells from healthy donor blood and
stimulated them with sub-optimal levels of plate-bound anti-CD3 and
recombinant human PVR-Fc fusion protein. In the presence of
isotype-matched control antibodies, PVR co-stimulation moderately
enhanced T cell stimulation and proliferation. Furthermore,
addition blocking anti-TIGIT antibodies significantly enhanced the
effects of PVR co-stimulation, consistent with TIGIT's effects on
primary murine CD8.sup.+ T cells (69% increase in proliferation,
P=0.0071, FIG. 29C). These data demonstrated a cell-intrinsic role
for TIGIT inhibition of CD226 function on primary murine and human
CD8.sup.+ T cells.
[0486] TR-FRET (Time-resolved Fluorescence Resonance Energy
Transfer) was used to determine the molecular mechanism by which
TIGIT impaired CD226 activity. First, we expressed and labeled
human ST-CD226 with non-permeant donor and acceptor fluorophores.
These cells yielded a strong FRET signal, confirming the ability of
CD226 to homodimerize (FIG. 29D). To monitor CD226 and TIGIT
interactions on the cell surface, we expressed ST-CD226 in absence
or in presence of human HA-TIGIT that we labeled with the SNAP-tag
substrate and an anti-HA antibody, respectively. Strikingly,
co-expression of increasing amounts of TIGIT (monitored by ELISA)
attenuated the CD226/CD226 FRET signal, indicating that TIGIT could
disrupt CD226 homodimerization (FIG. 29E). Indeed, acceptor CD226
and donor TIGIT also resulted in a significant FRET signal,
indicating a direct interaction between these two proteins (FIG.
29F). This interaction was further confirmed by
co-immunoprecipitation (FIG. 29G). These data demonstrated that
TIGIT and CD226 directly interact at the cell surface, and that
this interaction can impair CD226 homodimerization.
[0487] To test the effects of TIGIT antibody blockade on
TIGIT-CD226 interaction, we again co-expressed human ST-CD226 and
HA-TIGIT, this time in the presence or absence of blocking
antibodies against human TIGIT. The addition of anti-TIGIT to the
cell cultures significantly reduced the ability of TIGIT and CD226
to associate (FIG. 2911). These data suggested that anti-TIGIT
treatment can limit TIGIT's interaction with CD226, and are
consistent with the notion that suppression of CD226 activity is a
key mechanism of action by which TIGIT enforces CD8.sup.+ T cell
exhaustion. This is also consistent with the ability of anti-TIGIT
antibodies to enhance CD226 co-stimulation.
[0488] Next, we confirmed the capacity of endogenous TIGIT and
CD226 to interact (FIG. 30). Primary human T cells were stimulated
in vitro with anti-CD3 and anti-CD28 antibodies, sorted on the
basis of TIGIT expression, rested, re-stimulated, and labeled with
antibodies against endogenous TIGIT and CD226 that were conjugated
to fluorophores compatible with TR-FRET. TIGIT-expressing T cells
labeled with donor-conjugated anti-TIGIT and acceptor-conjugated
anti-CD226 antibodies yielded a strong FRET signal (FIG. 30). In
contrast, only a negligible FRET signal was detected on T cells
that did not express TIGIT or that were labeled with
donor-conjugated anti-TIGIT and acceptor-conjugated anti-HVEM
antibodies (FIG. 30), confirming the specificity of the detected
interaction between endogenous TIGIT and CD226.
[0489] These results demonstrate that endogenous TIGIT and CD226
can directly interact at the cell surface, and that this
interaction impairs CD226 homodimerization. Given the role of CD226
as a co-stimulator of T cell responses in vivo, and without wishing
to be bound by theory, it is believed that suppression of CD226 may
be a key mechanism of action by which TIGIT enforces CD8.sup.+ T
cell exhaustion during chronic viral infection and cancer.
[0490] Materials and Methods
[0491] Time-resolved Fluorescence Resonance Energy Transfer with
Transfected Cell Lines. CHO cells were transfected with N-terminus
SNAP-tagged (ST) CD226 and N-terminus HA-TIGIT using Lipofectamine
2000 (Life Technologies) and seeded in a white 96-well plate
(Costar) at 100,000 cells per well. 24 hours later, cells were
labeled to measure TR-FRET either between SNAP-donor/SNAP-acceptor
or between SNAP-acceptor/anti-HA donor. 1) SNAP-donor/SNAP-acceptor
labeling: Cells were incubated with 100 nM of donor-conjugated
benzyl-guanine SNAP-Lumi-4Tb (Cisbio) and 1 .mu.M
acceptor-conjugated benzyl-guanine SNAP-A647 (New England Biolabs)
diluted in DMEM 10% FCS for 1 h at 37.degree. C., 5% CO2. Cells
were then washed three times in PBS before reading of the FRET
signal. 2) SNAP-acceptor/anti-HA donor: Cells were incubated with 1
.mu.M acceptor-conjugated benzyl-guanine SNAP-A647 diluted in DMEM
10% FCS for 1 h at 37.degree. C., 5% CO2. After three washes in
PBS, cells were incubated for 2 hours with 2 nM anti-HA Lumi-4Tb
(Cisbio) in PBS+0.2% BSA at room temperature. The FRET signal was
then recorded at 665 nm for 400 .mu.s after a 60 .mu.s delay
following laser excitation at 343 nm using a Safire2 plate reader
(Tecan). When anti-TIGIT was tested at 10 .mu.g/ml final, the FRET
signal was also recorded after a 15 min incubation. For the
Flag-ST-CD226/Flag-ST-CD226 interaction, the FRET ratio was
calculated as the FRET intensity divided by the donor emission at
620 nm, which is proportional to the CD226 expression. The FRET
intensity being: (signal at 665 nm from cells labeled with
SNAP-donor and acceptor)--(signal at 665 nm from the same batch of
transfected cells labeled with SNAP-donor only). For the
Flag-ST-CD226/HA-TIGIT interaction, the FRET ratio represents the
FRET intensity divided by the Flag-ST-CD226 expression as measured
by an anti-Flag ELISA. In that case, the FRET intensity=(signal at
665 nm from cells labeled with SNAP-acceptor and anti-HA
donor)--(signal at 665 nm from mock transfected cells labeled with
SNAP-acceptor and anti-HA donor).
[0492] Time-resolved Fluorescence Resonance Energy Transfer with
Human T cells. Human anti-TIGIT (Genentech clone 1F4), anti-CD226
(Santa Cruz Biotechnology), and anti-HVEM (eBioscience) antibodies
were conjugated fluorophores compatible with TR-FRET (Cisbio).
Primary human T cells were MACS-enriched from blood, stimulated in
vitro with plate bound anti-CD3 and anti-CD28 for 72 hours.
TIGIT-expressing and non-expressing T cells (all expressing CD226)
were then sorted, rested without stimulation for 72 hours, and
re-stimulated for 48 hours. Each population was then washed once
with Tris-KREBS buffer (20 mM Tris pH 7.4, 118 mM NaCl, 5.6 mM
glucose, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.7 mM KCl, 1.8 mM CaCl2) and
cultured under the following conditions, in triplicate: 1)
Anti-TIGIT Ab-Lumi4-Tb (5 .mu.g/ml), 2) Anti-TIGIT Ab-Lumi4-Tb (5
.mu.g/ml)+anti-HVEM-d2 (10 .mu.g/ml), 3) Anti-TIGIT Ab-Lumi4-Tb (5
.mu.g/ml)+anti-CD226 (10 .mu.g/ml), 4) Anti-TIGIT Ab-Lumi4-Tb (5
.mu.g/ml)+anti-CD226 (10 .mu.g/ml)+cold anti-TIGIT Ab (clone 1F4)
(50 .mu.g/ml). The indicated concentrations were optimized to
ensure the highest FRET signal. Cells were incubated for 2 hours at
room temperature on a rotator and then washed 3 times in Tris-KREBS
buffer. T cells were then seeded at 400,000 cells/well in a white
96-well plate (Costar) and TR-FRET was recorded at 665 nm for 400
.mu.s after a 60 .mu.s delay following laser excitation at 343 nm
using a PHERAstar plate reader (BMG Labtech). FRET intensity was
expressed as the signal at 665 nm from cells labeled with
Ab-Lumi4-Tb+Ab-d2 minus the signal at 665 nm from the same batch of
cells labeled with Ab-Lumi4-Tb alone. The non-specific FRET signal
was given by the T cells incubated with Lumi4Tb+d2+ an excess of
cold Ab.
[0493] Co-immunoprecipitation. Briefly, COS 7 Cells in 15 cm plates
were co-transfected with expression plasmids containing the cDNA
for either TIGIT-HA (5 ng) or CD226-Flag (10 ng) tagged proteins,
or a control plasmid (pRK). 23 hrs after transfection the cells
were washed with PBS and harvested in 4 ml of ice cold PBS and
centrifuged at 300.times.g for 5 min and cell pellets were
re-suspended in 2 ml of Lysis buffer at 4.degree. C. The cells were
lysed over 50 min with vortexing every 15 min and subsequently
centrifuged at 10,00.times.g for 15 min at 4.degree. C. The
resultant supernatant was pre-cleared with 160 .mu.l of CL6B
sepahrose slurry by rotating for 30 min at 4.degree. C., and
centrifuged for 2 min at 3000.times.g. The supernatant was equally
split into two tubes and immuno-precipitated with either an anti-HA
or an anti-flag using standard procedures. The immune-precipitated
proteins were subjected to SDS-PAGE and western blotted. Western
blots were probed with either anti-Flag-HRP or anti-HA-HRP.
[0494] All patents, patent applications, documents, and articles
cited herein are herein incorporated by reference in their
entireties.
Sequence CWU 1
1
49117PRTArtificial SequenceSynthetic Construct 1Lys Ser Ser Gln Ser
Leu Tyr Tyr Ser Gly Val Lys Glu Asn Leu Leu1 5 10
15Ala26PRTArtificial SequenceSynthetic Construct 2Ala Ser Ile Arg
Phe Thr1 539PRTArtificial SequenceSynthetic Construct 3Gln Gln Gly
Ile Asn Asn Pro Leu Thr1 5410PRTArtificial SequenceSynthetic
Construct 4Gly Phe Thr Phe Ser Ser Phe Thr Met His1 5
10517PRTArtificial SequenceSynthetic Construct 5Phe Ile Arg Ser Gly
Ser Gly Ile Val Phe Tyr Ala Asp Ala Val Arg1 5 10
15Gly610PRTArtificial SequenceSynthetic Construct 6Arg Pro Leu Gly
His Asn Thr Phe Asp Ser1 5 10716PRTArtificial SequenceSynthetic
Construct 7Arg Ser Ser Gln Ser Leu Val Asn Ser Tyr Gly Asn Thr Phe
Leu Ser1 5 10 1587PRTArtificial SequenceSynthetic Construct 8Gly
Ile Ser Asn Arg Phe Ser1 599PRTArtificial SequenceSynthetic
Construct 9Leu Gln Gly Thr His Gln Pro Pro Thr1 51010PRTArtificial
SequenceSynthetic Construct 10Gly Tyr Ser Phe Thr Gly His Leu Met
Asn1 5 101117PRTArtificial SequenceSynthetic Construct 11Leu Ile
Ile Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys1 5 10
15Gly1210PRTArtificial SequenceSynthetic Construct 12Gly Leu Arg
Gly Phe Tyr Ala Met Asp Tyr1 5 1013114PRTArtificial
SequenceSynthetic Construct 13Asp Ile Val Met Thr Gln Ser Pro Ser
Ser Leu Ala Val Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Lys
Ser Ser Gln Ser Leu Tyr Tyr Ser 20 25 30Gly Val Lys Glu Asn Leu Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Ser Pro Lys Leu Leu Ile
Tyr Tyr Ala Ser Ile Arg Phe Thr Gly Val 50 55 60Pro Asp Arg Phe Thr
Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr65 70 75 80Ile Thr Ser
Val Gln Ala Glu Asp Met Gly Gln Tyr Phe Cys Gln Gln 85 90 95Gly Ile
Asn Asn Pro Leu Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile 100 105
110Lys Arg14112PRTArtificial SequenceSynthetic Construct 14Asp Val
Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Phe Gly1 5 10 15Asp
Gln Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Asn Ser 20 25
30Tyr Gly Asn Thr Phe Leu Ser Trp Tyr Leu His Lys Pro Gly Gln Ser
35 40 45Pro Gln Leu Leu Ile Phe Gly Ile Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile65 70 75 80Ser Thr Ile Lys Pro Glu Asp Leu Gly Met Tyr Tyr
Cys Leu Gln Gly 85 90 95Thr His Gln Pro Pro Thr Phe Gly Pro Gly Thr
Lys Leu Glu Val Lys 100 105 11015119PRTArtificial SequenceSynthetic
Construct 15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Thr Gln Pro
Gly Lys1 5 10 15Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe
Ser Ser Phe 20 25 30Thr Met His Trp Val Arg Gln Ser Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Phe Ile Arg Ser Gly Ser Gly Ile Val Phe
Tyr Ala Asp Ala Val 50 55 60Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Leu Leu Phe65 70 75 80Leu Gln Met Asn Asp Leu Lys Ser
Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Arg Pro Leu Gly His
Asn Thr Phe Asp Ser Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val
Ser Ser 11516119PRTArtificial SequenceSynthetic Construct 16Glu Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr1 5 10 15Ser
Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly His 20 25
30Leu Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile
35 40 45Gly Leu Ile Ile Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys
Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser Ala
Val Tyr Phe Cys 85 90 95Ser Arg Gly Leu Arg Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Ser Val Thr Val Ser Ser
1151710PRTArtificial SequenceSynthetic Construct 17Gly Phe Thr Phe
Ser Asp Ser Trp Ile His1 5 101818PRTArtificial SequenceSynthetic
Construct 18Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val1 5 10 15Lys Gly199PRTArtificial SequenceSynthetic Construct
19Arg His Trp Pro Gly Gly Phe Asp Tyr1 52011PRTArtificial
SequenceSynthetic Construct 20Arg Ala Ser Gln Asp Val Ser Thr Ala
Val Ala1 5 10217PRTArtificial SequenceSynthetic Construct 21Ser Ala
Ser Phe Leu Tyr Ser1 5229PRTArtificial SequenceSynthetic Construct
22Gln Gln Tyr Leu Tyr His Pro Ala Thr1 523118PRTArtificial
SequenceSynthetic Construct 23Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ala 11524108PRTArtificial SequenceSynthetic
Construct 24Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val
Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 1052525PRTArtificial SequenceSynthetic
Construct 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20
252613PRTArtificial SequenceSynthetic Construct 26Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val1 5 102732PRTArtificial
SequenceSynthetic Construct 27Arg Phe Thr Ile Ser Ala Asp Thr Ser
Lys Asn Thr Ala Tyr Leu Gln1 5 10 15Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 302811PRTArtificial
SequenceSynthetic Construct 28Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ala1 5 102923PRTArtificial SequenceSynthetic Construct 29Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys 203015PRTArtificial SequenceSynthetic
Construct 30Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr1 5 10 153132PRTArtificial SequenceSynthetic Construct 31Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
303211PRTArtificial SequenceSynthetic Construct 32Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg1 5 103310PRTArtificial
SequenceSynthetic ConstructVARIANT(6)..(6)Xaa = Asp or Gly 33Gly
Phe Thr Phe Ser Xaa Ser Trp Ile His1 5 103418PRTArtificial
SequenceSynthetic ConstructVARIANT(4)..(4)Xaa = Ser or
LeuVARIANT(10)..(10)Xaa = Thr or Ser 34Ala Trp Ile Xaa Pro Tyr Gly
Gly Ser Xaa Tyr Tyr Ala Asp Ser Val1 5 10 15Lys
Gly3511PRTArtificial SequenceSynthetic ConstructVARIANT(5)..(5)Xaa
= Asp or ValVARIANT(6)..(6)Xaa = Val or IleVARIANT(7)..(7)Xaa = Ser
or AsnVARIANT(9)..(9)Xaa = Ala or PheVARIANT(10)..(10)Xaa = Val or
Leu 35Arg Ala Ser Gln Xaa Xaa Xaa Thr Xaa Xaa Ala1 5
10367PRTArtificial SequenceSynthetic ConstructVARIANT(4)..(4)Xaa =
Phe or ThrVARIANT(6)..(6)Xaa = Tyr or Ala 36Ser Ala Ser Xaa Leu Xaa
Ser1 5379PRTArtificial SequenceSynthetic
ConstructVARIANT(3)..(3)Xaa = Tyr, Gly, Phe, or
SerVARIANT(4)..(4)Xaa = Leu, Tyr, Phe or TrpVARIANT(5)..(5)Xaa =
Tyr, Asn, Ala, Thr, Gly, Phe or IleVARIANT(6)..(6)Xaa = His, Val,
Pro, Thr or IleVARIANT(8)..(8)Xaa = Ala, Trp, Arg, Pro or Thr 37Gln
Gln Xaa Xaa Xaa Xaa Pro Xaa Thr1 538336DNAArtificial
SequenceSynthetic Construct 38gatgttgtgt tgactcaaac tccactctcc
ctgtctgtca gctttggaga tcaagtttct 60atctcttgca ggtctagtca gagtcttgta
aacagttatg ggaacacctt tttgtcttgg 120tacctgcaca agcctggcca
gtctccacag ctcctcatct ttgggatttc caacagattt 180tctggggtgc
cagacaggtt cagtggcagt ggttcaggga cagatttcac actcaagatc
240agcacaataa agcctgagga cttgggaatg tattactgct tacaaggtac
gcatcagcct 300cccacgttcg gtcctgggac caagctggag gtgaaa
33639357DNAArtificial SequenceSynthetic Construct 39gaggtccagc
tgcaacagtc tggacctgag ctggtgaagc ctggaacttc aatgaagata 60tcctgcaagg
cttctggtta ctcattcact ggccatctta tgaactgggt gaagcagagc
120catggaaaga accttgagtg gattggactt attattcctt acaatggtgg
tacaagctat 180aaccagaagt tcaagggcaa ggccacattg actgtagaca
agtcatccag cacagcctac 240atggagctcc tcagtctgac ttctgatgac
tctgcagtct atttctgttc aagaggcctt 300aggggcttct atgctatgga
ctactggggt caaggaacct cagtcaccgt ctcctca 35740122PRTArtificial
SequenceSynthetic Construct 40Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115
12041118PRTArtificial SequenceSynthetic Construct 41Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln
Gly Thr 100 105 110Leu Val Thr Val Ser Ser 1154211PRTArtificial
SequenceSynthetic Construct 42Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser1 5 104330PRTArtificial SequenceSynthetic Construct 43Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25
304414PRTArtificial SequenceSynthetic Construct 44Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala1 5 104511PRTArtificial
SequenceSynthetic Construct 45Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser1 5 104610PRTArtificial SequenceSynthetic Construct 46Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys1 5 104715PRTArtificial
SequenceSynthetic Construct 47Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys1 5 10 1548447PRTArtificial
SequenceSynthetic Construct 48Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Ala Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44549214PRTArtificial SequenceSynthetic Construct 49Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr
Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro
Ala 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210
* * * * *
References