RU2825390C2 - Methods of treating cancer using pd-1 axis binding antagonists and tigit inhibitors - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions can be used to treat cancer. Disclosed is a method of treating or slowing down the progression of cancer in an individual, comprising administering to the individual an effective amount of a PD-1 antagonist antibody and an effective amount of an anti-TIGIT antagonist antibody, wherein the anti-TIGIT antagonist antibody inhibits and/or blocks the interaction of CD226 with TIGIT, wherein the anti-TIGIT antagonist antibody inhibits the interaction of the PVR with TIGIT; and wherein the anti-PD-1 antagonist antibody inhibits the binding of PD-1 to PD-L1. Also disclosed are kits for treating or slowing down the progression of cancer in an individual.

EFFECT: invention provides a synergistic effect on T-cell responses and induction of tumor-specific immunity.

31 cl, 37 dwg

Description

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 61/846,941, filed July 16, 2013, U.S. Provisional Application No. 61/865,582, filed August 13, 2013, U.S. Provisional Application No. 61/950,754, filed March 10, 2014, U.S. Provisional Application No. 61/985,884, filed April 29, 2014, and U.S. Provisional Application No. 61/992,109, filed May 12, 2014, each of which is incorporated herein by reference in its entirety.

Representing a sequence list in an ASCII text file

The contents of the following ASCII text file representation are incorporated herein by reference in its entirety: machine-readable form (CRF) of the sequence listing (file name: 146392025940SEQLISTING.TXT, accessed: July 16, 2014, size: 25 kb).

State of the art

The delivery of two distinct signals to T cells is a widely accepted model for the 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 also distinguishes between self-tolerance and immunologic 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 transmitted through the T cell receptor (TCR) following recognition of a peptide antigen presented in the major histocompatibility complex (MHC). The secondary or costimulatory signal is transmitted to T cells by costimulatory molecules expressed on antigen-presenting cells (APCs) and induces T cells to undergo clonal expansion, secrete cytokines, and perform effector function. Lenschow et al., Ann. Rev. Immunol., 14:233 (1996). In the absence of costimulation, T cells can become unresponsive to antigen stimulation, resulting in a tolerogenic response to any foreign or endogenous antigen.

In the dual-signal model, T cells receive positive costimulatory and negative coinhibitory signals. Regulation of these positive and negative signals is critical to maximizing host defense responses while maintaining immune tolerance and preventing autoimmunity. Negative signals are likely required to induce T cell tolerance, whereas positive signals promote T cell activation.

Both costimulatory and coinhibitory signals are delivered to antigen-exposed T cells, and the interaction of costimulatory and coinhibitory signals is required to control the magnitude of the immune response. In addition, signals delivered to T cells change as the infection or immune challenge resolves, worsens, or persists, and these changes profoundly influence the responding T cells and alter the immune response.

The mechanism of costimulation is of interest from a therapeutic perspective, as manipulation of costimulatory signals has been shown to provide a means to enhance or suppress cellular immune responses. Recently, it has been shown that T cell dysfunction or anergy occurs concomitantly with induced and sustained expression of the inhibitory receptor, programmed death polypeptide 1 (PD-1). As a result, targeting PD-1 and other molecules that signal through interaction with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), is an area of increasing interest.

PD-L1 is overexpressed in many types of malignancies and is often associated with poor prognosis (Okazaki T et al., Intern. Immun., 2007 19(7):813; Thompson RH et al., Cancer Res, 2006, 66(7):3381). Interestingly, most tumor-infiltrating T cells preferentially express PD-1 in contrast to normal tissue T cells and peripheral blood T cells, suggesting that PD-1 upregulation on tumor-reactive T cells may contribute to the suppression of antitumor immune responses (Blood, 2009, 114(8):1537). This phenomenon may occur through the use of PD-L1 signaling, mediated by the interaction of PD-L1-expressing tumor cells with PD-1-expressing T cells, leading to attenuation of T cell activation and evasion of immune surveillance (Sharpe et al., Nat. Rev., 2002; Keir ME et al., 2008, Annu. Rev. Immunol., 26:677). Thus, inhibition of PD-L1/PD-1 interactions may enhance CD8+ T cell-mediated tumor clearance.

Inhibition of the PD-1 signaling axis via its direct ligands (e.g., PD-LL, PD-L2) has been proposed as a means to enhance T cell immunity for cancer treatment (e.g., antitumor immunity). In addition, similar enhancement of T cell immunity has been observed by inhibiting PD-L1 binding to its binding partner B7-1. Furthermore, combining inhibition of the PD-1 signaling pathway with other signaling pathways that are dysregulated in tumor cells may further enhance treatment efficacy. There is a need for such optimal therapy to treat, stabilize, prevent, and/or slow down the progression of various cancers.

All references, publications and patent applications cited in this document are incorporated herein by reference in their entirety.

The essence of the invention

The present invention relates to a combination therapy comprising an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT.

Methods are provided for treating or slowing the progression of cancer in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT.

Also provided are methods for reducing or inhibiting the recurrence or progression of cancer in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT.

Also provided are methods for treating or slowing the progression of an immune-related disease in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT.

Also provided are methods for reducing or inhibiting the progression of an immune-related disease in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT.

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 antigen stimulation. In some embodiments, the T-cell dysfunctional disorder is characterized by T-cell anergy or a decreased ability to secrete cytokines, proliferate, or perform 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 is selected from the group consisting of unresolved acute infection, chronic infection, and tumor immunity.

Also provided are methods for enhancing, enhancing, or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits TIGIT expression and/or activity.

Also provided are methods for treating or slowing the progression of cancer in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

Also provided are methods for reducing or inhibiting the recurrence and progression of cancer in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

Also provided are methods for treating or slowing the progression of an immune-related disease in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

Also provided are methods for reducing or inhibiting the progression of an immune-related disease in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

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 antigen stimulation. In some embodiments, the T-cell dysfunctional disorder is characterized by T-cell anergy or a decreased ability to secrete cytokines, proliferate, or perform 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 is selected from the group consisting of unresolved acute infection, chronic infection, and tumor immunity.

Also provided are methods for enhancing, enhancing, or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

In some embodiments, an agent that modulates the expression and/or activity of CD226 is capable of increasing or stimulating the expression and/or activity of CD226.

In some embodiments, the agent that modulates the expression and/or activity of CD226 is selected from an agent that inhibits and/or blocks the interaction of CD226 with TIGIT, an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, an agent that inhibits and/or blocks the interactions of TIGIT with PVR, an agent that inhibits and/or blocks intracellular signaling mediated by the binding of TIGIT to PVR.

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 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 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 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.

The present invention also relates to a combination therapy comprising an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that reduces or inhibits one or more additional immune coinhibitory receptors.

Methods are provided for increasing, enhancing, or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an agent that reduces or inhibits TIGIT expression and/or activity and an agent that reduces or inhibits one or more additional immune coinhibitory receptors.

In some embodiments, the one or more additional immune coinhibitory receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA, and VISTA. In some embodiments, the one or more additional immune coinhibitory receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, and TIM3.

The present invention also relates to a combination therapy comprising an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that increases or activates one or more additional immune costimulatory receptors.

Methods are provided for increasing, enhancing, or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that increases or activates one or more additional immune costimulatory receptors.

In some embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of CD226, OX-40, CD28, CD27, CD137, HVEM, and GITR. In some embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of CD226, OX40, CD27, CD137, HVEM, and GITR. In some embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of OX-40 and CD27.

In some embodiments, any of the above methods further comprises administering at least one chemotherapeutic agent.

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.

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 compared to a period prior to administration of the combination.

In some embodiments, the number of CD4 and/or CD8 T cells is increased compared to the period before the combination was administered. In some embodiments, the number of activated CD4 and/or CD8 T cells is increased compared to the period before the combination was administered. In some embodiments, the activated CD4 and/or CD8 T cells are characterized by CD4 and/or CD8 T cells producing IFN ⁺ γ and/or increased cytolytic activity compared to the period before the combination was administered. In some embodiments, the CD4 and/or CD8 T cells exhibit increased release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukins.

In some embodiments, the CD4 and/or CD8 T cell is a memory effector T cell. In some embodiments, the CD4 and/or CD8 memory effector T cell is characterized by CD4 and/or CD8 T cells producing γ-IFN ⁺ and/or increased cytolytic activity. In some embodiments, the CD4 and/or CD8 memory effector T cell is characterized by having CD44 ^high CD62L ^low expression.

In some embodiments, the malignant tumor in any of the above methods has increased levels of T cell infiltration.

In some embodiments, the agent that reduces or inhibits the expression and/or activity of TIGIT is selected from the group consisting of an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, and an agent that inhibits the interaction and/or intracellular signaling mediated by the binding of TIGIT to PVR.

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.

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.

In some embodiments, the agent that inhibits 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, an 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).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain comprising an amino acid sequence set forth in EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO:16).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14) and a heavy chain of the antibody comprising an amino acid sequence set forth in EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFK GKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO:16).

In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof is provided, wherein the antibody is selected from a humanized antibody, a chimeric antibody, a bispecific antibody, a heteroconjugate antibody, and an immunotoxin.

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises at least one HVR that is at least 90% identical to an HVR shown in any of 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).

In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a light chain and/or a heavy chain comprising amino acid sequences that are at least 90% identical to the amino acid sequences set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQ ID NO: 13) or DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO: 14) or EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFK GKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO:16) respectively.

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.

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 binding partner ligand. 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 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 the antibody MDX-1106 (nivolumab). In some embodiments, the PD-1-binding antagonist is the Merck antibody 3475 (lambrolizumab). In some embodiments, the PD-1-binding antagonist is the antibody CT-011 (pidilizumab). In some embodiments, the PD-1-binding antagonist is the antibody AMP-224.

In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody.

In some embodiments, the PD-L1 binding antagonist is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and 4736 MEDI.

In some embodiments, the anti-PD-L1 antibody comprises a heavy chain comprising the HVR-H1 sequence GFTFSDSWIH (SEQ ID NO:17), the HVR-H2 sequence AWISPYGGSTYYADSVKG (SEQ ID NO:18), and the HVR-H3 sequence RHWPGGFDY (SEQ ID NO:19); and a light chain comprising the HVR-L1 sequence RASQDVSTAVA (SEQ ID NO:20), the HVR-L2 sequence SASFLYS (SEQ ID NO:21), and the HVR-L3 sequence QQYLYHPAT (SEQ ID NO:22).

In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:23) and a light chain variable region comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24).

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.

In some embodiments, the cancer to be 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 cancers, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell cancer, and other malignant blood diseases.

In some embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered continuously. In some embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered intermittently. In some embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered prior to an antagonist that binds to the PD-1 axis. In some embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered concurrently with an antagonist that binds to the PD-1 axis. In some embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered after an antagonist that binds to the PD-1 axis.

Also provided are kits comprising an antagonist that binds to the PD-1 axis and a package insert containing instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT to treat or slow the progression of cancer in an individual.

Also provided are kits comprising an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert containing instructions for using the antagonist that binds to the PD-1 axis and the agent that reduces or inhibits the expression and/or activity of TIGIT to treat or slow the progression of cancer in an individual.

Also provided are kits comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert containing instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with an antagonist that binds to the PD-1 axis, to treat or slow the progression of cancer in an individual.

Also provided are kits comprising an antagonist that binds to the PD-1 axis and a package insert containing instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT to enhance the immune function of an individual having cancer.

Also provided are kits comprising an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert containing instructions for using the antagonist that binds to the PD-1 axis and the agent that reduces or inhibits the expression and/or activity of TIGIT to enhance the immune function of an individual having cancer.

Also provided are kits comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert containing instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with an antagonist that binds to the PD-1 axis, to enhance the immune function of an individual having cancer.

Also provided are kits comprising an antagonist that binds to the PD-1 axis and a package insert containing instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that modulates the expression and/or activity of CD226 to treat or slow the progression of cancer in an individual.

Also provided are kits comprising an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226, and a package insert containing instructions for using the antagonist that binds to the PD-1 axis and the agent that modulates the expression and/or activity of CD226 to treat or slow the progression of cancer in an individual.

Also provided are kits comprising an agent that modulates the expression and/or activity of CD226 and a package insert containing instructions for using the agent that modulates the expression and/or activity of CD226 in combination with an antagonist that binds to the PD-1 axis to treat or slow the progression of cancer in an individual.

Also provided are kits comprising an antagonist that binds to the PD-1 axis and a package insert containing instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that modulates the expression and/or activity of CD226 to enhance immune function in an individual with cancer.

Also provided are kits comprising an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226, and a package insert containing instructions for using the antagonist that binds to the PD-1 axis and the agent that modulates the expression and/or activity of CD226 to enhance immune function in an individual having cancer.

Also provided are kits comprising an agent that modulates the expression and/or activity of CD226 and a package insert containing instructions for using the agent that modulates the expression and/or activity of CD226 in combination with an antagonist that binds to the PD-1 axis to enhance immune function in an individual having cancer.

In some embodiments, the kits comprise an antagonist that binds to the PD-1 axis that is an anti-PD-L1 antibody. In some embodiments, the kits comprise an antagonist that binds to the PD-1 axis that is an anti-PD-1 antibody. In some embodiments, the kits comprise an agent that reduces or inhibits TIGIT expression and/or activity 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 interaction and/or intracellular signaling mediated by TIGIT binding to PVR. In some embodiments, the kits comprise an antagonist of TIGIT expression and/or activity that is an anti-TIGIT antibody or an antigen-binding fragment thereof.

In some embodiments, the kits comprise an agent that modulates the expression and/or activity of CD226, which is capable of increasing and/or promoting the expression and/or activity of CD226. In some embodiments, the kits comprise an agent that modulates the expression and/or activity of CD226, 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 intracellular signaling mediated by TIGIT binding to PVR. In some embodiments, the kits comprise an agent that inhibits and/or blocks the interaction of CD226 with TIGIT, and/or an antagonist of TIGIT expression and/or activity that is an anti-TIGIT antibody or an antigen-binding fragment thereof.

In some aspects, the present invention relates to a method of treating or slowing the progression of cancer in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT. In other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for treating or slowing the progression of cancer in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In yet other aspects, the present invention relates to the use of an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT in the manufacture of a medicament for treating or slowing the progression of cancer in an individual, wherein the agent that reduces or inhibits the expression and/or activity of TIGIT is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in the treatment or slowing down of cancer progression in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that reduces or inhibits the expression and/or activity of TIGIT for use in the treatment or slowing down of cancer progression in combination with an antagonist that binds to the PD-1 axis.

In still other aspects, the present invention relates to a method for reducing or inhibiting the recurrence or progression of cancer in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for reducing or inhibiting the recurrence or progression of cancer in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to the use of an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT in the manufacture of a medicament for reducing or inhibiting the recurrence or progression of cancer in an individual, wherein the agent that reduces or inhibits the expression and/or activity of TIGIT is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in reducing or inhibiting the recurrence or progression of cancer in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that reduces or inhibits the expression and/or activity of TIGIT for use in reducing or inhibiting the recurrence or progression of cancer in combination with an antagonist that binds to the PD-1 axis.

In still other aspects, the present invention relates to a method of treating or slowing the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for treating or slowing the progression of an immune-related disease in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to the use of an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT in the manufacture of a medicament for treating or slowing the progression of an immune-related disease in an individual, wherein the agent that reduces or inhibits the expression and/or activity of TIGIT is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in treating or slowing the progression of an immune-related disease, in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that reduces or inhibits the expression and/or activity of TIGIT for use in treating or slowing the progression of an immune-related disease, in combination with an antagonist that binds to the PD-1 axis.

In still other aspects, the present invention relates to a combination comprising an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT.

In still other aspects, the present invention relates to a method for reducing or inhibiting the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for reducing or inhibiting the progression of an immune-related disease in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to the use of an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT in the manufacture of a medicament for reducing or inhibiting the progression of an immune-related disease in an individual, wherein the agent that reduces or inhibits the expression and/or activity of TIGIT is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in reducing or inhibiting the progression of an immune-related disease in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that reduces or inhibits the expression and/or activity of TIGIT for use in reducing or inhibiting the progression of an immune-related disease in combination with an antagonist that binds to the PD-1 axis.

In some embodiments, which may be combined with any of the preceding embodiments, the immune-related disease is associated with a T-cell dysfunctional disorder. In some embodiments, which may be combined with any of the preceding embodiments, the immune-related disease is a viral infection. In some embodiments, which may be combined with any of the preceding embodiments, the viral infection is a chronic viral infection. In some embodiments, which may be combined with any of the preceding embodiments, the T-cell dysfunctional disorder is characterized by a decrease in responsiveness to antigen stimulation. In some embodiments, which may be combined with any of the preceding embodiments, the T-cell dysfunctional disorder is characterized by T-cell anergy or a decreased ability to secrete cytokines, proliferate, or perform cytolytic activity. In some embodiments, which may be combined with any of the preceding embodiments, the T-cell dysfunctional disorder is characterized by T-cell exhaustion. In some embodiments that can be combined with any of the preceding embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments that can 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.

In still other aspects, the present invention relates to a method for increasing, enhancing or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for increasing or stimulating an immune response or immune function in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to the use of an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT in the manufacture of a medicament for enhancing or stimulating an immune response or immune function in an individual, wherein the agent that reduces or inhibits the expression and/or activity of TIGIT is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in enhancing or stimulating an immune response or immune function in an individual in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that reduces or inhibits the expression and/or activity of TIGIT for use in enhancing or stimulating an immune response or immune function in an individual in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a combination comprising an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT.

In yet other aspects, the present invention relates to a method of treating or slowing the progression of cancer in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. In yet other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for treating or slowing the progression of cancer in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that modulates the expression and/or activity of CD226. In yet other aspects, the present invention relates to the use of an effective amount of an agent that modulates the expression and/or activity of CD226 in the manufacture of a medicament for treating or slowing the progression of cancer in an individual, wherein the agent that modulates the expression and/or activity of CD226 is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in the treatment or slowing down of cancer progression in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that modulates the expression and/or activity of CD226 for use in the treatment or slowing down of cancer progression in combination with an antagonist that binds to the PD-1 axis.

In still other aspects, the present invention relates to a method of reducing or inhibiting the recurrence or progression of cancer in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for reducing or inhibiting the recurrence or progression of cancer in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to the use of an effective amount of an agent that modulates the expression and/or activity of CD226 in the manufacture of a medicament for reducing or inhibiting the recurrence or progression of cancer in an individual, wherein the agent that modulates the expression and/or activity of CD226 is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in reducing or inhibiting the recurrence or progression of cancer in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that modulates the expression and/or activity of CD226 for use in reducing or inhibiting the recurrence or progression of cancer in combination with an antagonist that binds to the PD-1 axis.

In still other aspects, the present invention relates to a method of treating or slowing the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for treating or slowing the progression of an immune-related disease in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to the use of an effective amount of an agent that modulates the expression and/or activity of CD226 in the manufacture of a medicament for treating or slowing the progression of an immune-related disease in an individual, wherein the agent that modulates the expression and/or activity of CD226 is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in treating or slowing the progression of an immune-related disease in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that modulates the expression and/or activity of CD226 for use in treating or slowing the progression of an immune-related disease in combination with an antagonist that binds to the PD-1 axis.

In still further aspects, the present invention relates to a combination comprising an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

In still other aspects, the present invention relates to a method of reducing or inhibiting the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for reducing or inhibiting the progression of an immune-related disease in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to the use of an effective amount of an agent that modulates the expression and/or activity of CD226 in the manufacture of a medicament for reducing or inhibiting the progression of an immune-related disease in an individual, wherein the agent that modulates the expression and/or activity of CD226 is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in reducing or inhibiting the progression of an immune-related disease in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that modulates the expression and/or activity of CD226 for use in reducing or inhibiting the progression of an immune-related disease in combination with an antagonist that binds to the PD-1 axis.

In some embodiments, which may be combined with any of the preceding embodiments, the immune-related disease is associated with a T-cell dysfunctional disorder. In some embodiments, which may be combined with any of the preceding embodiments, the immune-related disease is a viral infection. In some embodiments, which may be combined with any of the preceding embodiments, the viral infection is a chronic viral infection. In some embodiments, which may be combined with any of the preceding embodiments, the T-cell dysfunctional disorder is characterized by a decrease in responsiveness to antigen stimulation. In some embodiments, which may be combined with any of the preceding embodiments, the T-cell dysfunctional disorder is characterized by T-cell anergy or a decreased ability to secrete cytokines, proliferate, or perform cytolytic activity. In some embodiments, which may be combined with any of the preceding embodiments, the T-cell dysfunctional disorder is characterized by T-cell exhaustion. In some embodiments that can be combined with any of the preceding embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments that can 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.

In still other aspects, the present invention relates to a method for increasing, enhancing or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to the use of an effective amount of an antagonist that binds to the PD-1 axis in the manufacture of a medicament for increasing or stimulating an immune response or immune function in an individual, wherein the antagonist that binds to the PD-1 axis is used in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to the use of an effective amount of an agent that modulates the expression and/or activity of CD226 in the manufacture of a medicament for enhancing or stimulating an immune response or immune function in an individual, wherein the agent that modulates the expression and/or activity of CD226 is used in combination with an antagonist that binds to the PD-1 axis. In still other aspects, the present invention relates to a pharmaceutical composition comprising an antagonist that binds to the PD-1 axis for use in enhancing or stimulating an immune response or immune function in combination with an agent that modulates the expression and/or activity of CD226. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that modulates the expression and/or activity of CD226 for use in enhancing or stimulating an immune response or immune function in combination with an antagonist that binds to the PD-1 axis. In still further aspects, the present invention relates to a combination comprising an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an agent that increases and/or stimulates the expression and/or activity of CD226. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an agent that increases and/or stimulates the interaction of CD226 with PVR. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an agent that increases and/or stimulates intracellular signaling mediated by the binding of CD226 to PVR. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is selected from the group consisting of an agent that inhibits and/or blocks the interaction of CD226 with TIGIT, an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, 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 intracellular signaling mediated by TIGIT binding to PVR, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVRL2, an agent that inhibits and/or blocks intracellular signaling, mediated by the binding of TIGIT to PVRL3, and combinations thereof. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an agent that inhibits and/or blocks the interaction of CD226 with TIGIT. In some embodiments, which 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, and an inhibitory polypeptide. In some embodiments, which 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 some embodiments, which 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 some embodiments, which may be combined with any of the preceding embodiments, the antisense polynucleotide is directed to TIGIT. In some embodiments, which may be combined with any of the preceding embodiments, the interfering RNA is directed to TIGIT. In some embodiments, which may be combined with any of the preceding embodiments, the catalytic RNA is directed to TIGIT. In some embodiments, which may be combined with any of the preceding embodiments, the RNA-DNA chimera is directed to TIGIT. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an antagonist of TIGIT expression and/or activity. In some embodiments, which 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 some embodiments, which 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 some 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 some 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 some 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 some embodiments, which 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 some embodiments, which 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 some embodiments, which may be combined with any of the preceding embodiments, the agent that inhibits and/or blocks 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, which 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 some embodiments, which 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 still other aspects, the present invention relates to a method for increasing, enhancing or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that reduces or inhibits one or more additional immune co-inhibitory receptors. In still other aspects, the present invention relates to the use of an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT in the manufacture of a medicament for increasing or stimulating an immune response or immune function in an individual, wherein the agent that reduces or inhibits the expression and/or activity of TIGIT is used in combination with an agent that reduces or inhibits one or more additional immune co-inhibitory receptors. In still other aspects, the present invention relates to the use of an effective amount of an agent that reduces or inhibits one or more additional immune coinhibitory receptors in the manufacture of a medicament for enhancing or stimulating an immune response or immune function in an individual, wherein the agent that reduces or inhibits one or more additional immune coinhibitory receptors is used in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that reduces or inhibits the expression and/or activity of TIGIT for use in enhancing or stimulating an immune response or immune function in combination with an agent that reduces or inhibits one or more additional immune coinhibitory receptors. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that reduces or inhibits one or more additional immune coinhibitory receptors for use in enhancing or stimulating an immune response or immune function in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a combination comprising an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that reduces or inhibits one or more additional immune coinhibitory receptors. In some embodiments, which may be combined with any of the preceding embodiments, the one or more additional immune coinhibitory receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4 and CD96. In some embodiments, which may be combined with any of the preceding embodiments, the one or more additional immune coinhibitory receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, and TIM3.

In still other aspects, the present invention relates to a method for increasing, enhancing or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that increases or activates one or more additional immune costimulatory receptors. In still other aspects, the present invention relates to the use of an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT in the manufacture of a medicament for increasing or stimulating an immune response or immune function in an individual, wherein the agent that reduces or inhibits the expression and/or activity of TIGIT is used in combination with an agent that increases or activates one or more additional immune costimulatory receptors. In still other aspects, the present invention relates to the use of an effective amount of an agent that increases or activates one or more additional immune costimulatory receptors in the manufacture of a medicament for enhancing or stimulating an immune response or immune function in an individual, wherein the agent that increases or activates one or more additional immune costimulatory receptors is used in combination with an agent that decreases or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that decreases or inhibits the expression and/or activity of TIGIT for use in enhancing or stimulating an immune response or immune function in combination with an agent that increases or activates one or more additional immune costimulatory receptors. In still other aspects, the present invention relates to a pharmaceutical composition comprising an agent that increases or activates one or more additional immune costimulatory receptors for use in enhancing or stimulating an immune response or immune function in combination with an agent that decreases or inhibits the expression and/or activity of TIGIT. In still other aspects, the present invention relates to a combination comprising an effective amount of an agent that decreases or inhibits the expression and/or activity of TIGIT and an agent that increases or activates one or more additional immune costimulatory receptors. In some embodiments, which may be combined with any of the preceding embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of CD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D and 2B4. In some embodiments that can be combined with any of the preceding embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of CD226, OX-40, CD27, CD137, HVEM, and GITR. In some embodiments that can be combined with any of the preceding embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of OX-40 and CD27.

In some embodiments, which may be combined with any of the preceding embodiments, the method further comprises administering at least one chemotherapeutic agent. In some embodiments, which may be combined with any of the preceding embodiments, the individual has cancer. In some embodiments, which may be combined with any of the preceding embodiments, the individual is a human. In some embodiments, which may be combined with any of the preceding embodiments, the CD4 and/or CD8 T cells in the individual have increased or enhanced priming, activation, proliferation, cytokine release, and/or cytolytic activity compared to before the combination was administered. In some embodiments, which may be combined with any of the preceding embodiments, the number of CD4 and/or CD8 T cells is increased compared to before the combination was administered. In some embodiments, which may be combined with any of the preceding embodiments, the number of activated CD4 and/or CD8 T cells is increased compared to before the combination was administered. In some embodiments, which may be combined with any of the preceding embodiments, the activated CD4 and/or CD8 T cells are characterized by CD4 and/or CD8 T cells producing γ-IFN ⁺ and/or increased cytolytic activity compared to the period before the combination was administered. In some embodiments, which 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-γ, TNF-α, and interleukins. In some embodiments, which may be combined with any of the preceding embodiments, the CD4 and/or CD8 T cells are effector memory T cells. In some embodiments, which may be combined with any of the preceding embodiments, the CD4 and/or CD8 effector memory T cells are characterized by CD4 and/or CD8 T cells producing γ-IFN ⁺ and/or increased cytolytic activity. In some embodiments, which may be combined with any of the preceding embodiments, the CD4 and/or CD8 effector memory T cells are characterized by having ^{CD44highCD62Llow} expression. In some embodiments, which may be combined with any of the preceding embodiments, the ^cancer has increased levels of T cell infiltration. In some embodiments, which may be combined with any of the preceding embodiments, the agent that reduces or inhibits the expression and/or activity of TIGIT is selected from the group consisting of an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, 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 intracellular signaling mediated by the binding of TIGIT to PVR, an agent that inhibits intracellular signaling mediated by the binding of TIGIT to PVRL2, an agent that inhibits intracellular signaling mediated by the binding of TIGIT to PVRL3, and combinations thereof. In some 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 some 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 some 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 some embodiments, which 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 some embodiments, which 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 some embodiments, which may be combined with any of the preceding embodiments, the agent that inhibits and/or blocks 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, which may be combined with any of the preceding embodiments, the agent that inhibits and/or blocks 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 some embodiments, which may be combined with any of the preceding embodiments, the agent that inhibits and/or blocks 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 some embodiments, which 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, interfering RNA, catalytic RNA, and an RNA-DNA chimera. In some embodiments, which may be combined with any of the preceding embodiments, the antisense polynucleotide is directed to TIGIT.

In some embodiments, which may be combined with any of the preceding embodiments, the interfering RNA is directed to TIGIT. In some embodiments, which may be combined with any of the preceding embodiments, the catalytic RNA is directed to TIGIT. In some embodiments, which may be combined with any of the preceding embodiments, the RNA-DNA chimera is directed to TIGIT. In some embodiments, which may be combined with any of the preceding embodiments, the antagonist of TIGIT expression and/or activity is an anti-TIGIT antibody or an antigen-binding fragment thereof. In some embodiments, which 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 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 some embodiments, which may be combined with any of the preceding embodiments, there is provided an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the light chain of the antibody comprises an amino acid sequence set forth in DIVMTQSPSSLAVSPGEKVTMTC KSSQSLYYSGVKENLLA WYQQKPGQSPKLLIYY ASIRFT GVPDRFTGSGSGTDYTLTITSVQAEDMGQYFC QQGINNPL TFGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISC RSSQSLVNSYGNTFLS WYLHKPGQSPQLLIF GISNRFS GVPDRFSGSGSGTDFTLKISTIKPEDLGMYYC LQGTHQPPT FGPGTKLEVK (SEQ ID NO:14). In some embodiments, which may be combined with any of the preceding embodiments, there is provided an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the heavy chain of the antibody comprises an amino acid sequence set forth in EVQLVESGGGLTQPGKSLKLSCEAS GFTFSSFTMH WVRQSPGKGLEWVA FIRSGSGIVFYADAVRG RFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR RPLGHNTFDS WGQGTLVTVSS (SEQ ID NO:15) or VQLQQSGPELVKPGTSMKISCKAS GYSFTGHLMN WVKQSHGKNLEWIG LIIPYNGGTSYNQKFKG KATLTVDKSSSTAYMELLSLTSDDSAVYFCSR GLRGFYAMDY WGQGTSVTVSS (SEQ ID NO:16). In some embodiments, which may be combined with any of the preceding embodiments, there is provided an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the light chain of the antibody comprises an amino acid sequence set forth in DIVMTQSPSSLAVSPGEKVTMTC KSSQSLYYSGVKENLLA WYQQKPGQSPKLLIYY ASIRFT GVPDRFTGSGSGTDYTLTITSVQAEDMGQYFC QQGINNPLT FGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISC RSSQSLVNSYGNTFLS WYLHKPGQSPQLLIF GISNRFS GVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), and the heavy chain of the antibody comprises an amino acid sequence set forth in EVQLVESGGGLTQPGKSLKLSCEAS GFTFSSFTMH WVRQSPGKGLEWVA FIRSGSGIVFYADAVRG RFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR RPLGHNTFDS WGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKAS GYSFTGHLMN WVKQSHGKNLEWIG LIIPYNGGTSYN QKFKG KATLTVDKSSSTAYMELLSLTSDDSAVYFCSR GLRGFYAMDY WGQGTSVTVSS SEQ ID NO:16). In some embodiments, which may be combined with any of the preceding embodiments, an 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 some embodiments, which 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 shown 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 some embodiments, which may be combined with any of the preceding embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a light chain comprising amino acid sequences that are at least 90% identical to the amino acid sequences set forth in DIVMTQSPSSLAVSPGEKVTMTC KSSQSLYYSGVKENLLA WYQQKPGQSPKLLIYY ASIRFT GVPDRFTGSGSGTDYTLTITSVQAEDMGQYFC QQGINNPLT FGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISC RSSQSLVNSYGNTFLS WYLHKPGQSPQLLIF GISNRFS GVPDRFSGSGSGTDFTLKISTIKPEDLGMYYC LQGTHQPPT FGPGTKLEVK (SEQ ID NO:14); and/or a heavy chain comprising amino acid sequences that are at least 90% identical to the amino acid sequences set forth in EVQLVESGGGLTQPGKSLKLSCEAS GFTFSSFTMH WVRQSPGKGLEWVA FIRSGSGIVFYADAVRG RFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR RPLGHNTFDS WGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKAS GYSFTGHLMN WVKQSHGKNLEWIG LIIPYNGGTSYNQKFKG KATLTVDKSSSTAYMELLSLTSDDSAVYFCSR GLRGFYAMDY WGQGTSVTVSS (SEQ ID NO:16). In some embodiments that can 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 some embodiments that can be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments that can be combined with any of the preceding embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its binding partner ligands. In some embodiments that can be combined with any of the preceding embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, which may be combined with any of the preceding embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, which 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 some embodiments, which may be combined with any of the preceding embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, which may be combined with any of the preceding embodiments, the PD-1 binding antagonist is the antibody MDX-1106. In some embodiments, which may be combined with any of the preceding embodiments, the PD-1 binding antagonist is the antibody MK-3475. In some embodiments that may be combined with any of the preceding embodiments, the PD-1 binding antagonist is the antibody CT-011. In some embodiments that may be combined with any of the preceding embodiments, the PD-1 binding antagonist is the antibody AMP-224. In some embodiments that may be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments that may be combined with any of the preceding embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to PD-1. In some embodiments that may be combined with any of the preceding embodiments, the PD-L1 binding antagonist inhibits PD-L1 binding to B7-1. In some embodiments that can 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 some embodiments that can be combined with any of the preceding embodiments, the PD-L1-binding antagonist is an anti-PD-L1 antibody. In some embodiments that can 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 some embodiments, which may be combined with any of the preceding embodiments, the anti-PD-L1 antibody comprises a heavy chain comprising the HVR-H1 sequence GFTFSDSWIH (SEQ ID NO:17), the HVR-H2 sequence AWISPYGGSTYYADSVKG (SEQ ID NO:18), and the HVR-H3 sequence RHWPGGFDY (SEQ ID NO:19); and a light chain comprising the HVR-L1 sequence RASQDVSTAVA (SEQ ID NO:20), the HVR-L2 sequence SASFLYS (SEQ ID NO:21), and the HVR-L3 sequence QQYLYHPAT (SEQ ID NO:22). In some embodiments, which 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 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:23), EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:40) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:41), and a light chain variable region comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24). In some embodiments, which can be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments, which may be combined with any of the preceding embodiments, the PD-L2-binding antagonist is an antibody. In some embodiments, which may be combined with any of the preceding embodiments, the PD-L2-binding antagonist is an immunoadhesin. In some embodiments that can 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 cancers, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell cancer and other malignant blood diseases. In some embodiments that can be combined with any of the preceding embodiments, the agent that reduces or inhibits TIGIT expression and/or activity is administered continuously. In some embodiments, which may be combined with any of the preceding embodiments, the agent that reduces or inhibits the expression and/or activity of TIGIT is administered periodically. In some embodiments, which may be combined with any of the preceding embodiments, the agent that reduces or inhibits the expression and/or activity of TIGIT is administered prior to the administration of the PD-1 axis binding antagonist. In some embodiments, which may be combined with any of the preceding embodiments, the agent that reduces or inhibits the expression and/or activity of TIGIT is administered simultaneously with the PD-1 axis binding antagonist. In some embodiments, which may be combined with any of the preceding embodiments, the agent that reduces or inhibits the expression and/or activity of TIGIT is administered after the administration of the PD-1 axis binding antagonist. In some embodiments, which may be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is administered prior to the agent that modulates the expression and/or activity of CD226. In some embodiments, which may be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is administered concurrently with the agent that modulates the expression and/or activity of CD226. In some embodiments, which may be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is administered after the agent that modulates the expression and/or activity of CD226. In some embodiments, which may be combined with any of the preceding embodiments, the agent that reduces or inhibits the expression and/or activity of TIGIT is administered prior to the agent that reduces or inhibits one or more additional immune coinhibitory receptors. In some embodiments, which may be combined with any of the preceding embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered simultaneously with an agent that reduces or inhibits one or more additional immune coinhibitory receptors. In some embodiments, which may be combined with any of the preceding embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered after an agent that reduces or inhibits one or more additional immune coinhibitory receptors. In some embodiments, which may be combined with any of the preceding embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered before an agent that increases or activates one or more additional immune costimulatory receptors. In some embodiments that can be combined with any of the preceding embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered simultaneously with an agent that increases or activates one or more additional immune costimulatory receptors. In some embodiments that can be combined with any of the preceding embodiments, an agent that reduces or inhibits the expression and/or activity of TIGIT is administered after an agent that increases or activates one or more additional immune costimulatory receptors.

In still other aspects, the present invention relates to a kit comprising an antagonist that binds to the PD-1 axis and a package insert containing instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT to treat or slow the progression of cancer in an individual.

In still other aspects, the present invention relates to a kit comprising an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert containing instructions for using the antagonist that binds to the PD-1 axis and the agent that reduces or inhibits the expression and/or activity of TIGIT to treat or slow the progression of cancer in an individual.

In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with an antagonist that binds to the PD-1 axis, to treat or slow the progression of cancer in an individual.

In still other aspects, the present invention relates to a kit comprising an antagonist that binds to the PD-1 axis and a package insert containing instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT to enhance the immune function of an individual having cancer.

In still other aspects, the present invention relates to a kit comprising an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert containing instructions for using the antagonist that binds to the PD-1 axis and the agent that reduces or inhibits the expression and/or activity of TIGIT to enhance immune function in an individual having cancer.

In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with an antagonist that binds to the PD-1 axis, to enhance immune function in an individual having cancer.

In still other aspects, the present invention relates to a kit comprising an antagonist that binds to the PD-1 axis and a package insert comprising instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that modulates the expression and/or activity of CD226 to treat or slow the progression of cancer in an individual.

In still other aspects, the present invention relates to a kit comprising an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226, and a package insert comprising instructions for using the antagonist that binds to the PD-1 axis and the agent that modulates the expression and/or activity of CD226 to treat or slow the progression of cancer in an individual.

In still other aspects, the present invention relates to a kit comprising an agent that modulates the expression and/or activity of CD226, and a package insert comprising instructions for using the agent that modulates the expression and/or activity of CD226, in combination with an antagonist that binds to the PD-1 axis, for treating or slowing the progression of cancer in an individual.

In still other aspects, the present invention relates to a kit comprising an antagonist that binds to the PD-1 axis and a package insert comprising instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that modulates the expression and/or activity of CD226 to enhance immune function in an individual having cancer.

In still other aspects, the present invention relates to a kit comprising an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226, and a package insert comprising instructions for using the antagonist that binds to the PD-1 axis and the agent that modulates the expression and/or activity of CD226 to enhance immune function in an individual having cancer.

In still other aspects, the present invention relates to a kit comprising an agent that modulates the expression and/or activity of CD226, and a package insert comprising instructions for using the agent that modulates the expression and/or activity of CD226, in combination with an antagonist that binds to the PD-1 axis, to enhance the immune function of an individual having cancer.

In some embodiments that may be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody. In some 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 some embodiments that may be combined with any of the preceding embodiments, the anti-PD-L1 antibody comprises a heavy chain comprising the HVR-H1 sequence GFTFSDSWIH (SEQ ID NO:17), the HVR-H2 sequence AWISPYGGSTYYADSVKG (SEQ ID NO:18), and the HVR-H3 sequence RHWPGGFDY (SEQ ID NO:19); and a light chain comprising the HVR-L1 sequence RASQDVSTAVA (SEQ ID NO:20), the HVR-L2 sequence SASFLYS (SEQ ID NO:21), and the HVR-L3 sequence QQYLYHPAT (SEQ ID NO:22). In some embodiments, which 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 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:23), EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:40) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:41), and a light chain variable region comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24). In some embodiments, which can be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is an anti-PD-1 antibody. In some 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 some embodiments that may be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is the AMP-224 antibody. In some embodiments that may be combined with any of the preceding embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments that may be combined with any of the preceding embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments that may be combined with any of the preceding embodiments, the PD-L2 binding antagonist is an immunoadhesin.

In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with an agent that reduces or inhibits one or more additional immune coinhibitory receptors, to treat or slow the progression of cancer in an individual. In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and an agent that reduces or inhibits one or more additional immune coinhibitory receptors, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, and the agent that reduces or inhibits one or more additional immune coinhibitory receptors, to treat or slow the progression of cancer in an individual. In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits one or more additional immune coinhibitory receptors, and a package insert comprising instructions for using the agent that reduces or inhibits one or more additional immune coinhibitory receptors, in combination with an agent that reduces or inhibits TIGIT expression and/or activity, to treat or slow the progression of cancer in an individual. In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits TIGIT expression and/or activity, and a package insert comprising instructions for using the agent that reduces or inhibits TIGIT expression and/or activity, in combination with an agent that reduces or inhibits one or more additional immune coinhibitory receptors, to enhance immune function in an individual having cancer. In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that reduces or inhibits one or more additional immune coinhibitory receptors, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT and the agent that reduces or inhibits one or more additional immune coinhibitory receptors to enhance immune function in an individual having cancer. In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits one or more additional immune coinhibitory receptors and a package insert comprising instructions for using the agent that reduces or inhibits one or more additional immune coinhibitory receptors, in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT, to enhance immune function in an individual having cancer. In some embodiments that can be combined with any of the preceding embodiments, the one or more additional immune coinhibitory receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and CD96. In some embodiments that can be combined with any of the preceding embodiments, the one or more additional immune coinhibitory receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, and TIM3.

In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with an agent that increases or activates one or more additional immune costimulatory receptors, to treat or slow the progression of cancer in an individual. In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and an agent that increases or activates one or more additional immune costimulatory receptors, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, and the agent that increases or activates one or more additional immune costimulatory receptors, to treat or slow the progression of cancer in an individual. In still other aspects, the present invention relates to a kit comprising an agent that increases or activates one or more additional immune costimulatory receptors, and a package insert comprising instructions for using the agent that increases or activates one or more additional immune costimulatory receptors in combination with an agent that decreases or inhibits the expression and/or activity of TIGIT, to treat or slow the progression of cancer in an individual. In still other aspects, the present invention relates to a kit comprising an agent that decreases or inhibits the expression and/or activity of TIGIT, and a package insert comprising instructions for using the agent that decreases or inhibits the expression and/or activity of TIGIT, in combination with an agent that increases or activates one or more additional immune costimulatory receptors, to enhance immune function in an individual having cancer. In still other aspects, the present invention relates to a kit comprising an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that increases or activates one or more additional immune costimulatory receptors, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT and the agent that increases or activates one or more additional immune costimulatory receptors to enhance immune function in an individual having cancer. In still other aspects, the present invention relates to a kit comprising an agent that increases or activates one or more additional immune costimulatory receptors and a package insert comprising instructions for using the agent that increases or activates one or more additional immune costimulatory receptors, in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT, to enhance immune function in an individual having cancer. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of CD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of CD226, OX-40, CD27, CD137, HVEM, and GITR. In some embodiments that may be combined with any of the preceding embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of OX-40 and CD27.

In some embodiments, which may be combined with any of the preceding embodiments, the individual is a human. In some embodiments, which may be combined with any of the preceding embodiments, the agent that reduces or inhibits the expression and/or activity of TIGIT is selected from the group consisting of an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, 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 intracellular signaling mediated by the binding of TIGIT to PVR, an agent that inhibits and/or blocks intracellular signaling mediated by the binding of TIGIT to PVL2, and an agent that inhibits and/or blocks intracellular signaling mediated by the binding of TIGIT to PVL3. In some embodiments, which may be combined with any of the preceding embodiments, the antagonist of TIGIT expression and/or activity is an anti-TIGIT antibody or an antigen-binding fragment thereof. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an agent that increases and/or stimulates the expression and/or activity of CD226. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an agent that increases and/or stimulates the interaction of CD226 with PVR. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an agent that increases and/or stimulates intracellular signaling mediated by the binding of CD226 to PVR. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is selected from the group consisting of an agent that inhibits and/or blocks the interaction of CD226 with TIGIT, an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, 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 intracellular signaling mediated by TIGIT binding to PVR, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVRL2, and an agent that inhibits and/or blocks intracellular signaling, mediated by the binding of TIGIT to PVRL3. In some embodiments, which may be combined with any of the preceding embodiments, the agent that modulates the expression and/or activity of CD226 is an agent that inhibits and/or blocks the interaction of CD226 with TIGIT. In some embodiments, which 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, and an inhibitory polypeptide. In some embodiments, which 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 some embodiments, which 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 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 some embodiments, which may be combined with any of the preceding embodiments, there is provided an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the light chain of the antibody comprises an amino acid sequence set forth in DIVMTQSPSSLAVSPGEKVTMTC KSSQSLYYSGVKENLLA WYQQKPGQSPKLLIYY ASIRFT GVPDRFTGSGSGTDYTLTITSVQAEDMGQYFC QQGINNPL TFGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISC RSSQSLVNSYGNTFLS WYLHKPGQSPQLLIF GISNRFS GVPDRFSGSGSGTDFTLKISTIKPEDLGMYYC LQGTHQPPT FGPGTKLEVK (SEQ ID NO:14). In some embodiments, which may be combined with any of the preceding embodiments, there is provided an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the heavy chain of the antibody comprises an amino acid sequence set forth in EVQLVESGGGLTQPGKSLKLSCEAS GFTFSSFTMH WVRQSPGKGLEWVA FIRSGSGIVFYADAVRG RFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR RPLGHNTFDS WGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKAS GYSFTGHLMN WVKQSHGKNLEWIG LIIPYNGGTSYNQKFKG KATLTVDKSSSTAYMELLSLTSDDSAVYFCSR GLRGFYAMDY WGQGTSVTVSS (SEQ ID NO:16). In some embodiments, which may be combined with any of the preceding embodiments, there is provided an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the light chain of the antibody comprises an amino acid sequence set forth in DIVMTQSPSSLAVSPGEKVTMTC KSSQSLYYSGVKENLLA WYQQKPGQSPKLLIYY ASIRFT GVPDRFTGSGSGTDYTLTITSVQAEDMGQYFC QQGINNPL TFGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISC RSSQSLVNSYGNTFLS WYLHKPGQSPQLLIF GISNRFS GVPDRFSGSGSGTDFTLKISTIKPEDLGMYYC LQGTHQPPT FGPGTKLEVK (SEQ ID NO:14), and the heavy chain of the antibody comprises an amino acid sequence set forth in EVQLVESGGGLTQPGKSLKLSCEAS GFTFSSFTMH WVRQSPGKGLEWVA FIRSGSGIVFYADAVRG RFTISRDNAKNLLFLQMNDLKSEDTAMYYCAR RPLGHNTFDS WGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKAS GYSFTGHLMN WVKQSHGKNLEWIG LIIPYNGGTSYN QKFKG KATLTVDKSSSTAYMELLSLTSDDSAVYFCSR GLRGFYAMDY WGQGTSVTVSS (SEQ ID NO:16).

Brief description of the figures

Figure 1 shows that TIGIT is expressed at high levels on depleted CD8+ and CD4+ T cells. Figure 1A shows MACS-enriched splenic CD8+ T cells from C57BL6/J mice that were stimulated with plate-immobilized anti-CD3 and anti-CD28 antibodies for 24 to 48 h in vitro. Flow cytometry histograms represent TIGIT expression (red) versus isotype staining (gray). Results of TIGIT MFI quantification are also shown. ***P < 0.001. Data represent two independent experiments; n = 3. In Figures 1B–1C, C57BL6/J mice were infected with the Armstrong strain of LCMV and splenocytes were analyzed 7 days post-infection. Data represent two independent experiments; n = 5. Figure 1B–1C 1B is a flow cytometric histogram showing TIGIT expression by ^naïve ( ^{CD44lowCD62Lhigh} ) and effector CD4+ and CD8+ memory T cells ( ^{CD44highCD62Llow} ). Results of TIGIT MFI quantification are also shown. ***P< ^0.001 . Fig. 1C is a flow cytometric histogram showing TIGIT PD- ^1high and PD- ^1low expression by effector CD8+ T cells. Results of TIGIT MFI quantification are also shown. ***P<0.001. Fig. 1D shows that C57BL6/J mice were rapidly depleted of CD4+ T cells and infected with LCMV clone 13. Splenocytes were analyzed 42 days postinfection. Flow cytometry histogram shows TIGIT expression by naïve CD8+ T cells (CD44 ^low CD62L ^high ), central memory CD8+ T cells (CD44 ^high CD62L ^high ), and effector (CD44 ^high CD62L ^low ) memory CD8+ T cells. TIGIT MFI quantification results are also shown. ***P < 0.001. Data represent two independent experiments; n = 5. Vertical lines show standard error of the mean.

Figure 2 shows the generation of the TIGIT ^loxP/loxP mouse. TIGIT exon 1 was flanked by loxP sites using standard techniques.

Figure 3 shows that TIGIT-deficient CD8 ⁺ and CD4 ⁺ T cells respond normally to acute viral infection. TIGIT ^fl/ flCD4 ^cre (CKO) and TIGIT ^fl/fl (WT) littermates were infected with the Armstrong strain of LCMV. Splenocytes were analyzed 7 days after infection. Data are representative of two independent experiments; n = 5. Figure 3A shows representative FACS plots gated on CD8 ⁺ T cells, with activated (CD44 ^high ) cells boxed. Resulting data show quantification of activated CD8 ⁺ T cells as a percentage of total CD8 ⁺ T cells. Figure 3B shows representative FACS plots gated on CD8 ⁺ T cells after in vitro stimulation, with IFNγ-producing cells boxed. Results of quantification of IFNγ-producing cells as a percentage of total CD8 ⁺ T cells. Figure 3C shows representative FACS plots gated on CD4 ⁺ T cells, where activated (CD44 ^high ) cells are boxed. Results of quantification of activated CD4 ⁺ T cells as a percentage of total CD4 ⁺ T cells. Figure 3D shows representative FACS plots gated on CD4 ⁺ T cells after in vitro stimulation, where IFNγ-producing cells are boxed. Results of quantification of IFNγ-producing cells as a percentage of total CD4 ⁺ T cells. Vertical lines represent standard deviation from the mean.

Figure 4 shows that TIGIT and PD-1 synergistically regulate exhausted T cell effector function in vivo. In Figures 4A–4E, TIGIT ^fl/fl CD4-cre- (WT) and TIGIT ^fl/fl CD4-cre+ (CKO) mice were rapidly depleted of CD4 ⁺ T cells and infected with LCMV clone 13. Viral titers in splenocytes and liver were determined 42 days post-infection. Data are representative of two independent experiments, and n=9 per group. Figure 4A shows representative FACS plots gated on CD8 ⁺ T cells, with activated (CD44 ^high CD62L ^low ) cells boxed. Results are quantification of activated CD8 ⁺ T cells as a percentage of total CD8 ⁺ T cells. Figure 4B shows representative FACS plots gated on CD8 ⁺ T cells following in vitro stimulation, where IFNγ-producing cells are boxed. The results quantify activated IFNγ-producing cells as a percentage of total CD8 ⁺ T cells. Figure 4C shows representative FACS plots gated on CD4 ⁺ T cells, where activated (CD44 ^high , CD62L ^low ) cells are boxed. The results quantify activated CD4 ⁺ T cells as a percentage of total CD4+ T cells. Figure 4D shows representative FACS plots gated on CD4 ⁺ T cells following in vitro stimulation, where IFNγ-producing cells are boxed. The results quantify IFNγ-producing cells as a percentage of total CD4 ⁺ T cells. Figure 4E shows the results of quantification of LCMV titers in liver. ***P < 0.0001. In Figures 4F–4H, C57BL6/J mice were rapidly depleted of CD4 ⁺ T cells and challenged with LCMV clone 13. Mice were treated with isotype-matched control antibodies, anti-PD-L1, anti-TIGIT, or anti-PD-L1+anti-TIGIT antibodies, beginning at day 28 post-challenge. Viral titers in splenocytes and liver were determined at day 42 post-challenge. Data represent two independent experiments; n=10 per group. Figure 4F shows representative FACS plots gated on CD8 ⁺ T cells, with activated (CD44 ^high, CD62L ^low ) CD8 ⁺ T cells boxed. Results of quantification of activated CD8 ⁺ T cells as a percentage of total CD8 ⁺ T cells. ***P < 0.0001. Figure 4G shows representative FACS plots gated on activated CD8 ⁺ T cells after in vitro stimulation, with IFNγ-producing cells in the box. Results of quantification of IFNγ-producing cells as a percentage of total CD8 ⁺ T cells. *P = 0.0352. **P = 0.0047. Figure 4H shows the results of quantification of LCMV titers in the liver. *P = 0.0106. **P = 0.0047. Vertical lines represent standard deviation from the mean.

Figure 5 shows that TIGIT/PD-L1 co-blockade enhanced CD4 ⁺ T cell effector function during chronic viral infection. C57BL6/J mice were depleted of CD8 ⁺ T cells and infected with LCMV clone 13. Mice were treated with isotype-matched control antibodies, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies, beginning at day 28 post-infection. Viral titers in splenocytes and liver were determined at day 42 post-infection. Data represent two independent experiments; n=10 per group. Figure 5A shows representative FACS plots gated on CD4 ⁺ T cells, with activated (CD44 ^high, CD62L ^low ) cells boxed. Results of quantification of activated CD4 ⁺ T cells as a percentage of total CD4 ⁺ T cells. Figure 5B shows representative FACS plots gated on CD4 ⁺ T cells after in vitro stimulation, with IFNγ-producing cells boxed. *P = 0.019. Vertical lines represent standard deviation from the mean.

Figure 6 shows that TIGIT expression is increased in human breast cancer and correlates with the expression of CD8 and inhibitory co-receptors. Breast cancer gene expression data from The Cancer Genome Atlas were analyzed using microarray analysis. Gene expression data were normalized and expressed as relative values (log2). Figure 6A shows TIGIT expression in normal and breast cancer samples (left) and in breast cancer subtypes (right). ***P = 6×10 ^-12 . Box and plots are shown. Figure 6B shows the correlation of TIGIT expression and CD3ε. R2 = 0.61. Figure 6C shows the correlation of TIGIT and CD8α (left, R2 = 0.80) or CD4 (right, R2 = 0.42). Figure Figure 6D shows the correlation of TIGIT and PD-1 (left, R2 = 0.87), LAG3 (center, R2 = 0.80), and CTLA4 (right, R2 = 0.76).

Figure 7 shows that TIGIT and PD-1 inhibit antitumor T cell responses. In Figures 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 ³ in size. Data represent the results of one experiment; n = 6. Figure 7A shows a flow cytometry histogram showing TIGIT expression by splenic CD8 ⁺ T cells and tumor-infiltrating CD8 ⁺ T cells. The results of TIGIT MFI quantification are also shown. **P = 0.0023. Figure 7B is a flow cytometry histogram showing TIGIT expression by splenic CD4 ⁺ T cells and tumor-infiltrating CD4 ⁺ T cells. Results of TIGIT MFI quantification are also shown. ***P = 0.0002. In Figs. 7C–7E, BALB/c mice were inoculated with CT26 colorectal carcinoma cells. When the tumors reached approximately 200 mm ³ in size, the mice were treated with isotype-matched control antibodies, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies for three weeks. Data represent the results of two independent experiments; n = 10–20 (Figs. 7C–7D) or 7–10 (Fig. 7E). Fig. 7C shows the mean CT26 tumor volumes over time. Fig. 7D shows the survival of mice. Fig. 7E shows that approximately 60 days after the initial inoculation, complete remission (CR) mice previously treated with anti-TIGIT + anti-PD-L1 antibodies and naïve BALB/c mice were inoculated with CT26 cells in the left thorax and inoculated with EMT6 mammary carcinoma cells in the mammary fat pad. Average (left) and individual (right) CT26 (squares) and EMT6 (triangles) tumor volumes are shown in CR (purple and green) and naïve mice (black and orange). Fig. 7F shows that mice were inoculated with CT26 tumors and treated as in Fig. 7C. Tumor-infiltrating T cells and tumor-draining lymph node resident T cells were analyzed by flow cytometry. Representative FACS plots of CD8 ⁺ TIL after in vitro stimulation, with IFNγ-producing cells in the box. Results of quantification of IFNγ-producing CD8 ⁺ TIL as a percentage of total CD8 ⁺ TIL. ***P = 0.0003. Data represent two independent experiments; n = 5. Vertical lines represent standard deviation from the mean.

Figure 8 shows that TIGIT expression by CT26 tumor-infiltrating lymphocytes correlates 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 reached approximately 200 mm ³ in size. Data represent one experiment; n = 6. Figure 8A shows a representative histogram of TIGIT expression by splenic CD8 ⁺ T cells and tumor-infiltrating CD8 ⁺ T cells. Results of TIGIT MFI quantification. **P = 0.0026. Figure 8B shows a representative histogram of TIGIT expression by splenic CD4 ⁺ T cells and tumor-infiltrating CD4 ⁺ T cells. Results of TIGIT MFI quantification. ***P < 0.0001. Vertical lines represent standard deviation from the mean.

Figure 9 shows that TIGIT expression by MC38 tumor-infiltrating lymphocytes correlates 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 reached a size of approximately 200 mm ³ . Data represent results from one experiment; n = 5. Figure 9A shows a representative histogram of TIGIT expression by splenic CD8 ⁺ T cells and tumor-infiltrating CD8 ⁺ T cells. Results of TIGIT MFI quantification. ***P < 0.0001. Figure 9B shows a representative histogram of TIGIT expression by splenic CD4 ⁺ T cells and tumor-infiltrating CD4 ⁺ T cells. Results of TIGIT MFI quantification. *P = 0.0136. **P = 0.0029. Vertical lines represent standard deviation from the mean.

Figure 10 shows CT26 tumor growth in mice treated with anti-PD-L1 and/or anti-TIGIT antibodies. Naive BALB/c mice were inoculated with CT26 tumor cells and treated with anti-PD-L1 and/or anti-TIGIT antibodies or isotype-matched control antibodies as shown in Figures 4D–4F. Tumor volumes over time are shown for individual mice in each treatment group. Data represent results from two independent experiments.

Figure 11 shows flow cytometric analysis of CD4 ⁺ TILs and tumor-draining lymph node T cells. BALB/c mice were inoculated with CT26 colorectal carcinoma cells. When tumors reached approximately 200 mm ³ in size, mice were treated with isotype-matched control antibodies, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies for 7 days. Tumors and tumor-draining lymph nodes were collected. Data represent the results of two independent experiments; n = 5. Representative FACS histograms gated on tumor-draining lymph node CD8 ⁺ T cells after in vitro stimulation are shown, with IFNγ-producing cells boxed. Results of quantification of IFNγ-producing cells as a percentage of total CD8 ⁺ T cells. ***P < 0.001. Results of quantification of CD8 ⁺ T cells as a percentage of total TILs. **P = 0.0065. Results of quantification of activated (CD44 ^high CD62L ^low ) CD8 ⁺ T cells as a percentage of total CD8 ⁺ TILs. *P = 0.012. Results of quantification of CD8 ⁺ T cells as a percentage of total cells in tumor-draining lymph nodes. Results of quantification of activated CD8 ⁺ T cells as a percentage of total CD8 ⁺ T cells in tumor-draining lymph nodes. *P < 0.05. Figure 11C shows the results of quantification of CD4 ⁺ T cells as a percentage of total TILs. *P = 0.016. Figure 11D shows the results of quantification of activated CD4 ⁺ T cells as a percentage of total CD4 ⁺ TILs. Fig. 11E shows the results of quantification of CD4 ⁺ T cells as a percentage of total cells in tumor draining lymph nodes. Fig. 11F shows the results of quantification of activated CD4 ⁺ T cells as a percentage of total CD4 ⁺ T cells in tumor draining lymph nodes. Fig. 11A shows the results of quantification of IFNγ-producing cells as a percentage of total CD4 ⁺ TILs after in vitro stimulation. Fig. 11B shows the results of quantification of IFNγ-producing cells as a percentage of total CD4 ⁺ TILs in tumor draining lymph nodes after in vitro stimulation. Vertical lines represent standard deviation from the mean.

Figure 12 shows additional flow cytometric analysis of CD8 ⁺ TILs. BALB/c mice were inoculated with CT26 colorectal carcinoma cells and treated with isotype-matched control antibodies, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies as described in Figure 4. Tumors were collected 7 days after treatment and analyzed by flow cytometry. Data represent two independent experiments; n = 5. Figure 12A shows the results of quantification of TNFα ⁺ cells as a percentage of total CD8 ⁺ TILs. **P < 0.01. Figure 12B shows the results of quantification of CD8 ⁺ TILs as a percentage of total TILs. **P < 0.01. Figure 12C shows the results of quantification of activated (CD44 ^high CD62L ^low ) CD8 ⁺ TILs as a percentage of total CD8 ⁺ TILs. *P < 0.05. Vertical lines represent standard deviation from the mean.

Figure 13 shows flow cytometric analysis of resident CD8 ⁺ T cells in tumor-draining lymph nodes. BALB/c mice were inoculated with CT26 colorectal carcinoma cells and treated with isotype-matched control antibodies, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies as described in Figure 4. Tumor-draining lymph nodes were collected 7 days after treatment and analyzed by flow cytometry. Data represent the results of two independent experiments; n = 5. Figure 13A shows representative FACS histograms gated on resident CD8 ⁺ T cells in tumor-draining lymph nodes after in vitro stimulation, with IFNγ-producing cells boxed. Results of quantification of IFNγ-producing ⁺ cells as a percentage of total CD8 ⁺ T cells. ***P < 0.001. Figure 13B shows the results of quantification of CD8 ⁺ T cells as a percentage of total cells in tumor-draining lymph nodes. Figure 13C shows the results of quantification of activated (CD44 ^high CD62L ^low ) CD8 ⁺ T cells as a percentage of total CD8 ⁺ T cells. *P < 0.05. Vertical lines represent standard deviation from the mean. Figure 13D shows the results of quantification of TNFα-producing ⁺ cells as a percentage of total CD8 ⁺ T cells in tumor-draining lymph nodes.

Figure 14 shows the coexpression 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 a size of approximately 200 mm ³ . Representative histograms of CD226 expression by splenic B cells (gray), CD8 ⁺ T cells (blue), and TIGT+ tumor-infiltrating CD8 ⁺ T cells (red). Data represent two independent experiments; n=5.

Figure 15 shows that CD226 and TIGIT co-immunoprecipitate (co-IP) on transfected cells. COS7 cells were co-transfected with expression plasmids containing cDNAs of the tagged TIGIT-HA (5 ng) or CD226-Flag (10 ng) proteins or with a control plasmid (pRK). After transfection, the cells were washed and centrifuged, and the cell pellet was lysed. The resulting supernatant was precleared and centrifuged, and then divided equally into two tubes and immunoprecipitated with anti-HA or anti-Flag antibodies using standard procedures. The immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting. Western blots were probed with anti-FLAG-HRP or anti-HA-HRP antibodies.

Figure 16 shows that TIGIT and CD226 interact on primary CD8 ⁺ T cells. MACS-enriched splenic CD8 ⁺ T cells from C57BL6/J mice were stimulated with plate-fixed anti-CD3 and anti-CD28 antibodies and recombinant IL-2 for 48 h and lysed. Cell lysates were immunoprecipitated with anti-TIGIT antibody and probed with anti-CD226 antibody. Lanes: molecular weight marker (1), input (2), flow-through coimmunoprecipitation (3), and coimmunoprecipitate. Arrow indicates the predicted molecular weight of CD226.

Figure 17 shows the detection of TIGIT/CD226 interaction using TR-FRET methodology. Figure 17A shows the dissociation of Flag-ST-CD226 homodimers by HA-TIGIT. The Flag-ST-CD226 FRET ratio was measured in COS-7 cells expressing a constant amount of Flag-ST-CD226 and increasing concentrations of HA-TIGIT. Figure 17B shows the Flag-ST-CD226 FRET ratio recorded after 15 min incubation with PBS (white bar) or anti-TIGIT antibody (black bar). Figure 17C shows the association of Flag-ST-CD226 with HA-TIGIT. FRET intensity between Flag-ST-CD226 and HA-TIGIT over Flag-ST-CD226 expression as determined by anti-Flag ELISA on the same batch of transfected COS-7 cells. Figure 17D shows the change in FRET between Flag-ST-CD226 and HA-TIGIT after 15 min incubation with PBS (white bar) or anti-TIGIT antibody (black bar). Data in Figures A and C represent four independent experiments, each performed in triplicate. Data in Figures B and D represent two independent experiments, each performed in triplicate.

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 three independent experiments, each performed in triplicate.

Figure 19 shows that blocking CD226 abolishes the enhanced antiviral T cell response induced by TIGIT/PD-L1 co-blocking. In Figures 19A–19D, C57BL6/J mice were rapidly depleted of CD4 ⁺ T cells and infected with LCMV clone 13. Mice were treated with isotype-matched control antibodies, anti-CD226, anti-PD-L1 + anti-TIGIT, or anti-PD-L1 + anti-TIGIT + anti-CD226 antibodies, beginning 28 days post-infection. Viral titers in splenocytes and liver were determined 42 days post-infection. Figure 19A shows the results of CD8 ⁺ T cell quantification as a percentage of splenocytes. Figure 19B shows the results of quantification of CD8 ⁺ T cells as a percentage of total CD8 ⁺ T cells. ***P < 0.001. Fig. 19C shows the results of quantification of IFNg-producing cells as a percentage of total activated CD8 ⁺ T cells. ***P < 0.001. Fig. 19D shows the results of quantification of LCMV titers in the liver. ***P < 0.001. Vertical lines represent standard deviation from the mean.

Figure 20 shows that TIGIT expression is increased in human cancer and highly correlates with CD8 and PD-1 expression. Gene expression analysis in various human cancer types was performed as described in Example 11. Scatterplots show per gene data normalized to library size. Box and box plots show the stabilized variance of the TIGIT to CD3e expression ratio. Figure 20A shows the correlation of TIGIT and CD3e RNA expression in LUSC (gray) and normal lung (black). ρ = 0.86. The results of quantification of TIGIT/CD3e expression ratios are also shown. The ratio was increased in LUSC = 372%. ***P = 1.46×10 ^-46 . Figure 20B shows the correlation of TIGIT and CD3e RNA expression in COAD (gray) and normal colon (black). ρ = 0.83. The results of quantification of TIGIT/CD3e expression ratios are also shown. Increase in ratio in COAD = 116%. ***P = 3.66×10 ^-6 . Fig. 20C shows the correlation of TIGIT and CD3e RNA expression in UCEC (gray) and normal uterine endometrium (black). ρ = 0.87. The results of quantification of TIGIT/CD3e expression ratios are also shown. Increase in ratio in UCEC = 419%. ***P = 7.41×10 ^-5 . Fig. 20D shows the correlation of TIGIT and CD3e RNA expression in BRCA (gray) and normal breast (black). ρ = 0.82. The results of quantification of TIGIT/CD3e expression ratios are also shown. Increase in ratio in BRCA = 313%. ***P = 4.6×10 ^-44 . Figure 20E shows the correlation of TIGIT and CD3e RNA expression in clear renal cell carcinoma (gray) and normal kidneys (black). ρ = 0.94. The results of quantification of TIGIT/CD3e expression ratios are also shown. Figure 20F shows the correlation of TIGIT and CD8A (left) or TIGIT and CD4 (right) in lung squamous cell carcinoma (gray) and normal lungs (black). ρ = 0.77 and 0.48, respectively. Figure 20G shows the correlation of TIGIT and PD-1 (Pdcd1) in lung squamous cell carcinoma (gray) and normal lungs (black). ρ = 0.82. Figure 20H shows the correlation of TIGIT and CD226 in lung squamous cell carcinoma (red) and normal lung (black). ρ= 0.64.

Figure 21 shows the expression analysis of T cell-associated genes in lung squamous cell carcinoma (LUSC). Gene expression in LUSC and normal tissue samples was analyzed as described in Example 11, and a gene heat map was prepared that best correlated with the gene signature in LUSC. Genes and samples were clustered by hierarchical clustering using Ward's method on a Euclidean distance matrix for centered and scaled expression data.

Figure 22 shows that TIGIT and PD-1 are coordinately expressed by tumor-infiltrating lymphocytes in humans and mice. Figures 22A-22C show analysis of lymphocytes from a freshly resected human NSCLC tumor, tumor-matched peripheral blood, and healthy donor peripheral blood. The data represent results from two independently analyzed tumors. Figure 22A shows representative FACS histograms of TIGIT expression by peripheral and tumor-infiltrating CD8 ⁺ T cells, with TIGIT ⁺ cells boxed. Figure 22B shows representative FACS histograms of TIGIT expression by peripheral and tumor-infiltrating CD4 ⁺ T cells, with TIGIT ⁺ cells boxed. Figure 22C is a flow cytometry histogram showing TIGIT expression by PD- ^1high (red) and PD- ^1low (blue) NSCLC-infiltrating CD8 ⁺ (left) and CD4 ⁺ (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 ³ in size. Data represent the results of two independent experiments; n=5–6. Fig. 22D shows representative FACS histograms of TIGIT expression by tumor-infiltrating CD8 ⁺ T cells, with TIGIT ⁺ cells boxed. Fig. 22E shows representative FACS histograms of TIGIT expression by tumor-infiltrating CD4 ⁺ T cells, with TIGIT ⁺ cells boxed. Quantification of TIGIT ⁺ T cell frequency as a percentage of total T cells is also shown. *P = 0.0134. ***P < 0.0001. Fig. 22F shows a flow cytometry histogram showing TIGIT PD- ^1high and PD- ^1low expression by tumor-infiltrating CD8 ⁺ T cells and splenic CD8 ⁺ T cells. Quantification of TIGIT MFI is also shown. **P = 0.0023. Fig. 22G shows a flow cytometry histogram showing the expression of TIGIT PD- ^1high and PD- ^1low by tumor-infiltrating CD4 ⁺ T cells and splenic CD4 ⁺ T cells. The results of TIGIT MFI quantification are also shown. ***P = 0.0002. Vertical lines represent standard deviation of the mean.

Figure 23 shows the characterization of TIGIT expression by human tumor-infiltrating T cells. Figures 23A-23B are FACS histograms showing TIGIT expression by NSCLC-infiltrating CD8 ⁺ and CD4 ⁺ T cells (Figure 23A) and by PBMC CD8 ⁺ and CD4 ⁺ T cells from matched donors (Figure 23B), where TIGIT ⁺ cells are boxed. Figures 23C-23D are FACS histograms showing TIGIT expression by CRC-infiltrating CD8 ⁺ and CD4 ⁺ T cells (Figure 23C) and by PBMC CD8 ⁺ and CD4 ⁺ T cells from matched donors (Figure 23D), where TIGIT ⁺ cells are boxed.

Figure 24 shows that the TIGIT:CD226 interaction is not regulated by the PVR TIGIT:CD226 and TIGIT Q56R:CD226 interactions as detected using TR-FRET methodology, and the FRET ratio between Flag-ST-CD226 and HA-TIGIT or HA-TIGIT Q56R indicates that WT and Q56R TIGIT bind CD226 with equal efficiency. Data represent the results of three independent experiments performed in triplicate.

Figure 25 shows the co-blocking efficacy of TIGIT/PD-L1 antibodies in MC38 tumor-bearing mice. In Figures 25A-25C, MC38 tumor-bearing mice were generated as described 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 antibodies alone, anti-TIGIT antibodies alone) or 20 (anti-TIGIT antibodies + anti-PD-L1 antibodies). Figure 25A shows mean (left) and individual (right) MC38 tumor volumes over time. Figure 25B shows MC38 tumor volumes after 14 days of antibody treatment. ***P = 0.0005. **P = 0.0093. *P = 0.0433. Fig. 25C shows the survival data of mice over time. The vertical lines represent the standard deviation from the mean.

Figure 2B shows further characterization of TIGIT expression by murine tumor-infiltrating T cells. Figure 26A shows that splenic CD8 ⁺ T cells from C57BL6/J mice were enriched using MACS and cultured with plate-immobilized agonist anti-CD3 and anti-CD28 antibodies. Representative histograms of TIGIT (red) and isotype-matched control antibody (solid gray line) staining over time. Results of TIGIT MFI quantification. ***P < 0.001. Stimulated cells inducibly expressed PD-1 and constitutively expressed CD226 (data not shown). Data represent results from two independent experiments; n = 5. Figure 2B 26B-26E, wild-type C57BL6/J mice were inoculated subcutaneously with syngeneic MC38 colorectal carcinoma cells. Tumors were allowed to grow undisturbed until they reached a size of 150-200 ^mm3 . Data represent two independent experiments; n=5. Fig. 26B shows a representative FACS histogram of tumor-infiltrating CD8 ⁺ T cells, with TIGIT ⁺ cells boxed. Results of quantification of TIGIT ⁺ cell frequencies as a percentage of total tumor-infiltrating CD8 ⁺ T cells or splenic CD8 ⁺ T cells. ***P<0.0001. Fig. 26C shows a representative FACS histogram of tumor-infiltrating CD4 ⁺ T cells, with TIGIT ⁺ cells boxed. Results of quantification of TIGIT ⁺ cell frequencies as a percentage of total tumor-infiltrating CD4 ⁺ T cells or splenic CD4 ⁺ T cells. ***P < 0.0001. Fig. 26D shows a representative histogram of TIGIT PD- ^1high and PD- ^1low expression by tumor-infiltrating CD8 ⁺ T cells (red and blue, respectively) and splenic CD8 ⁺ T cells (gray). Results of TIGIT MFI quantification. ***P < 0.0001. Fig. 26E shows a representative histogram of TIGIT PD- ^1high and PD- ^1low expression by tumor-infiltrating CD4 ⁺ T cells (red and blue, respectively) and splenic CD4 ⁺ T cells (gray). Results of TIGIT MFI quantification. *P = 0.0136. **P = 0.0029. The vertical lines represent the standard deviation from the mean.

Figure 27 shows that tumor-infiltrating CD8 ⁺ and CD4 ⁺ T cells maintain high levels of CD226 expression. Wild-type BALB/c mice were inoculated with CT26 tumor cells as described herein. After tumors reached approximately 150-200 ^mm3 in size, tumors and spleens were analyzed by flow cytometry. Figure 27A shows the results of quantification of CD226 ⁺ CD8 ⁺ T cells, CD4 ⁺ T cells, and non-T cells as a percentage of total CD8 ⁺ T cells, CD4 ⁺ T cells, and non-T cells, respectively. Figure 27B shows representative histograms of CD226 expression in tumor and spleen. Data are representative of two independent experiments; n=5. Vertical lines represent standard deviations from the mean.

Figure 28 shows that TIGIT suppression of CD8 ⁺ T cell responses is dependent on CD226. BALB/c mice were inoculated subcutaneously with CT26 colorectal carcinoma cells in the right thorax. When tumors reached approximately 200 ^mm3 in size, mice were treated with isotype-matched control antibodies (black), anti-CD226 antibody (orange), anti-PD-L1 antibody (red), anti-TIGIT- + anti-PD-L1 antibodies (purple), or anti-TIGIT- + anti-PD-L1- + anti-CD226 antibodies (green) for three weeks. Data represent single experiments; n=10 (A–B) or 5 (C–F). Figure 28A shows mean (left) and individual (right) CT26 tumor volumes over time. Figure 28B shows the survival of mice over time. In Figures 28C-28F, after 7 days of treatment, resident lymphocytes in tumor-draining lymph nodes were analyzed by flow cytometry. Figure 28C shows the quantification of IFNγ-producing CD8 ⁺ TILs as a percentage of total CD8 ⁺ TILs after in vitro stimulation. **P < 0.01. Figure 28D shows the quantification of IFNγ-producing cells as a percentage of total CD8 ⁺ T cells after in vitro stimulation. *P < 0.05. Figure 28E shows the quantification of CD8 ⁺ TILs as a percentage of total TILs. **P < 0.01. Figure 28F shows the quantification of CD8 ⁺ T cells as a percentage of total resident lymphocytes in tumor-draining lymph nodes. The vertical lines represent the standard deviation from the mean.

Figure 29 shows that TIGIT suppresses CD226 function by directly disrupting CD226 homodimerization. Figure 29A shows that CD8 ⁺ T cells from TIGIT ^fl/fl CD4 ^cre (WT) and TIGIT ^fl/fl CD4 ^wt littermates were enriched using MACS and stimulated in the presence of anti-CD226 antibodies or isotype-matched control antibodies as indicated. ^H3 -thymidine uptake is shown as the ratio of cells cultured with anti-CD3 antibody + PVR-Fc to cells cultured with anti-CD3 antibody alone. **P = 0.0061. ***P < 0.0001. Data represent the results of two independent experiments; n = 5. Figure 29A 29B shows that CD8 ⁺ T cells from wild-type C57BL6/J mice were enriched using MACS and stimulated in the presence of anti-TIGIT antibody, anti-CD226 antibody, or isotype-matched control antibodies, as indicated. ^H3 -thymidine uptake is shown as the ratio of cells cultured with anti-CD3 antibody + PVR-Fc to cells cultured with anti-CD3 antibody alone. ***P < 0.001 in paired t-tests. Fig. 29C shows that CD8 ⁺ T cells were enriched using MACS and stimulated with suboptimal concentrations of plate-fixed anti-CD3 antibody in the presence or absence of human recombinant PVR-Fc. Anti-TIGIT antibody or isotype-matched control antibodies were added as indicated. ^3H -thymidine uptake quantification results. **P = 0.0071 and 0.0014, respectively. Figure 29D shows CHO cells were transiently transfected with increasing concentrations of the FLAG-ST-CD226 acceptor and donor, as indicated. Results of quantification of FRET intensity relative to donor emission. Data are representative of three independent experiments; n = 3. In Figures 29E–29F, CHO cells were transiently transfected with FLAG-ST-CD226 and increasing concentrations of HA-TIGIT, as indicated. Data are representative of two or more independent experiments; n = 4. Data were normalized to the maximal signal. Figure 29E shows the results of quantification of the FRET ratio of TIGIT:CD226 (FRET ratio 1). Figure 29F shows the results of quantification of the FRET ratio of TIGIT:CD226 (FRET ratio 2). Figure Figure 29G shows anti-Flag (left) and anti-HA (right) immunoblots obtained on anti-FLAG or anti-HA immunoprecipitates prepared from COS-7 cells transfected with the empty pRK vector or the combination of Flag-CD226 and HA-TIGIT. Data represent the results of two independent experiments. Figure 29H shows the results of FRET quantification of the TIGIT:CD226 ratio after incubation with PBS (white) or anti-TIGIT antibody (red). ***P < 0.001. Data represent the results of four independent experiments; n = 3. Vertical lines represent the standard deviation of the mean.

Figure 30 shows that primary human T cells were enriched from blood using MACS and stimulated with anti-CD3 and anti-CD28 antibodies. TIGIT ⁺ and ^TIGIT− cells were sorted, maintained, restimulated, and FRET labeled with the indicated antibodies. Data represent the results of two independent experiments. ***P < 0.001. Vertical lines represent standard deviations from the mean.

Figure 31 shows that co-blocking TIGIT and PD-1 did not restore the effector function of exhausted CD4 ⁺ T cells during chronic viral infection. Figure 31A shows the results of quantification of CD8 ⁺ T cells as a percentage of total splenocytes. Figure 31B shows the results of quantification of gp33 Pentamer ⁺ cells as a percentage of total splenic CD8 ⁺ T cells. **P = 0.0040. Figure 31C shows representative FACS histograms gated on gp33 Pentamer ⁺ CD8 ⁺ T cells after in vitro stimulation, with IFNγ ⁺ cells boxed. Results of quantification of IFNγ-producing cells as a percentage of total gp33 Pentamer ⁺ CD8 ⁺ cells. *P = 0.0319. **P = 0.0030. Vertical lines represent standard deviation from the mean.

Figure 32 shows that the efficacy of TIGIT/PD-L1 co-blockade is CD8 ⁺ T cell dependent. In Figures 32A–32B, wild-type BALB/c mice were inoculated with CT26 tumors as described in Figure 7. When tumors reached 100–150 mm ³ in size, mice were transiently depleted of CD8 ⁺ T cells and treated with anti-TIGIT- + anti-PD-L1 antibodies. Data represent results from one experiment; n=10/group. Figure 32A shows mean (left) and individual (right) CT26 tumor volumes over time. Figure 32B shows quantification of CT26 tumor volumes 17 days after treatment initiation. ***P=0.0004. Figure In Fig. 32C, wild-type BALB/c mice were inoculated with CT26 tumors and treated with anti-TIGIT- + anti-PD-L1 antibodies, and then re-inoculated with CT26 tumors that were transiently depleted of CD8 ⁺ T cells at the time of re-inoculation. Data represent two independent experiments; n = 5. Fig. 32C shows mean (left) and individual (right) CT26 tumor volumes over time. Vertical lines represent standard deviations from the mean.

Figure 33 shows that PVR expression on tumor cells is not required for the efficacy of TIGIT/PD-L1 co-blockade. Wild-type BALB/c mice were inoculated with wild-type or PVR-deficient (PVR.KO) tumors as described. When tumors reached 150-200 mm ³ in size, mice were treated with anti-TIGIT- + anti-PD-L1 antibodies or isotype-matched control antibodies. Data represent the results of a single experiment; n=10/group. Figure 33 shows mean (left) and individual (right) CT26 tumor volumes over time.

Figure 34 shows the co-blocking efficacy of TIGIT/PD-L1 antibody in mice bearing EMT6 tumors. Mice bearing EMT6 tumors were generated as described 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 antibody alone, anti-TIGIT antibody alone) or 20 (anti-TIGIT + anti-PD-L1 antibodies). Figure 34 shows mean (left) and individual (right) EMT26 tumor volumes over time.

Figure 35 shows that TIGIT regulates the effector function of tumor-infiltrating CD8 ⁺ T cells. BALB/c mice were inoculated subcutaneously with CT26 colorectal carcinoma cells in the right thorax and treated with anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies as shown in Figure 7. Tumor-draining lymph node (dLN) resident T cells and tumor-infiltrating T cells were analyzed by flow cytometry 7 days after treatment. Data represent two independent experiments; n = 5. Figure 35A shows the quantification of dual IFNγ/TNF producer CD8 ⁺ and CD4 ⁺ T cells as a percentage of total dLN resident CD8 ⁺ and CD4 ⁺ T cells, respectively. Production of two cytokines by unstimulated T cells is also shown. **P = 0.002, 0.003, 0.001, respectively. Figure 35B shows the results of quantification of tumor-infiltrating CD8 ⁺ and CD4 ⁺ dual IFNγ/TNF producer T cells as a percentage of total tumor-infiltrating CD8 ⁺ and CD4 ⁺ T cells, respectively. Production of two cytokines by unstimulated T cells is also shown. ***P < 0.0001. Vertical lines represent standard deviation from the mean.

Figure 36 shows an analysis of lymphocytes from resected human NSCLC tumors, tumor-matched peripheral blood, and healthy donor peripheral blood. Data from three independent sample collections were pooled. Figure 36A shows the results of quantification of TIGIT ⁺ cells as a percentage of total CD8 ⁺ T cells. *P < 0.05. Figure 36B shows the results of quantification of TIGIT ⁺ cells as a percentage of total CD4 ⁺ T cells.

Figure 37 shows the characterization of TIGIT expression in human tumors. Figure 37A shows representative flow cytometric histograms of TIGIT expression by resident lymphocytes in NSCLC (red, CD45 ⁺ FSC ^low ), myeloid cells (blue, CD45 ⁺ FSC ^high ), and nonhematopoietic cells (green, ^CD45− ) relative to staining of isotype-matched subsets (gray). Figure 37B shows the gating strategy for PD-1 ^high and PD-1 ^low NSCLC tumor-infiltrating CD8 ⁺ and CD4 ⁺ T cells.

Detailed description of the invention

I. General methods

The techniques and procedures described or referred to in this specification are generally well known and commonly used by those skilled in the art using conventional methodologies described, for example, 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)); 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

The term "PD-1 axis binding antagonist" is a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, in order to reverse T cell dysfunction resulting from PD-1 axis signaling, thereby restoring or enhancing T cell function (e.g., proliferation, cytokine production, induced target cell death). As used herein, the term "PD-1 axis binding antagonist" includes a PD-1-binding antagonist, a PD-L1-binding antagonist, and a PD-L2-binding antagonist.

The term "PD-1 binding antagonists" represents a molecule that reduces, blocks, inhibits, abolishes or interferes with signaling 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 particular 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 reduce, block, inhibit, abolish or interfere with signaling resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, the PD-1-binding antagonist reduces the negative costimulatory signal mediated or induced by cell surface proteins expressed on T lymphocytes that mediate signaling through PD-1 such that dysfunctional T cells are restored to less dysfunctional ones (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, the PD-1-binding antagonist is the MDX-1106 antibody described herein. In another specific aspect, the PD-1-binding antagonist is the Merck 3745 antibody described herein. In another specific aspect, the PD-1-binding antagonist is the CT-011 antibody described herein. In another specific aspect, the PD-1 binding antagonist is the AMP-224 antibody described herein.

The term "PD-L1-binding antagonists" is a molecule that reduces, blocks, inhibits, abolishes or interferes with signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, B71. In some embodiments, the PD-L1-binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a particular aspect, the PD-L1-binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B71. In some embodiments, PD-L1-binding antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, abolish or interfere with signaling 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 a negative costimulatory signal mediated or induced by cell surface proteins expressed on T lymphocytes that mediate signaling through PD-L1 in a manner that restores dysfunctional T cells to less dysfunctional ones (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L1-binding antagonist is an anti-PD-L1 antibody. In a specific aspect, the anti-PD-L1 antibody is the YW243.55.S70 antibody described herein. In another specific aspect, the anti-PD-L1 antibody is the MDX-1105 antibody described herein. In yet another specific aspect, the anti-PD-L1 antibody is the MPDL3280A antibody described herein. In yet another specific aspect, the anti-PD-L1 antibody is the MEDI 4736 antibody described herein.

The term "PD-L2-binding antagonists" represents a molecule that reduces, blocks, inhibits, abolishes or interferes with signaling resulting from the interaction of PD-L2 with 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 particular aspect, a PD-L2-binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2-binding antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that reduce, block, inhibit, abolish or interfere with signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, the PD-L2-binding antagonist reduces the negative costimulatory signal mediated or induced by cell surface proteins expressed on T lymphocytes that mediate signaling through PD-L2 in a manner that restores dysfunctional T cells to less dysfunctional ones (e.g., enhancing effector responses to antigen recognition). In some embodiments, the PD-L2-binding antagonist is an immunoadhesin.

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 may specifically bind to a TIGIT polypeptide, or to a molecule in a signaling pathway that modulates the expression of TIGIT. The preparation and therapeutic use of aptamers are well known in the art. See, for example, U.S. Patent No. 5,475,096, and the therapeutic efficacy of ^Macugen® (Eyetech, New York) for the treatment of age-related macular degeneration.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of a native polypeptide disclosed herein. Likewise, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of a native polypeptide disclosed herein. Suitable agonist or antagonist molecules include, but are not limited to, agonist or antagonist antibodies or antibody fragments, amino acid sequence fragments or variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a polypeptide may include contacting the polypeptide with a candidate agonist or antagonist molecule and determining a detectable change in one or more biological activities normally associated with the polypeptide.

The terms "TIGIT antagonist" and "antagonist of TIGIT activity or TIGIT expression" are used interchangeably and refer to a compound that disrupts the normal functioning of TIGIT, either by reducing the transcription or translation of a nucleic acid encoding TIGIT, or by inhibiting or blocking the activity of a TIGIT polypeptide, or both. Examples of TIGIT antagonists include, but are not limited to, antisense polynucleotides, interfering RNA, catalytic RNA, RNA-DNA chimeras, TIGIT-specific aptamers, anti-TIGIT antibodies, TIGIT-binding fragments of anti-TIGIT antibodies, small molecules that bind to TIGIT, TIGIT-binding peptides, and other polypeptides that specifically bind to 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 decrease or abrogation of TIGIT activity or expression. It will be appreciated by those of ordinary skill in the art that in some instances, a TIGIT antagonist may antagonize one activity of TIGIT without affecting another activity of TIGIT. For example, a desirable TIGIT antagonist for use in some of the methods described herein is a TIGIT antagonist that antagonizes TIGIT activity with respect to only the PVR interaction, the PVRL3 interaction, or the PVRL2 interaction, for example, while having no or minimal effect on other TIGIT interactions.

The terms "PVR antagonist" and "antagonist of PVR activity or PVR expression" are used interchangeably and refer to a compound that disrupts the normal functioning of PVR, either by reducing the transcription or translation of a nucleic acid encoding PVR, or by inhibiting or blocking the activity of a PVR polypeptide, 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, small molecules that bind to PVR, PVR-binding peptides, and other polypeptides that specifically bind to 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 decrease in or abrogation of PVR activity or expression. Those of ordinary skill in the art will appreciate that in some instances, a PVR antagonist may antagonize only one activity of PVR without affecting another activity of PVR. For example, a desirable PVR antagonist for use in some of the methods described herein is a PVR antagonist that antagonizes PVR activity with respect to interaction with TIGIT only, without affecting PVR-CD96 and/or PVR-CD226 interactions.

The term "dysfunction" in the context of immune dysfunction refers to a state of decreased immune responsiveness to antigen stimulation. The term includes general elements of exhaustion and/or anergy in which antigen recognition may occur but the subsequent immune response is ineffective in controlling infection or tumor growth.

As used herein, the term “dysfunctional” includes refractory or unresponsive antigen recognition, in particular an impaired ability to translate antigen recognition downstream to T cell effector functions such as proliferation, cytokine production (e.g., IL-2), and/or induction of target cell death.

The term anergy refers to a state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals via the T cell receptor (e.g., elevated intracellular Ca ⁺² in the absence of ras activation). T cell anergy can also result from antigen stimulation in the absence of costimulation, resulting in the cell becoming unresponsive to subsequent antigen activation even with costimulation. The unresponsive state can often be overcome in the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and/or acquire effector functions.

The term exhaustion refers to T cell depletion, a state of T cell dysfunction resulting from continuous TCR signaling that occurs in many chronic infections and cancers. It differs from anergy in that it does not result from incomplete or deficient signaling but from prolonged signaling. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion interferes with optimal infection and tumor control. Exhaustion can be induced by extrinsic negative regulatory pathways (e.g., immunoregulatory cytokines) as well as by intrinsic negative regulatory (costimulatory) pathways of the cell (PD-1, B7-H3, B4-H7, etc.).

"Enhancement of T cell function" means inducing, providing or stimulating T cells to have prolonged or enhanced biological function, or restoring or reactivating exhausted or inactive T cells. Examples of enhancement of T cell function include: increased secretion of γ-interferon by CD8 ⁺ T cells, increased proliferation, increased reactivity to antigens (e.g., elimination of a virus, pathogen or tumor) compared to such levels before the intervention. In one embodiment, the degree of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. One of ordinary skill in the art is familiar with methods for measuring this enhancement.

"T-cell dysfunctional disorder" is a disorder or condition of T cells characterized by decreased responsiveness to antigen stimulation. In a specific embodiment, the T-cell dysfunctional disorder is a disorder that is specifically associated with inappropriate increased signaling involving PD-1. In another embodiment, the T-cell dysfunctional disorder is a disorder in which T cells are anergic or have a decreased ability to secrete cytokines, proliferate, or lack 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.

“Tumor immunity” refers to the process by which tumors evade immunological recognition and elimination. Thus, from a therapeutic perspective, tumor immunity is “cured” when such evasion is reduced and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor elimination.

“Immunogenicity” refers to the ability of a given substance to induce an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity is aimed at eliminating tumor cells by the immune response. Examples of enhancing tumor immunogenicity include, but are not limited to, treatment with an antagonist that binds to the PD-1 axis (e.g., an anti-PD-L1 antibody) and a TIGIT inhibitor (e.g., an anti-TIGIT antibody).

"A durable response" refers to a prolonged effect on reducing tumor growth after treatment is stopped. For example, the tumor size may remain the same or become smaller compared to its size at the beginning of the treatment phase. In some embodiments, a durable response has a duration of at least the same as the duration of treatment, at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times the duration of treatment.

The term "antibody" includes monoclonal antibodies (including full-length antibodies that contain the Fc region of an immunoglobulin), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, dimers, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab') ₂ , and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody."

The basic four-chain structural unit of an antibody is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units plus an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2-5 basic 4-chain structural units that can polymerize to form multivalent assemblies in combination with the J chain). In the case of IgG, the 4-chain structural unit typically has a molecular weight of approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while 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 a variable region (VH) at the N-terminus followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable region (VL) at the N-terminus followed by a constant domain (CL) at the other end. V _L is aligned with VH, and _CL is aligned with the first constant domain of the heavy chain ( _CH 1). Certain amino acid residues are thought to form an interface between the variable regions of the light chain and the heavy chain. The pairing of VH and VL results in a single antigen-binding site. For a discussion of the structure and properties of the different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6. The L chain of different vertebrate species can be classified into one of two 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 (CH), immunoglobulins can be classified into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, σ, γ, ε, and μ, respectively. Classes γ and α are further subdivided into subclasses based on relatively minor differences in CH sequence and function, for example, the following subclasses are expressed in humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

The term "variable region" or "variable domain" of an antibody refers to the amino-terminal regions of the heavy or light chain of an antibody. The variable regions of the heavy chain and light chain may be referred to as "VH" and "VL", respectively. These regions are typically the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen-binding sites.

The term "variable" refers to the fact that some segments of the variable regions of antibodies vary greatly in sequence among some antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, variability is not distributed evenly across the entire variable region. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in the variable regions of both the light and heavy chains. The more highly conserved portions of the variable regions are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FRs, predominantly in a beta-sheet configuration linked by three HVRs that form loops that link, and in some cases form part of, the beta-sheet structures. The HVRs in each chain are held together in close proximity by FR regions and, with HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant regions are not directly involved in antibody-antigen binding but exhibit various effector functions, such as involvement of the antibody in antibody-dependent cellular toxicity.

As used herein, the term "monoclonal antibody" 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-translational modifications (e.g., isomerization, amidation) 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, monoclonal antibodies have the advantage of being synthesized by 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 process. For example, monoclonal antibodies to be used according to the present invention can be obtained by various methods, 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^n.d.ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, 563-681 (Elsevier, N.Y., 1981)), recombinant DNA techniques (see, e.g., U.S. Patent 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); Fellowes, 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 fragments or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, 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. Patents 5,545,807; 5545806; 5569825; 5625126; 5633425 and 5661016; 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).

The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radioactive label.

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. In particular, whole antibodies include antibodies with heavy and light chains, including the Fc region. Constant regions may be native sequence constant regions (e.g., native sequence human constant regions) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

An "antibody fragment" includes a fragment of an intact antibody, preferably the antigen-binding portion and/or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng., 8(10):1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies derived from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, termed "Fab" fragments, and a residual "Fc" fragment, its designation reflecting its ability to crystallize readily. The Fab fragment consists of the entire L chain plus the variable region of the H chain (VH) and the first constant domain of one heavy chain (CH1). Treatment of the antibody with pepsin yields a single large F(ab') ₂ fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and still capable of antigen cross-linking. Fab' fragments differ from Fab fragments by the presence of several additional residues at the carboxy terminus of the _CH 1 domain, including one or more cysteine residues from the hinge region of the antibody. Fab'-SH is used herein to designate Fab' in which the cysteine residue(s) of the constant region bear a free thiol group. F(ab') ₂ antibody fragments were originally produced as a pair of Fab' fragments that have hinge cysteine residues between them. Other examples of binding of antibody fragments by chemical bonds are also known.

The Fc region contains the carboxyl-terminal portions of both H chains linked by a disulfide bond. The effector functions of antibodies are determined by sequences in the Fc region, the region recognized by Fc receptors (FcRs) present on some cell types.

"Fv" represents the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region and one light-chain variable region tightly linked by a non-covalent bond. The folding of these two domains results in six hypervariable loops (3 loops from each of the H and L chains) that are included in the amino acid residues involved in antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable region (or half of the Fv, containing only the three HVRs specific for the antigen) is capable of recognizing and binding antigen, although with lower affinity than the entire binding site.

A "single chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment that comprises the VH and VL regions of an antibody linked into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL regions that facilitates the formation of a structure by the sFv fragment necessary for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

"Functional fragments" of the antibodies of the invention comprise a fragment of an intact antibody, typically comprising an antigen-binding site or variable region of an intact antibody or an Fc region of an antibody that retains or has a modified ability to bind to an FcR. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies derived from antibody fragments.

The term "diabodies" refers to small antibody fragments obtained by constructing sFv fragments (see previous paragraph) with short linkers (approximately 5-10 residues) between the V _H and V _L domains so as to achieve interchain rather than intrachain pairing of the V domains, resulting in a bivalent fragment, i.e., a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossed" sFv fragments in which the V _H and V _L domains of the two antibodies are on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The monoclonal antibodies of the present invention particularly include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to a different class or subclass of antibodies, 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 to the present invention include primatized antibodies ( ^PRIMATIZED® ), wherein the antigen-binding portion of the antibody is derived from an antibody obtained, for example, by immunizing macaques with the antigen of interest. As used herein, the term "humanized antibody" includes a subset of "chimeric antibodies."

"Humanized" forms of non-human antibodies (e.g., murine) are chimeric antibodies that contain minimal sequence derived from a non-human antibody. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR (defined below) of the recipient are replaced with residues from an HVR from a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and/or activity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced with corresponding residues of a non-human immunoglobulin. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody characteristics, such as binding affinity. Typically, a "humanized" antibody will comprise substantially all or at least one, typically two, variable domains in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin sequence and all or substantially all of the FR regions are the FR regions of a human immunoglobulin sequence, although the FR regions may include one or more substitutions of individual FR residues that improve characteristics of the antibody such as binding affinity, isomerization, immunogenicity, etc. Typically, the number of such amino acid substitutions in the FRs is no more than 6 in the H chain and no more than 3 in the L chain. Optionally, a humanized antibody will comprise at least a fragment 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); Riechmaim 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. Patents 6,982,321 and 7,087,409.

A "human antibody" is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and/or has been produced using any of the methods for producing human antibodies described herein. This definition of a human antibody specifically excludes a humanized antibody having non-human antigen-binding residues. Human antibodies can be produced using a variety of methods 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 production of human monoclonal antibodies are the methods described by 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). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be produced by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen stimulation but whose endogenous loci have been blocked, such as immunized xeno-mice (see, e.g., U.S. Patents 6,075,181 and 6,150,584, covering the XENOMOUSE technology^TM). See also, for example, the publication by Li et al., Proc. Natl. Acad. Sci. USA, 103:3557–3562 (2006) on human antibodies produced using human B-cell hybridoma technology.

As used herein, the term "hypervariable region," "HVR," or "HV" refers to the regions of the variable region of an antibody that are hypervariable in sequence and/or form structurally defined loops. Antibodies typically contain six HVRs; three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the greatest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring fine antibody specificity. 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, NJ, 2003). Indeed, naturally occurring camelid heavy-chain-only antibodies are functional and stable in the absence of a light chain. See, for example, Hamers-Casterman et al., Nature, 363:446–448 (1993); Sheriff et al., Nature Struct. Biol., 3:733–736 (1996).

A number of HVR definitions are used and covered in this document. Kabat complementarity determining regions (CDRs) are based on sequence variability and are generally the most widely used (Kabat et al., Sequences of Proteins of Immunological Interest, ^5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). In contrast, Chothia defines hypervariable regions based on the location of structural loops (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's antibody AbM modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are noted below.

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 H1 H31- H35 H26-H35 H26-H32 H30- H35 H2 H50- H65 H50-H58 H53-H55 H47- H58 H3 H95- H102 H95-H102 H96-H101 H93- H101

HVR regions may include "extended HVRs" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. Residues in the variable domains are numbered according to Kabat et al., supra, for each of these definitions.

The expressions "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and variations thereof refer to the numbering system used for the heavy chain variable regions or light chain variable regions in the construction of antibodies in the publication by Kabat et al. cited above. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a truncation or insertion in the FR or HVR of the variable region. For example, the variable region of the heavy chain may include an insertion of one amino acid (residue 52a according to Kabat) after residue 52 of the H2 region and insertions of residues (e.g., 82a, 82b, and 82c, etc. according to Kabat) after residue 82 of the heavy chain FR. Kabat residue numbering can be determined for a given antibody by aligning homologous regions of the antibody sequence with a "standard" Kabat numbered sequence.

"Framework region" or "FR" residues represent variable region residues other than the HVR residues defined herein.

The "human consensus framework" or "acceptor human framework" is the framework region that contains the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is made from a subset of variable region sequences. Typically, the subset of sequences is a subgroup according to Kabat et al., Sequences of Proteins of Immunological Interest, ^5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples of a subgroup for VL may include subgroup kappa I, kappa II, kappa III, or kappa IV, as given in Kabat et al., supra. Additionally, a subgroup for VH may include subgroup I, subgroup II, or subgroup III, as given in Kabat et al., supra. Alternatively, a human consensus sequence can be obtained from the above sequence in which certain residues, such as human framework residues, are selected based on their homology to a donor framework by aligning the donor framework sequence with a number of different human framework sequences. A human acceptor framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence, or it can comprise pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid sequence changes is 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.

"VH subgroup III consensus framework" comprises a consensus sequence derived from the amino acid sequences in the variable heavy chain subgroup III of Kabat et al., supra. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least a fragment of or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (HC-FR1) (SEQ ID NO:25), WVRQAPGKGLEWV (HC-FR2) (SEQ ID NO:26), RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (HC-FR3, SEQ ID NO:27), WGQGTLVTVSA (HC-FR4), (SEQ ID NO:28).

"VL subgroup I consensus framework sequence" includes a consensus sequence derived from the amino acid sequences in the variable light region of kappa subgroup I of Kabat et al. In one embodiment, the VL subgroup I consensus framework amino acid sequence includes at least a portion of or all of each of the following sequences: DIQMTQSPSSLSASVGDRVTITC (LC-FR1) (SEQ ID NO:29), WYQQKPGKAPKLLIY (LC-FR2) (SEQ ID NO:30), GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (LC-FR3) (SEQ ID NO:31), FGQGTKVEIKR (LC-FR4) (SEQ ID NO:32).

The term "amino acid modification" at a specified position, for example, the Fc region, refers to a substitution or deletion of said residue or an insertion of at least one amino acid residue adjacent to said residue. An insertion "adjacent" to said residue means an insertion of one to two residues. The insertion may be N-terminal or C-terminal to said residue. A preferred amino acid modification herein is a substitution.

The term "affinity matured" antibody is an antibody with one or more alterations in one or more HVRs that results in an increase in the affinity of the antibody for the antigen compared to a parent antibody that does not possess such alteration(s). In one embodiment, an affinity matured antibody has an affinity at the nanomolar or even picomolar level for the target antigen. Affinity matured antibodies are prepared using procedures known in the art. For example, Marks et al., Bio/Technology, 10:779-783 (1992) describe affinity maturation by shuffling the VH and VL domains. Random mutagenesis of HVR residues and/or framework regions is described, for example, by 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).

As used herein, the term "specifically binds to" or "specific for" refers to a detectable and reproducible interaction, such as binding of a target and an antibody, that is determined by 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 may be an epitope) is an antibody that binds to that target with greater affinity, avidity, greater ease, and/or 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 a related target, as determined, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In some embodiments, the antibody specifically binds to an epitope on a protein that is a conserved protein among different species. In another embodiment, specific binding may include, but is not required to include, binding that excludes binding to other targets.

As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding specificity of a heterologous protein ("adhesin") with the effector functions of immunoglobulin constant regions. Structurally, immunoadhesins are a hybrid of an amino acid sequence with the desired binding specificity, which is other than the antigen recognition and antibody binding site (i.e., is "heterologous"), and an immunoglobulin constant region sequence. The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence containing at least a receptor or ligand binding site. The immunoglobulin constant region sequence of the immunoadhesin can be obtained from any immunoglobulin, such as the IgG1, IgG2 (including IgG2a and IgG2b), IgG3 or IgG4 subtypes of IgA (including IgA1 and IgA2), IgE, IgD or IgM. The hybrid Igs preferably comprise the replacement of at least one variable region within an Ig molecule with a domain of a polypeptide or antibody of the present invention. In a particularly preferred embodiment, the hybrid immunoglobulin comprises the hinge region, CH2 and CH3, or the hinge region, CH1, CH2 and CH3 of an IgG1 molecule. For information on the preparation of immunoglobulin hybrids, see also U.S. Patent No. 5,428,130, issued June 27, 1995. For example, useful immunoadhesins used as second drugs useful herein for combination therapy include polypeptides that comprise an extracellular or PD-1-binding fragment of PD-L1 or PD L2, or an extracellular or PD-L1 or PD-L2-binding fragment of PD-1 fused to an immunoglobulin constant region sequence, such as PD-L1 ECD-Fc, PD-L2 ECD-Fc, and PD-1 ECD-Fc, respectively. In some cases, immunoadhesin combinations of Ig Fc and ECD cell surface receptors are sometimes referred to as soluble receptors.

"Hybrid protein" and "hybrid polypeptide" refer to a polypeptide having two fragments covalently linked to each other, where each fragment is a polypeptide having different properties. The property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two fragments may be linked directly by a single peptide bond or through a peptide linker, but both are in frame with each other.

"PD-1 oligopeptide", "PD-L1 oligopeptide", or "PD-L2 oligopeptide" represent 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 pathway component, respectively, as described herein. Such oligopeptides can be chemically synthesized using known oligopeptide synthesis methodology or can be produced and purified using recombinant technology. Such oligopeptides are typically 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 or more. Such oligopeptides can be identified using well-known methods. In this regard, it should be noted that methods for screening oligopeptide libraries to identify oligopeptides that are capable of specifically binding to a target polypeptide are well known in the art (see, for example, U.S. Patents 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 Publications 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).

A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of an antigen. Anti-PD-L1 antibodies of the invention block signaling through PD-1, with restoration of the functional response of T cells (e.g., proliferation, cytokine production, induction of target cell death) from a dysfunctional state to antigen stimulation.

The term "agonist" or activating antibody is an antibody that enhances or initiates signaling by an antigen to which it binds. In some embodiments, agonist antibodies induce or activate signaling without the presence of a natural ligand.

The term "Fc region" is used herein to define the 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 may vary, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 of its carboxyl terminus. 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 a nucleic acid encoding the antibody heavy chain may be recombinantly engineered. Accordingly, an intact antibody composition may include populations of antibodies with all K447 residues removed, populations of antibodies without any K447 residues removed, and populations of antibodies comprising a mixture of populations of antibodies with and without K447 residues removed. Suitable native sequence Fc regions for use in the antibodies of the invention include human IgG1, IgG2 (IgG2a, IgG2b), IgG3 and IgG4.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Further preferred FcR is a receptor that binds to an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITAM) in its cytoplasmic domain (see, e.g., M. Daëron, Annu. Rev. Immunol., 15:203–234 (1997). FcRs are reviewed by Ravetch and Kinet, Annu. Rev. Immunol., 9:457–92 (1991); Capel et al., Immunomethods, 4:25–34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330–41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein.

The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG antibodies to the fetus. Guyer et al., J. Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994). Methods for quantifying 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.)). In vivo FcRn binding and human serum half-life of high-affinity FcRn-binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides containing a variant Fc region. WO 2004/42072 (Presta) describes antibody variants that have improved or reduced FcR binding. See also, for example, Shields et al., J. Biol. Chem., 9(2):6591-6604 (2001).

As used herein, the expression "significantly reduced" or "significantly different" denotes a sufficiently high degree of difference between two numerical values (typically one relating to the molecule being determined and the other relating to a standard/reference molecule) that one skilled in the art would consider the difference between the two values to be at a level of statistical significance in the context of the biological characteristic defined by said values (e.g., a Kd value). 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% compared to the value for the standard/reference molecule.

As used herein, the expression "substantially similar" or "substantially the same" refers to a sufficiently high degree of similarity between two numerical values (typically one for the molecule being determined and the other for a standard/reference molecule) that one skilled in the art would consider the difference between the two values to be insignificant or of no biological and/or statistical significance in the context of the biological characteristic measured by the values (e.g., a Kd value). The difference between the 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% compared to the value for the standard/reference molecule.

As used herein, the term "carriers" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being treated therewith at the dosages and concentrations employed. Often, a physiologically acceptable carrier is an aqueous solution with a buffered pH. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptide (less than about 10 residues); 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 non-ionic surfactants such as Tween ^™ , polyethylene glycol (PEG) and PLURONICS™.

"Package insert" refers to the instructions that are typically included in commercial packages of medicinal products that contain information on the indications, usage, dosage, administration, contraindications, other medicinal products that are used in conjunction with the packaged product and/or warnings concerning the use of such medicinal products, etc.

As used herein, the term "treatment" refers to a clinical intervention that is intended to alter a natural process in the individual or cell being treated for a clinical disorder. Desirable effects of treatment include a decrease in the rate of disease progression, alleviation or amelioration of the disease state, and remission or a more favorable prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are reduced or eliminated, including, but not limited to, a decrease in the proliferation (or death) of cancer cells, an amelioration of symptoms resulting from the disease, an improvement in the quality of life of individuals suffering from the disease, a decrease in the dosage of other drugs needed to treat the disease, a slowing of the progression of the disease, and/or an increase in the survival of individuals.

As used herein, the term "slowing down the progression of a disease" means slowing down, preventing, slowing down, stopping, stabilizing, and/or delaying the progression of a disease (e.g., cancer). Such slowing down may be of varying lengths of time, depending on the history of the disease and/or the individual being treated. As will be apparent to those skilled in the art, sufficient or significant slowing down may, in fact, include preventing the disease, including a situation where the subject does not develop the disease. For example, late-stage cancer, such as the development of metastases, may be delayed.

As used herein, the term "reducing or inhibiting the recurrence of cancer" means reducing or inhibiting the recurrence of a tumor or cancer or the progression of a tumor or cancer.

As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. This definition includes benign and malignant tumors, as well as "dormant" tumors or micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell lung cancer), abdominal cancer, hepatocellular carcinoma, gastric cancer or gastric carcinoma (including gastrointestinal cancers), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney cancer or renal cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, and various head and neck malignancies, and B-cell lymphoma (including poorly differentiated/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediately differentiated/follicular NHL; intermediately differentiated diffuse NHL; well-differentiated immunoblastic NHL; well-differentiated lymphoblastic NHL; well-differentiated small noncleaved cell NHL; massive involvement NHL; AIDS-related mantle cell lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myelogenous 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.

As used here, the term metastasis refers to the spread of cancer from its primary site to other sites in the body. Cancer cells can break away from the primary tumor, enter lymphatic and blood vessels, circulate in the bloodstream, and grow at a distant site (metastasize) in normal tissues elsewhere in the body. Metastasis may be local or distant. Metastasis is a sequential process that depends on tumor cells breaking away from the primary tumor, traveling through the bloodstream, and landing 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 in the tumor cell regulate this process, and interactions between the tumor cell and the host cell at the distant site are also important.

The term "effective amount" means at least the minimum concentration necessary to achieve a detectable improvement in or prevention of a particular disease. An effective amount may vary depending on factors such as the disease state, age, sex, and body weight of the patient, as well as the ability of the antibody to elicit a desired response in the individual. An effective amount also represents the amount at which any toxic or negative effects of treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as elimination or reduction in the risk of, attenuation of the severity of, or delay in the onset of disease, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as a reduction in one or more symptoms resulting from a disease, an improvement in the quality of life of an individual suffering from the disease, a reduction in the dose of other drugs needed to treat the disease, an enhancement of the effect of another drug, such as by targeting, a slowing of the progression of the disease, and/or an increase in survival. In the case of cancer or a tumor, an effective amount of a drug may have the effect of reducing the number of cancer cells; reducing the size of the tumor; inhibiting (i.e., slowing to some extent, or preferably stopping) the infiltration of cancer cells into peripheral organs; inhibiting (i.e., slowing to some extent, and preferably stopping) tumor metastasis; inhibiting to some extent tumor growth; and/or alleviating to some extent one or more symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment directly or indirectly. As is known in clinical practice, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Thus, an "effective amount" may be considered taking into account the administration of one or more therapeutic agents, and one agent may be considered to be administered in an effective amount if, in combination with one or more other agents, the desired result can be or is achieved.

As used herein, the expression "in combination with" refers to the administration of one medicinal product in combination with another medicinal product. Thus, the expression "in combination with" refers to the administration of one medicinal product before, during or after the administration of another medicinal product to an individual.

As used herein, the term "subject" means 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.

As used herein, the term “complete response” or “CR” refers to the disappearance of all target lesions; “partial response” or “PR” refers to at least a 30% reduction in the sum of longest diameters (SLD) of target lesions, using the baseline SLD as the standard; and “stable disease” or “SD” refers to a state in which there is neither a sufficient reduction in target lesions to qualify as a PR nor a sufficient increase in them to qualify as a PD, using the lowest SLD since the start of treatment as the standard.

As used herein, the term “progressive disease” or “PD” refers to at least a 20% increase in the SLD of target lesions, using the nadir of the SLD since the start of treatment as the standard, or the formation of one or more new lesions.

As used here, the term progression-free survival (PFS) refers to the length of time during and after treatment during which the disease being treated (eg, cancer) does not worsen. Progression-free survival can include the period of time when patients have a complete response or partial response, as well as the period of time when patients have stable disease.

As used here, the term overall response rate (ORR) refers to the overall complete response rate (CR) and the partial response rate (PR).

As used here, the term "overall survival" refers to the percentage of individuals in a group who are likely to be alive after a given period of time.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of tumors. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ^® ); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (notably bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL ^® ); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analog topotecan (HYCAMTIN ^® ); CPT-11 (irinotecan, CAMTOSAR ^® ), acetylcamptothecin, scolectin, and 9-aminocamptothecin); bryostatin; pemetrexed; callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmucin (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an orally administered alpha-4 integrin inhibitor; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ^ADRIAMYCIN® , morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, and liposomal deoxydoxorubicin HCl for injection ( ^DOXIL® 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; antimetabolites such as methotrexate, gemcitabine (GEMZAR ^® ), tegafur (UFTORAL ^® ), capecitabine (XELODA ^® ), epothilone, and 5-fluorouracil (5-FU); folate analogues such as denopterin, methotrexate, pteropterin, trimethotrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and imatinib (a 2-phenylaminopyrimidine derivative), and other c-Kit inhibitors; Antiadrenaline agents 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; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamytocins; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerin; Pentostatin; Fenamet; Pirarubicin; Losoxantrone; 2-Ethylhydrazide; Procarbazine; PSK ^® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (including T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine (ELDISINE ^®, FILDESIN ^®) ; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside ("Ara-C");thiotepa; taxoids such as paclitaxel TAXOL ^® , a nanoparticle albumin-based formulation of paclitaxel (ABRAXANE ^TM ), and doxetaxel TAXOTERE ^® ; chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine (VELBAN ^® ); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN ^® ); oxaliplatin; leucovina; vinorelbine (NAVELBINE ^® ); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above drugs; and combinations of two or more of the above drugs, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for the treatment regimen of oxaliplatin (ELOXATIN ^TM ) in combination with 5-FU and leucovorin.

Also included in this definition are antihormonal agents that regulate, reduce, block, or inhibit the action of hormones that may promote tumor growth and are often used as systemic or whole-body treatment. They may themselves be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including ^NOLVADEX® tamoxifen), raloxifene ( ^EVISTA® ), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene ( ^FARESTON® ); antiprogesterones; estrogen receptor downregulators (ERDs); agents that act by suppressing or blocking ovarian function, such as luteinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate ( ^LUPRON® and ^ELIGARD® ), goserelin acetate, buserelin acetate, and tripterelin; antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE ^® ), exemestane (AROMASIN ^® ), formestane, fadrozole, vorozole (RIVISOR ^® ), letrozole (FEMARA ^® ) and anastrozole (ARIMIDEX ^® ). In addition, the definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g. BONEFOS ^® or OSTAC ^® ), etidronate (DIDROCAL ^® ), NE-58095, zoledronic acid/zoledronate (ZOMETA ^® ), alendronate (FOSAMAX ^® ), pamidronate (AREDIA ^® ), tiludronate (SKELID ^® ), or risedronate (ACTONEL ^® ); as well as troxacitabine (a 1,3-dioxolane-based cytosine nucleoside analogue); antisense oligonucleotides, in particular oligonucleotides that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATORE ^® vaccine and gene therapy vaccines such as ALLOVECTIN ^® vaccine, LEUVECTIN ^® vaccine and VAXID ^® vaccine; a topoisomerase 1 inhibitor (e.g., LUPTOTECAN ^® ); an antiestrogen such as fulvestrant; a Kit inhibitor such as imatinib or EXEL-0862 (a tyrosine kinase inhibitor); an EGFR inhibitor such as erlotinib or cetuximab; a VEGF inhibitor such as bevacizumab; arinotecan; rmRH (e.g., ABARELIX ^® ); lapatinib and lapatinib ditosylate (a small molecule dual ErbB-2 and EGFR tyrosine kinase inhibitor, also known as GW572016); 17AAG (a derivative of geldanamycin, which is a poison for heat shock protein (Hsp)90) and pharmaceutically acceptable salts, acids or derivatives of any of the above mentioned drugs.

As used herein, the term "cytokine" generally refers to proteins released by one population of cells that act on another cell as intercellular mediators or that exert an autocrine effect on protein-producing cells. Examples of such cytokines are lymphokines, monokines; interleukins ("ILs") such as IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18-IL-29 (such as IL-23), IL-31, including rIL-2 PROLEUKIN ^® ; tumor necrosis factor such as TNF-α or TNF-β, TNF-β1-3; and other polypeptide factors including leukemia inhibitory factor ("LIF"), ciliary neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"), cardiotrophin ("CT") and kit ligand (KL).

As used here, 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 angiogenesis, inflammation, wound healing, and tumorigenesis. Exemplary chemokines include IL-8, the human homolog of mouse keratinocyte chemoattractant (KC).

In this description and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

A description of a value or parameter by the term "about" includes (and describes) variations that relate to that value or parameter as such. For example, a description that refers to "about X" includes the description of "X."

As used herein, the expression "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of a compound of the present invention. Examples of salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, hydrogen phosphate, isonicotinate, lactate, salicylate, hydrogen 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)), alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., magnesium salts), and ammonium salts. A pharmaceutically acceptable salt may comprise an inclusion of another molecule, such as an acetate ion, a succinate ion, or another counterion. The counterion may be any organic or inorganic group that stabilizes the charge on the parent compound. In addition, its pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where several charged atoms are part of the pharmaceutically acceptable salt, the compound may have several counterions. Therefore, its pharmaceutically acceptable salt may have one or more charged atoms and/or one or more counterions.

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, by treating 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 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 pyranosidic 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.

If the compound of the invention is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or an 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.

The term "pharmaceutically acceptable" means that the compound or composition must be compatible chemically and/or toxicologically with the other ingredients of the drug included in the drug and/or the mammal to be treated with them.

It should be understood that aspects and embodiments of the invention described herein include “consisting of” and/or “consisting essentially of” aspects and embodiments.

III. Methods

In one aspect, a method of treating or slowing the progression of cancer in an individual is provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that reduces or inhibits the expression and/or activity of TIGIT.

In another aspect, there is provided a method for reducing or inhibiting the recurrence or progression of cancer in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. As disclosed herein, recurrence and/or progression of cancer includes, but is not limited to, metastasis of cancer.

In another aspect, there is provided a method for treating or slowing the progression of an immune-related disease in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT.

In another aspect, a method is provided for reducing or inhibiting the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT.

In some embodiments, the immune-related disease is associated with a T-cell dysfunctional disorder. In some embodiments, the immune-related disease is a viral infection. In some embodiments, the viral infection is a chronic viral infection. In some embodiments, the T-cell dysfunctional disorder is characterized by decreased responsiveness to antigen stimulation. In some embodiments, the T-cell dysfunctional disorder is characterized by T-cell anergy or a decreased ability to secrete cytokines, proliferate, or perform 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 is selected from the group consisting of unresolved acute infection, chronic infection, and tumor immunity.

In another aspect, a method is provided for increasing, enhancing, or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT.

In another aspect, a method of treating or slowing the progression of cancer in an individual is provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

In another aspect, a method is provided for reducing or inhibiting the recurrence or progression of cancer in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

In another aspect, a method of treating or slowing the progression of an immune-related disease in an individual is provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

In another aspect, a method is provided for reducing or inhibiting the progression of an immune-related disease in an individual comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

In some embodiments, the immune-related disease is associated with a T-cell dysfunctional disorder. In some embodiments, the immune-related disease is a viral infection. In some embodiments, the viral infection is a chronic viral infection. In some embodiments, the T-cell dysfunctional disorder is characterized by decreased responsiveness to antigen stimulation. In some embodiments, the T-cell dysfunctional disorder is characterized by T-cell anergy or a decreased ability to secrete cytokines, proliferate, or perform 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 is selected from the group consisting of unresolved acute infection, chronic infection, and tumor immunity.

In another aspect, a method is provided for enhancing, enhancing, or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

In some embodiments, an agent that modulates the expression and/or activity of CD226 is capable of increasing and/or stimulating the expression and/or activity of CD226; increasing and/or stimulating the interaction of CD226 with PVR, PVRL2, and/or PVRL3; and increasing and/or stimulating intracellular signaling mediated by the binding of CD226 to PVR, PVRL2, and/or PVRL3. As used herein, the term "an agent that is capable of increasing and/or stimulating the expression and/or activity of CD226" includes, but is not limited to, agents that increase and/or stimulating the expression and/or activity of CD226. As used herein, the term "an agent that is capable of enhancing and/or stimulating the interaction of CD226 with PVR, PVRL2, and/or PVRL3" includes, but is not limited to, agents that enhance and/or stimulating the interaction of CD226 with PVR, PVRL2, and/or PVRL3. As used herein, the term "an agent that is capable of enhancing and/or stimulating intracellular signaling mediated by the binding of CD226 to PVR, PVRL2, and/or PVRL3" includes, but is not limited to, agents that enhance and/or stimulating intracellular signaling mediated by the binding of CD226 to PVR, PVRL2, and/or PVRL3.

In some embodiments, the agent that modulates the expression and/or activity of CD226 is selected from an agent that inhibits and/or blocks the interaction of CD226 with TIGIT, an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, 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 intracellular signaling mediated by TIGIT binding to PVR, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVRL2, an agent that inhibits and/or blocks 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 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.

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 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.

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.

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.

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.

In some embodiments, the agent that inhibits and/or blocks 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 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.

In some embodiments, the agent that inhibits and/or blocks 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.

In some embodiments, the agent that inhibits and/or blocks 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.

In another aspect, a method is provided for increasing, enhancing or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that reduces or inhibits one or more additional immune coinhibitory receptors. In some embodiments, the one or more additional immune coinhibitory receptors are selected from PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4 and CD96. In some embodiments, the one or more additional immune coinhibitory receptors are selected from PD-1, CTLA-4, LAG3 and TIM3.

In another aspect, there is provided a method for increasing, enhancing or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that increases or activates the expression and/or activity of one or more additional immune costimulatory receptors. In some embodiments, the one or more additional immune costimulatory receptors are selected from CD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D and 2B4. In some embodiments, the one or more additional immune costimulatory receptors are selected from CD226, OX40, CD28, CD27, CD137, HVEM and GITR. In some embodiments, the one or more additional immune costimulatory receptors are selected from the group consisting of OX-40 and CD27.

The methods of the present invention may find use in the treatment of conditions where increased immunogenicity is required, such as increased tumor immunogenicity for the treatment of cancer or T-cell dysfunctional disorders.

Different types of cancer can be treated or their progression can be slowed down.

In some embodiments, the individual has non-small cell lung cancer. The non-small cell lung cancer may be at an early stage or at an advanced stage. In some embodiments, the individual has small cell lung cancer. The small cell lung cancer may be at an early stage or at an advanced stage. In some embodiments, the individual has renal cell cancer. The renal cell cancer may be at an early stage or at an advanced stage. In some embodiments, the individual has colorectal cancer. The colorectal cancer may be at an early stage or at an advanced stage. In some embodiments, the individual has ovarian cancer. The ovarian cancer may be at an early stage or at an advanced stage. In some embodiments, the individual has breast cancer. The breast cancer may be at an early stage or at an advanced stage. In some embodiments, the individual has pancreatic cancer. The pancreatic cancer may be at an early stage or at an advanced stage. In some embodiments, the individual has gastric carcinoma. The gastric carcinoma may be at an early stage or at an advanced stage. In some embodiments, the individual has bladder cancer. The bladder cancer may be in an early stage or in a late stage. In some embodiments, the individual has esophageal cancer. The esophageal cancer may be in an early stage or in a late stage. In some embodiments, the individual has mesothelioma. The mesothelioma may be in an early stage or in a late stage. In some embodiments, the individual has melanoma. The melanoma may be in an early stage or in a late stage. In some embodiments, the individual has head and neck cancers. The head and neck cancers may be in an early stage or in a late stage. In some embodiments, the individual has thyroid cancer. The thyroid cancer may be in an early stage or in a late stage. In some embodiments, the individual has sarcoma. The sarcoma may be in an early stage or in a late stage. In some embodiments, the individual has prostate cancer. The prostate cancer may be in an early stage or in a late stage. In some embodiments, the individual has glioblastoma. The glioblastoma may be in an early stage or in a late stage. In some embodiments, the individual has cervical cancer. The cervical cancer may be in an early stage or in a late stage. In some embodiments, the individual has thymic carcinoma. The thymic carcinoma may be in an early stage or in a late stage. In some embodiments, the individual has leukemia. The leukemia may be in an early stage or in a late stage. In some embodiments, the individual has lymphoma. The lymphoma may be in an early stage or in a late stage. In some embodiments, the individual has myeloma. The myeloma may be in an early stage or in a late stage. In some embodiments, the individual has mycosis fungoides. Mycosis fungoides may be in an early stage or in a late stage. In some embodiments, the individual has Merkel cell cancer. Merkel cell cancer may be in an early stage or in a late stage. In some embodiments, the individual has malignant blood diseases. The malignant blood diseases may be in an early stage or in a late stage. In some embodiments, the individual is a human.

In some embodiments of the methods of the present invention, CD4 and/or CD8 T cells in the individual have increased or enhanced priming, activation, proliferation, cytokine release, and/or cytolytic activity compared to the period before the combination was administered.

In some embodiments of the methods of the present invention, the number of CD4 and/or CD8 T cells is increased compared to the period before the combination is administered. In some embodiments of the methods of the present invention, the number of activated CD4 and/or CD8 T cells is increased compared to the period before the combination is administered.

In some embodiments of the methods of the present invention, the activated CD4 and/or CD8 T cells are characterized by CD4 and/or CD8 T cells producing γ-IFN ⁺ and/or increased cytolytic activity compared to the period before the combination was administered.

In some embodiments of the methods of the present invention, CD4 and/or CD8 T cells exhibit increased release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukins.

In some embodiments of the methods of the present invention, the CD4 and/or CD8 T cell is a memory effector T cell. In some embodiments of the methods of the present invention, the CD4 and/or CD8 memory effector T cell is characterized by CD4 and/or CD8 T cells producing γ-IFN ⁺ and/or increased cytolytic activity. In some embodiments of the methods of the present invention, the CD4 and/or CD8 memory effector T cell is characterized by having CD44 ^high CD62L ^low expression.

In some embodiments of the methods of the present invention, the malignant tumor has elevated levels of T cell infiltration.

In some embodiments, the methods of the present invention may further comprise the use of 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 thereof. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of agents that reduce side effects (e.g., agents designed to reduce the development and/or severity of side effects resulting from treatment, such as antiemetics, 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 provided above.

Any of the PD-1 axis binding antagonists and agents that reduce or inhibit TIGIT expression and/or activity described below can be used in the methods of the present invention.

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

A method of treating or slowing the progression of cancer in an individual is provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. A method of reducing or inhibiting the recurrence or progression of cancer in an individual is provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. A method of treating or slowing the progression of an immune-related disease in an individual is provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. A method is provided for reducing or inhibiting the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis, in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. A method is provided for increasing, enhancing or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis, in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT.

For example, an antagonist that binds to the PD-1 axis includes a PD-1-binding antagonist, a PD-L1-binding antagonist, and a PD-L2-binding antagonist.

In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to a ligand of its binding partners. In a particular aspect, the ligand of the PD-1 binding partners is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a particular aspect, the binding partners of PD-L1 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 particular aspect, the binding partner of PD-L2 is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

In some embodiments, the PD-1 binding antagonist is selected from MDX-1106 (nivolumab), Merck 3475 (lambrolizumab), CT-011 (pidilizumab), and AMP-224. In some embodiments, the PD-L1 binding antagonist is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI 4736. In some embodiments, the PD-L2 binding antagonist is the antibody AMP-224. In some embodiments, the PD-1 binding antagonist is the antibody AMP-224. The MDX-1105 antibody, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007/005874. The YW243.55.S70 antibody (SEQ ID NO:20) is an anti-PD-L1 antibody described in WO 2010/077634 A1 and U.S. Patent No. 8,217,149, which are incorporated herein by reference. The MDX-1106 antibody, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO2006/121168. The Merck 3745 antibody, also known as MK 3475, MK-3475, SCH-900475, or lambrolizumab, is an anti-PD-1 antibody described in WO2009/114335. The CT-011 antibody, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1 antibody described in WO2009/101611. The AMP-224 antibody, also known as B7-DCIg, is a soluble PD-L2-Fc receptor fusion described in WO2010/027827 and WO2011/066342.

Examples of anti-PD-L1 antibodies useful in the methods of the present invention and methods for making them are described in PCT Patent Application WO 2010/077634 A1 and U.S. Patent No. 8,217,149, which are incorporated herein by reference.

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 the binding of PD-L1 and PD-1 and/or 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') 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.

Anti-PD-L1 antibodies useful in the present invention, including compositions comprising such antibodies, such as those described in WO 2010/077634 A1 and U.S. Patent No. 8,217,149, can be used in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT, with or without additional therapy (e.g., chemotherapy), to treat cancer or an immune-related disease (e.g., T-cell dysfunction disorder, viral infection, chronic viral infection, etc.).

In one embodiment, an anti-PD-L1 antibody comprises a heavy chain variable region polypeptide comprising the sequence HVR-H1, HVR-H2, and HVR-H3, wherein:

(a) the HVR-H1 sequence is GFTFSX ₁ SWIH (SEQ ID NO:33);

(b) the HVR-H2 sequence is AWIX ₂ PYGGSX ₃ YYADSVKG (SEQ ID NO:34);

(c) the HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:19);

where further: X ₁ represents D or G, X ₂ represents S or L; X ₃ represents T or S.

In one particular aspect, X ₁ is D; X ₂ is S, and X ₃ is T. In yet another aspect, the polypeptide further comprises heavy chain variable region framework sequences located 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 framework consensus sequences. In a further aspect, the framework sequences represent a VH subgroup III consensus framework. In yet another aspect, at least one of the framework sequences is as follows:

HC-FR1 represents EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:25)

HC-FR2 represents WVRQAPGKGLEWV (SEQ ID NO:26)

HC-FR3 represents RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

HC-FR4 is WGQGTLVTVSA (SEQ ID NO:28).

In another aspect, the heavy chain polypeptide is further combined with a light chain variable region comprising HVR-L1, HVR-L2 and HVR-L3, wherein:

(a) the HVR-L1 sequence is RASQX ₄ X ₅ X ₆ TX ₇ X ₈ A (SEQ ID NO:35);

(b) the HVR-L2 sequence is SASX ₉ LX ₁₀ S, (SEQ ID NO:36);

(c) the HVR-L3 sequence is QQX ₁₁ X ₁₂ X ₁₃ X ₁₄ PX ₁₅ T (SEQ ID NO:37);

wherein further: X ₄ represents D or V; X ₅ represents V or I; X ₆ represents S or N; X ₇ represents A or F; X ₈ represents V or L; X ₉ represents F or T; X ₁₀ represents Y or A; X ₁₁ represents Y, G, F or S; X ₁₂ represents L, Y, F or W; X ₁₃ represents Y, N, A, T, G, F or I; X ₁₄ represents H, V, P, T or I; X ₁₅ represents A, W, R, P or T.

In yet a further aspect, X ₄ is D; X ₅ is V; X ₆ is S; X ₇ is A; X ₈ is V; X ₉ is F; X ₁₀ is Y; X ₁₁ is Y; X ₁₂ is L; X ₁₃ is Y; X ₁₄ is H; X ₁₅ is A. In yet a further aspect, the light chain further comprises light chain variable region framework sequences located between the HVRs according to the formula: (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 framework consensus sequences. In a further aspect, the framework sequences represent a VL kappa I consensus framework. In yet another aspect, at least one of the framework sequences is as follows:

LC-FR1 represents DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:29)

LC-FR2 represents WYQQKPGKAPKLLIY (SEQ ID NO:30)

LC-FR3 represents GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

LC-FR4 represents FGQGTKVEIKR (SEQ ID NO:32).

In another embodiment, there is provided an isolated anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain and light chain variable region sequence, wherein:

(a) The heavy chain contains HVR-H1, HVR-H2, and HVR-H3, where:

(i) the HVR-H1 sequence is GFTFSX ₁ SWIH; (SEQ ID NO:33)

(ii) the HVR-H2 sequence is AWIX ₂ PYGGSX ₃ YYADSVKG (SEQ ID NO:34)

(iii) the HVR-H3 sequence is RHWPGGFDY and (SEQ ID NO:19)

(b) The light chain contains HVR-L1, HVR-L2 and HVR-L3, where:

(i) the HVR-L1 sequence is RASQX ₄ X ₅ X ₆ TX ₇ X ₈ A (SEQ ID NO:35)

(ii) the HVR-L2 sequence is SASX ₉ LX ₁₀ S; and (SEQ ID NO:36)

(iii) the HVR-L3 sequence is QQX ₁₁ X ₁₂ X ₁₃ X ₁₄ PX ₁₅ T; (SEQ ID NO:37)

wherein further: X ₁ represents D or G; X ₂ represents S or L; X ₃ represents T or S; X ₄ represents D or V; X ₅ represents V or I; X ₆ represents S or N; X ₇ represents A or F; X ₈ represents V or L; X ₉ represents F or T; X ₁₀ represents Y or A; X ₁₁ represents Y, G, F or S; X ₁₂ represents L, Y, F or W; X ₁₃ represents Y, N, A, T, G, F or I; X ₁₄ represents H, V, P, T or I; X ₁₅ represents A, W, R, P or T.

In a particular aspect, X ₁ represents D; X ₂ represents S, and X ₃ represents T. In yet another aspect, X ₄ represents D; X ₅ represents V; X ₆ represents S; X ₇ represents A; X ₈ represents V; X ₉ represents F; X ₁₀ represents Y; X ₁₁ represents Y; X ₁₂ represents L; X ₁₃ represents Y; X ₁₄ represents H; X ₁₅ represents A. In yet another aspect, X ₁ represents D; X ₂ represents S; and X ₃ represents T; X ₄ represents D; X ₅ represents V; X ₆ represents S; X ₇ represents A; X ₈ represents V; X ₉ represents F; X ₁₀ represents Y; X ₁₁ represents Y; X ₁₂ represents L; X ₁₃ represents Y; X ₁₄ represents H, and X ₁₅ represents A.

In yet another further aspect, the variable region of the heavy chain comprises one or more framework region sequences located between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the variable regions of the light chain comprise one or more framework region sequences located between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework region sequences are derived from human framework region consensus sequences. In yet another aspect, the heavy chain framework region sequences are derived from a Kabat subgroup I, II, III sequence. In yet another further aspect, the heavy chain framework region sequence is a VH subgroup III consensus framework. In yet another further aspect, one or more heavy chain framework region sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:25)

HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:26)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

HC-FR4 WGQGTLVTVSA (SEQ ID NO:28).

In yet another aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III, or IV sequence. In yet another aspect, the light chain framework sequences are a VL kappa subgroup I consensus framework. In yet another aspect, the one or more light chain framework sequences are as follows:

LC-FR1 DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:29)

LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:30)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

LC-FR4 FGQGTKVEIKR (SEQ ID NO:32).

In yet another further specific aspect, the antibody further comprises a human or mouse constant region. In yet another further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet another further specific aspect, the human constant region is IgG1. In yet another further aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3. In yet another further aspect, the mouse constant region is IgG2a. In yet another further specific aspect, the antibody has reduced or minimal effector function. In yet another further specific aspect, the minimal effector function is the result of an "Fc region effector function reduction mutation" or aglycosylation. In yet another further embodiment, the mutation reducing Fc region effector function is N297A or a D265A/N297A substitution in the constant region.

In another embodiment, there is provided an anti-PD-L1 antibody comprising a heavy chain and light chain variable region sequence wherein:

(a) the heavy chain further comprises a HVR-H1, HVR-H2 and 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

(b) the light chain further comprises an HVR-L1, HVR-L2 and 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,

(c) in a particular 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 variable region of the heavy chain comprises one or more framework region sequences located between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the variable regions of the light chain comprise one or more framework region sequences located between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework region sequences are derived from human framework region consensus sequences. In a still further aspect, the heavy chain framework region sequences are derived from a Kabat subgroup I, II or III sequence. In a still further aspect, the heavy chain framework region sequence is a VH subgroup III consensus framework. In another aspect, one or more heavy chain framework region sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:25)

HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:26)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

HC-FR4 WGQGTLVTVSA (SEQ ID NO:28).

In yet another aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III, or IV sequence. In yet another aspect, the light chain framework sequences are a VL kappa subgroup I consensus framework. In yet another aspect, the one or more light chain framework sequences are as follows:

LC-FR1 DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:29)

LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:30)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

LC-FR4 FGQGTKVEIKR (SEQ ID NO:32).

In yet another further specific aspect, the antibody further comprises a human or mouse constant region. In yet another further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet another further specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3. In yet another further aspect, the mouse constant region is IgG2a. In yet another further specific aspect, the antibody has reduced or minimal effector function. In yet another further specific aspect, the minimal effector function is the result of an "Fc region effector function reduction mutation" or aglycosylation. In yet another further embodiment, the mutation reducing Fc region effector function is N297A or a D265A/N297A substitution in the constant region.

In another embodiment, there is provided an anti-PD-L1 antibody comprising a heavy chain and light chain variable region sequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity with the heavy chain sequence of:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA (SEQ ID NO:23), EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISA DTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:40) or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:41), or

(b) the light chain sequence has at least 85% sequence identity with the light chain sequence of:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24).

In a particular 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 located between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprise one or more framework sequences located between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework sequences are derived from human framework consensus 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 yet another further aspect, one or more heavy chain framework region sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:25)

HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:26)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

HC-FR4 WGQGTLVTVSA (SEQ ID NO:28).

In yet another further aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III or IV sequence. In yet another further aspect, the light chain framework sequences represent a VL kappa subgroup I consensus framework. In yet another further aspect, the one or more light chain framework sequences represent the following:

LC-FR1 DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:29)

LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:30)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

LC-FR4 FGQGTKVEIKR (SEQ ID NO:32).

In yet another further specific aspect, the antibody further comprises a human or mouse constant region. In yet another further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet another further specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3. In yet another further aspect, the mouse constant region is IgG2a. In yet another further specific aspect, the antibody has reduced or minimal effector function. In yet another further specific aspect, the minimal effector function is the result of production in prokaryotic cells. In yet another further specific aspect, the minimal effector function is the result of an "Fc region effector function reduction mutation" or aglycosylation. In yet another further embodiment, the mutation reducing Fc region effector function is N297A or a D265A/N297A substitution in the constant region.

In yet another further embodiment, the present invention relates to compositions comprising any of the above-described anti-PD-L1 antibodies in combination with at least one pharmaceutically acceptable carrier.

In yet another further embodiment, there is provided an isolated nucleic acid encoding a variable region of a light chain or a heavy chain of an anti-PD-L1 antibody, wherein:

(a) the heavy chain further comprises a HVR-H1, HVR-H2 and 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

(b) the light chain further comprises a HVR-L1, HVR-L2 and 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.

In a specific embodiment, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In another aspect, the variable region of the heavy chain comprises one or more framework region sequences located between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the variable regions of the light chain comprise one or more framework region sequences located between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework region sequences are derived from human framework region consensus sequences. In a still further aspect, the heavy chain framework region sequences are derived from a Kabat subgroup I, II or III sequence. In a still further aspect, the heavy chain framework region sequence is a VH subgroup III consensus framework. In yet another further aspect, one or more heavy chain framework region sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:25)

HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:26)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

HC-FR4 WGQGTLVTVSA (SEQ ID NO:28).

In yet another aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III, or IV sequence. In yet another aspect, the light chain framework sequences are a VL kappa subgroup I consensus framework. In yet another aspect, the one or more light chain framework sequences are as follows:

LC-FR1 DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:29)

LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:30)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

LC-FR4 FGQGTKVEIKR (SEQ ID NO:32).

In yet another further specific aspect, the antibody further comprises a human or mouse constant region. In yet another further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet another further specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3. In yet another further aspect, the mouse constant region is IgG2a. In yet another further specific aspect, the antibody has reduced or minimal effector function. In yet another further specific aspect, the minimal effector function is the result of production in prokaryotic cells. In yet another further specific aspect, the minimal effector function is the result of an "Fc region effector function reduction mutation" or aglycosylation. In yet another further embodiment, the mutation reducing Fc region effector function is N297A or a D265A/N297A substitution in the constant region.

In yet another further embodiment, there is provided an anti-PD-L1 antibody comprising a heavy chain and light chain variable region sequence wherein:

(a) the heavy chain sequence has at least 85% sequence identity with the heavy chain sequence of:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO:41), or

(b) the light chain sequence has at least 85% sequence identity with the light chain sequence of:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24).

In a particular 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 region sequences located between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprise one or more framework region sequences located between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework region sequences are derived from human framework region consensus sequences. In a further aspect, the heavy chain framework region sequences are derived from a Kabat subgroup I, II or III sequence. In a still further aspect, the heavy chain framework region sequence is a VH subgroup III consensus framework. In yet another further aspect, one or more heavy chain framework region sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:25)

HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:26)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

HC-FR4 WGQGTLVTVSS (SEQ ID NO:42).

In yet another further aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III or IV sequence. In yet another further aspect, the light chain framework sequences represent a VL kappa subgroup I consensus framework. In yet another further aspect, the one or more light chain framework sequences represent the following:

LC-FR1 DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:29)

LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:30)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

LC-FR4 FGQGTKVEIKR (SEQ ID NO:32).

In yet another further specific aspect, the antibody further comprises a human or mouse constant region. In yet another further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet another further specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3. In yet another further aspect, the mouse constant region is IgG2a. In yet another further specific aspect, the antibody has reduced or minimal effector function. In yet another further specific aspect, the minimal effector function is the result of production in prokaryotic cells. In yet another further specific aspect, the minimal effector function is the result of an "Fc region effector function reduction mutation" or aglycosylation. In yet another further embodiment, the mutation reducing Fc region effector function is N297A or a D265A/N297A substitution in the constant region.

In a further aspect, the heavy chain variable region comprises one or more framework region sequences located between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprise one or more framework region sequences located between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet a further aspect, the framework region sequences are derived from human framework consensus sequences. In a further aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II or III sequence. In yet a further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In yet another further aspect, one or more heavy chain framework region sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAASGTFFS (SEQ ID NO:43)

HC-FR2 WVRQAPGKGLEWVA (SEQ ID NO:44)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

HC-FR4 WGQGTLVTVSS (SEQ ID NO:45).

In yet another further aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III or IV sequence. In yet another further aspect, the light chain framework sequences represent a VL kappa subgroup I consensus framework. In yet another further aspect, the one or more light chain framework sequences represent the following:

LC-FR1 DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:29)

LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:30)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

LC-FR4 FGQGTKVEIK (SEQ ID NO: 46).

In yet another further specific aspect, the antibody further comprises a human or mouse constant region. In yet another further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet another further specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3. In yet another further aspect, the mouse constant region is IgG2a. In yet another further specific aspect, the antibody has reduced or minimal effector function. In yet another further specific aspect, the minimal effector function is the result of an "Fc region effector function reduction mutation" or aglycosylation. In yet another further embodiment, the mutation reducing Fc region effector function is N297A or a D265A/N297A substitution in the constant region.

In another embodiment, there is provided an anti-PD-L1 antibody comprising a heavy chain and light chain variable region sequence wherein:

(d) the heavy chain further comprises a HVR-H1, HVR-H2 and 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

(e) the light chain further comprises an HVR-L1, HVR-L2 and 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.

In a particular 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 variable region of the heavy chain comprises one or more framework region sequences located between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the variable regions of the light chain comprise one or more framework region sequences located between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the framework region sequences are derived from human framework region consensus sequences. In a still further aspect, the heavy chain framework region sequences are derived from a Kabat subgroup I, II or III sequence. In a still further aspect, the heavy chain framework region sequence is a VH subgroup III consensus framework. In yet another further aspect, one or more heavy chain framework region sequences are as follows:

HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:25)

HC-FR2 WVRQAPGKGLEWV (SEQ ID NO:26)

HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

HC-FR4 WGQGTLVTVSSASTK (SEQ ID NO: 47).

In yet another further aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III or IV sequence. In yet another further aspect, the light chain framework sequences represent a VL kappa subgroup I consensus framework. In yet another further aspect, the one or more light chain framework sequences represent the following:

LC-FR1 DIQMTQSPSSLSSASVGDRVTITC (SEQ ID NO:29)

LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO:30)

LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

LC-FR4 FGQGTKVEIKR (SEQ ID NO:32).

In yet another further specific aspect, the antibody further comprises a human or mouse constant region. In yet another further aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In yet another further specific aspect, the human constant region is IgG1. In yet another aspect, the mouse constant region is selected from the group consisting of IgG1, IgG2a, IgG2b, IgG3. In yet another further aspect, the mouse constant region is IgG2a. In yet another further specific aspect, the antibody has reduced or minimal effector function. In yet another further specific aspect, the minimal effector function is the result of an "Fc region effector function reduction mutation" or aglycosylation. In yet another further embodiment, the mutation reducing Fc region effector function is N297A or a D265A/N297A substitution in the constant region.

In yet another further embodiment, there is provided an anti-PD-L1 antibody comprising a heavy chain and light chain variable region sequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity with the heavy chain sequence of:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK (SEQ ID NO:40), or

(b) the light chain sequence has at least 85% sequence identity with the light chain sequence of:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO:24).

In some embodiments, there is provided 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, there is provided 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, there is provided 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 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 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 yet another further embodiment, there is provided an anti-PDL1 antibody comprising a heavy chain and a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity with the heavy chain sequence of:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:48), or

(b) the light chain sequence has at least 85% sequence identity with the light chain sequence of:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:49).

In some embodiments, there is provided 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, there is provided 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, there is provided 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 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 yet another further aspect, the nucleic acid further comprises a vector suitable for expressing a nucleic acid encoding any of the above-described anti-PD-L1 antibodies. In yet another further specific aspect, the vector further comprises a host cell suitable for expressing the nucleic acid. In yet another further specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In yet another further specific aspect, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

An anti-PD-L1 antibody or antigen-binding fragment thereof can be produced using methods known in the art, for example, a method comprising culturing a host cell containing a nucleic acid encoding any of the above-described anti-PD-L1 antibodies or antigen-binding fragment thereof in a form suitable for expression, under conditions suitable for producing such an antibody or fragment, and isolating the antibody or fragment.

In yet another further embodiment, the present invention relates to a composition comprising an anti-PD-L1 antibody or antigen-binding fragment thereof described herein and at least one pharmaceutically acceptable carrier.

Agents that reduce or inhibit TIGIT expression and/or activity

A method of treating or slowing the progression of cancer in an individual is provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. A method of reducing or inhibiting the recurrence or progression of cancer in an individual is also provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. A method of treating or slowing the progression of an immune-related disease in an individual is also provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. Also provided is a method for reducing or inhibiting the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. Also provided is a method for increasing, enhancing or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis in combination with an agent that reduces or inhibits the expression and/or activity of TIGIT. Also provided is a method for increasing, enhancing or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that reduces or inhibits one or more additional immune coinhibitory receptors. Also provided is a method for increasing, enhancing or stimulating an immune response or immune function in an individual, comprising administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that increases or activates one or more additional immune costimulatory receptors. For example, an agent that reduces or inhibits the expression and/or activity of TIGIT includes an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, 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 intracellular signaling mediated by TIGIT binding to PVR, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVRL2, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVRL3, and combinations thereof.

In some embodiments, an antagonist of TIGIT expression and/or activity comprises 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 comprises 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, an agent that inhibits and/or blocks the interaction of TIGIT with PVR comprises 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, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL2 comprises 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, an agent that inhibits and/or blocks the interaction of TIGIT with PVRL3 comprises 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, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVR comprises 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, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVRL2 comprises 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, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVRL3 comprises 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 inhibitory nucleic acid selected from the group consisting of an antisense polynucleotide, an interfering RNA, a catalytic RNA, and an RNA-DNA chimera.

In some embodiments, the antagonist of TIGIT expression and/or activity is an anti-TIGIT antibody or antigen-binding fragment thereof.

Anti-TIGIT antibodies useful in the present invention, including compositions comprising such antibodies, such as those described in WO 2009/126688, can be used in combination with antagonists that bind to the PD-1 axis.

Anti-TIGIT antibodies

The present invention relates to anti-TIGIT antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies. One of ordinary skill in the art will appreciate that the invention also relates to antibodies to other polypeptides (i.e., anti-PVR antibodies), and that any description herein relating to a method for creating, producing, varieties, uses, or other aspects of anti-TIGIT antibodies will be applicable to antibodies specific for other polypeptides other than TIGIT.

Polyclonal antibodies

Anti-TIGIT antibodies may include polyclonal antibodies. Methods for producing polyclonal antibodies are known to those skilled in the art. The production of polyclonal antibodies may be induced 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 is administered to the mammal by several subcutaneous or intraperitoneal injections. The immunizing agent may include a TIGIT polypeptide or a fusion protein thereof. It may be advantageous 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 that may be used include complete Freund's adjuvant and MPL-TDM (monophosphoryl lipid A, synthetic trehalose dicornomycolate) adjuvant. The immunization protocol can be selected by one skilled in the art without undue experimentation.

Monoclonal antibodies

Anti-TIGIT antibodies may alternatively be monoclonal antibodies. Monoclonal antibodies may be produced using hybridoma techniques such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma technique, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to induce the production of lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

The immunizing agent typically includes a TIGIT polypeptide or a fusion protein thereof. Typically, peripheral blood lymphocytes ("PBL") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if a non-human mammalian source is 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 Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63]. Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are typically used. Hybridoma cells may be cultured in a suitable culture medium, which 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 phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas will typically include hypoxanthine, aminopterin, and thymidine ("HAT medium"), where these substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high levels of antibody expression by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have also been described [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are grown is then assayed for the presence of monoclonal antibodies to the polypeptide. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent 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 assay described by Munson and Pollard, Anal. Biochem., 107:220 (1980).

Once the desired hybridoma cells have been identified, the clones can be subcloned by limiting dilution procedures and cultured by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo in ascites fluid from a mammal.

Monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be produced by recombinant DNA techniques, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors that are then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to allow the synthesis of monoclonal antibodies in the recombinant host cells. The DNA may also be modified, for example, by replacing the sequence encoding the constant regions of the heavy and light chains with homologous murine sequences [U.S. Patent No. 4,816,567; Morrison et al., supra] or by covalently attaching to the coding sequence of an immunoglobulin all or part of the coding sequence for a polypeptide other than an immunoglobulin. Such a polypeptide other than an immunoglobulin may be replaced by the constant region of an antibody of the invention, or may be replaced by the variable region of one antigen-binding site of an antibody of the invention to produce a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for producing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. Typically, the heavy chain is truncated at any point in the Fc region to prevent cross-linking of the heavy chain. Alternatively, the corresponding cysteine residues are replaced by another amino acid residue or are deleted in a manner that prevents cross-linking.

Methods for producing monovalent antibodies in vitro are also suitable. Cleavage of antibodies to obtain fragments, particularly Fab fragments, can be carried out using conventional methods known in the art.

Human and humanized antibodies

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 (e.g., Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding subsequences of antibodies) that comprise a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (the recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from the CDR of a non-human species (the donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and activity. In some cases, residues of the Fv framework region of a human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are not found in the recipient antibody or in the imported CDR or framework sequences. 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 CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions from a human immunoglobulin consensus sequence. A humanized antibody optimally also comprises 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)].

Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody will comprise one or more amino acid residues introduced therein from a source that is not human. Such "non-human" amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable region. Humanization can generally be accomplished following the method of Winter et al. [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 replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies typically represent human antibodies in which some CDR residues and possibly some FR residues have been replaced by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using a variety of methods 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 methods of Cole et al. and Boerner et al. are also available. [to produce 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 produced by introducing human immunoglobulin loci into transgenic animals, such as mice, in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon stimulation, the production of human antibody is observed, closely resembling that observed in humans in all respects, including gene rearrangement, antibody assembly, and repertoire. This approach is described, for example, in U.S. Patents 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).

Such antibodies may also be affinity matured using known selection and/or mutagenesis techniques, as described above. Preferred affinity matured antibodies have an affinity that is 5-fold, more preferably 10-fold, even more preferably 20 or 30-fold higher than the affinity of the parent antibody (usually murine humanized or human) from which the mature antibody is derived.

Bispecific antibodies

Bispecific antibodies are monoclonal, preferably human or humanized antibodies, which have binding specificity for at least two different antigens. In this case, one of the binding specificities is specificity for TIGIT, the other for any other antigen, and preferably for a protein or a receptor or a subunit of a cell surface receptor.

Methods for producing bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. By randomly assorting immunoglobulin heavy and light chains, such hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is typically accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

The variable regions of antibodies with the desired binding specificity (antibody-antigen binding sites) can be fused to immunoglobulin constant region sequences. The fusion is preferably with an immunoglobulin heavy chain constant region comprising at least part of the hinge region, CH2 and CH3. 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 fusion constructs. DNAs encoding the immunoglobulin heavy chain and, optionally, immunoglobulin light chain fusion constructs are inserted into separate expression vectors and cotransfected into a suitable host organism. For further information on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach, described in WO 96/27011, the interface between a pair of antibody molecules can be designed to maximize the percentage of heterodimers that are recovered from recombinant cell culture. A preferred interface comprises at least a portion of the CH3 domain of the constant region of the antibody. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced by larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the relatively large side chain(s) are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer relative to undesirable end products such as homodimers.

Bispecific antibodies can be prepared as full-length antibodies or as antibody fragments (e.g., F(ab') ₂ bispecific antibodies). Methods for preparing bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical coupling. Brennan et al., Science, 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to yield F(ab') ₂ fragments. These fragments are reduced in the presence of sodium arsenite, a complex with dithiols, to stabilize the vicinal dithiol groups and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One such derivative, Fab'-TNB, is then converted back to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibodies can be used as agents for selective immobilization of enzymes.

Fab’ fragments can also be directly isolated from E. coli and chemically coupled to produce bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217–225 (1992) describe the production of a molecule fully humanized bispecific antibody F(ab’)₂. Each Fab’ fragment is separately secreted from E. coli and subjected to direct chemical coupling in vitro to form a bispecific antibody. The resulting bispecific antibody is capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, and also “triggers” the lytic activity of human cytotoxic lymphocytes against a target human breast cancer.

Various techniques for the production and isolation of bispecific antibody fragments directly from recombinant cell culture have been described in the literature. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547–1553 (1992)). Leucine zippers from the Fos and Jun proteins are linked to the Fab’ fragments of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to yield antibody heterodimers. This method can also be used to produce antibody homodimers. The diabody technology described by Hollinger et al., Proc. Natl Acad. Sci. USA, 90:6444–6448 (1993) represents an alternative mechanism for producing bispecific antibody fragments. The fragments comprise a heavy chain variable region (VH) linked to a light chain variable region (VL) via a linker that is too short to allow pairing between the two domains of the same chain. Accordingly, the VH and VL regions of one fragment are forced to pair with the complementary VL and VH regions of the other fragment, thereby forming two antigen-binding sites. Another strategy for generating bispecific antibody fragments has also been described using single-chain Fv (sFv) dimers (see Gruber et al, J. Immunol, 152:5368 (1994)).

Antibodies having more than two valencies are also contemplated. As one non-limiting example, trispecific antibodies can be produced (Tuft et al. J. Immunol. 147: 60 (1991)).

Exemplary bispecific antibodies can bind to two different epitopes on a TIGIT polypeptide of the present invention. Alternatively, an anti-TIGIT polypeptide arm can be combined with an arm that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), so as to focus the cell's defense mechanisms toward a cell expressing a particular TIGIT polypeptide. Bispecific antibodies can also be used to deliver cytotoxic agents to cells that express a particular TIGIT polypeptide. Such antibodies have a TIGIT-binding arm and an arm that binds a cytotoxic agent or radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the TIGIT polypeptide and also binds tissue factor (TF).

Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have, for example, been proposed for targeting immune system cells to unwanted cells [U.S. Patent No. 4,676,980] and for treating HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including methods involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or thioether bond formation. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate and the reagents described, for example, in U.S. Patent No. 4,676,980.

Construction of effector function

It may be desirable to modify the antibody of the present invention with respect to its effector function in order to enhance the efficacy of the antibody in the treatment of cancer. For example, one or more cysteine residues can be introduced into the Fc region to promote the formation of an interchain disulfide bond at that site. The resulting homodimeric antibody may have enhanced internalization and/or enhanced complement-mediated cell lysis and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med., 176:1191-1195 (1992); and Shopes, B. J. Immunol., 148:2918-2922 (1992)). Homodimeric antibodies with enhanced antitumor activity can also be generated using the heterobifunctional cross-linkers described by Wolff et al. Cancer Research, 53:2560-2565 (1993)). Alternatively, the antibody can be engineered to have dual Fc regions and thus have enhanced complement-dependent lysis and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

In some embodiments, anti-TIGIT antibodies are obtained that are 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 antibody 10A7 were determined using standard techniques. The light chain sequence of the antibody is: DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQ ID NO:13) and the heavy chain sequence of this antibody is: EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15), where the complementarity determining regions (CDRs) of each chain are shown in bold. Thus, CDR1 of the light chain of antibody 10A7 has the sequence KSSQSLYYSGVKENLLA (SEQ ID NO:1), CDR2 of the light chain of antibody 10A7 has the sequence ASIRFT (SEQ ID NO:2), and CDR3 of the light chain of antibody 10A7 has the sequence QQGINNPLT (SEQ ID NO:3). CDR1 of the heavy chain of antibody 10A7 has the sequence GFTFSSFTMH (SEQ ID NO:4), CDR2 of the heavy chain of antibody 10A7 has the sequence FIRSGSGIVFYADAVRG (SEQ ID NO:5), and CDR3 of the heavy chain of antibody 10A7 has the sequence RPLGHNTFDS (SEQ ID NO:6).

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: DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14) and the heavy chain sequence of this antibody is: EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO:16), where the complementarity determining regions (CDRs) from each chain are shown in bold. Thus, CDR1 of the light chain of antibody 1F4 has the sequence RSSQSLVNSYGNTFLS (SEQ ID NO:7), CDR2 of the light chain of antibody 1F4 has the sequence GISNRFS (SEQ ID NO:8), and CDR3 of the light chain of antibody 1F4 has the sequence LQGTHQPPT (SEQ ID NO:9). CDR1 of the heavy chain of antibody 1F4 has the sequence GYSFTGHLMN (SEQ ID NO:10), CDR2 of the heavy chain of antibody 1F4 has the sequence LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and CDR3 of the heavy chain of antibody 1F4 has the sequence GLRGFYAMDY (SEQ ID NO:12).

The nucleotide sequence encoding the light chain of antibody 1F4 was determined: GATGTTGTGTTGACTCAAACTCCACTCTCCCTGTCTGTCAGCTTTGGAGATCAAGTTTCTATCTCTTGCAGGTCTAGTCAGAGTCTTGTAAACAGTTATGGGAACACCTTTTTGTCTTGGTACCTGCACAAGCCTGGCCAGTCTCCACAGCTCCTCATCTTTGGGATTTCCAACAGATTTTCTGGGGTGCCAGACAGGTTCAGTGGCAGTGGTTCAGGGACAGATTTCACACTCAAGATCAGCACAATAAAGCCTGAGGACTTGGGAATGTATTACTGCTTACAAGGTACGCATCAGCCTCCCACGTTCGGTCCTGGGACCAAGCTGGAGGTGAAA (SEQ ID NO:38) and the nucleotide sequence encoding the heavy chain of antibody 1F4: GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGAACTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCCATCTTATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAGTGGATTGGACTTATTATTCCTTACAATGGTGGTACAAGCTATAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCATCCAGCACAGCCTACATGGAG CTCCTCAGTCTGACTTCTGATGACTCTGCAGTCTATTTCTGTTCAAGAGGCCTTAGGGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO:39).

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), (SEQ ID NO:13), and (SEQ ID NO:14), (SEQ ID NO:15), (SEQ ID NO:16), and (SEQ ID NO:17), (SEQ ID NO:18), (SEQ ID NO:19), and (SEQ ID NO:20), (SEQ ID NO:21), (SEQ ID NO:22), and (SEQ ID NO:23), (SEQ ID NO:24), (SEQ ID NO:25), and (SEQ ID NO:26), (SEQ ID NO:27), (SEQ ID NO:28), and (SEQ ID NO:29), (SEQ ID NO:30), (SEQ ID NO:31), and (SEQ ID NO:32), (SEQ ID NO:33), (SEQ ID NO:34), (SEQ ID NO:35), (SEQ ID NO:36), (SEQ ID NO:37), (SEQ ID NO:38), (SEQ ID NO:39), (SEQ ID NO:40), (SEQ ID NO:41), and ( NO:12).

In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof is provided, wherein the light chain of the antibody comprises an amino acid sequence set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14).

In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof is provided, wherein the heavy chain of the antibody comprises an amino acid sequence set forth in EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO:16).

In some embodiments, there is provided an anti-TIGIT antibody or antigen-binding fragment thereof, wherein the light chain of the antibody comprises an amino acid sequence set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQS PKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), and the heavy chain of the antibody comprises an amino acid sequence set forth in EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFK GKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO:16).

In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof is provided, wherein the antibody is selected from a humanized antibody, a chimeric antibody, a bispecific antibody, a heteroconjugate antibody, and an immunotoxin.

In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof comprises at least one HVR that is at least 90% identical to an HVR shown 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 some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a light chain and/or a heavy chain comprising amino acid sequences that are at least 90% identical to the amino acid sequences set forth in DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLIYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKR (SEQ ID NO:13) or DVVLTQTPLSLSVSFGDQVSISCRSSQSLVNSYGNTFLSWYLHKPGQSPQLLIFGISNRFSGVPDRFSGSGSGTDFTLKISTIKPEDLGMYYCLQGTHQPPTFGPGTKLEVK (SEQ ID NO:14), or EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSS (SEQ ID NO:15) or EVQLQQSGPELVKPGTSMKISCKASGYSFTGHLMNWVKQSHGKNLEWIGLIIPYNGGTSYNQKFK GKATLTVDKSSSTAYMELLSLTSDDSAVYFCSRGLRGFYAMDYWGQGTSVTVSS (SEQ ID NO:16) respectively.

Agents that modulate CD226 expression and/or activity

A method of treating or slowing the progression of cancer in an individual is provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. A method of reducing or inhibiting the recurrence or progression of cancer in an individual is also provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. A method of treating or slowing the progression of an immune-related disease in an individual is also provided, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. Also provided is a method for reducing or inhibiting the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226. Also provided is a method for increasing, enhancing or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226.

For example, agents that modulate the expression and/or activity of CD226 are agents that are capable of increasing or promoting the expression and/or activity of CD226, increasing and/or promoting the interaction of CD226 with PVR, PVRL2 and/or PVRL3, and increasing and/or promoting intracellular signaling mediated by the binding of CD226 to PVR, PVRL2 and/or PVRL3. In some embodiments, agents that are capable of increasing and/or promoting the expression and/or activity of CD226 are agents that increase and/or promote the expression and/or activity of CD226. In some embodiments, agents that are capable of increasing and/or promoting the interaction of CD226 with PVR, PVRL2 and/or PVRL3 are agents that increase and/or promote the interaction of CD226 with PVR, PVRL2 and/or PVRL3. In some embodiments, agents capable of enhancing and/or stimulating intracellular signaling mediated by CD226 binding to PVR, PVRL2 and/or PVRL3 are agents that enhance and/or stimulating intracellular signaling mediated by CD226 binding to PVR, PVRL2 and/or PVRL3.

In some embodiments, the agent that modulates the expression and/or activity of CD226 is selected from an agent that inhibits and/or blocks the interaction of CD226 with TIGIT, an antagonist of the expression and/or activity of TIGIT, an antagonist of the expression and/or activity of PVR, 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 intracellular signaling mediated by TIGIT binding to PVR, an agent that inhibits and/or blocks intracellular signaling mediated by TIGIT binding to PVRL2, an agent that inhibits and/or blocks 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.

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 an 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, an agent that inhibits and/or blocks 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, an agent that inhibits and/or blocks 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, an agent that inhibits and/or blocks 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.

In some embodiments, an antagonist of TIGIT expression and/or activity comprises 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, an antagonist of PVR expression and/or activity comprises 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, an agent that inhibits and/or blocks 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

In addition to specific antigen recognition via the TCR, T cell activation is regulated by the balance of positive and negative signals from costimulatory receptors. These surface proteins are typically members of the TNF or B7 receptor superfamilies. Activating costimulatory receptors include CD226, CD28, OX40, GITR, CD137, CD27, HVEM, MICA, ICOS, NKG2D, and 2B4. Inhibitory costimulatory receptors include CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7H4, and CD96. Agonist antibodies directed against activating costimulatory molecules and blocking antibodies against negative costimulatory molecules can enhance T cell stimulation, promoting tumor lysis.

A method is provided for increasing, enhancing or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that reduces or inhibits one or more additional immune coinhibitory receptors. In some embodiments, the one or more additional immune coinhibitory receptors are selected from PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4 and CD96. In some embodiments, the one or more additional immune coinhibitory receptors are selected from PD-1, CTLA-4, LAG3 and TIM3.

Also provided is a method for increasing, enhancing or stimulating an immune response or immune function in an individual by administering to the individual an effective amount of an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that increases or activates one or more additional immune costimulatory receptors. In some embodiments, the one or more additional immune costimulatory receptors are selected from CD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D and 2B4. In some embodiments, the one or more additional immune costimulatory receptors are selected from CD226, OX-40, CD27, CD137, HVEM and GITR. In some embodiments, the one or more additional immune costimulatory receptors are selected from OX-40 and CD27.

IV Sets

In another aspect, a kit is provided 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 reduces or inhibits TIGIT expression and/or activity to treat or slow the progression of cancer in an individual or to enhance immune function in an individual having cancer. Any of the PD-1 axis binding antagonists and/or agents that reduce or inhibit TIGIT expression and/or activity described herein may be included in the kit.

In another aspect, a kit is provided comprising a PD-1 axis binding antagonist and an agent that reduces 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 reduces or inhibits TIGIT expression and/or activity to treat or slow the progression of cancer in an individual or to enhance immune function in an individual having cancer. Any of the PD-1 axis binding antagonists and/or agents that reduce or inhibit TIGIT expression and/or activity described herein may be included in the kit.

In another aspect, a kit is provided comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with a PD-1 axis binding antagonist, to treat or slow the progression of cancer in an individual or to enhance immune function in an individual having cancer. Any of the PD-1 axis binding antagonists and/or agents that reduce or inhibit the expression and/or activity of TIGIT described herein can be included in the kit.

In another aspect, a kit is provided 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 expression and/or activity of CD226 to treat or slow the progression of cancer in an individual. Any of the PD-1 axis binding antagonists and/or agents that modulate the expression and/or activity of CD226 described herein can be included in the kit.

In another aspect, a kit is provided comprising an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226, and a package insert comprising instructions for using the antagonist that binds to the PD-1 axis and the agent that modulates the expression and/or activity of CD226 to treat or slow the progression of cancer in an individual. Any of the antagonists that bind to the PD-1 axis and/or agents that modulate the expression and/or activity of CD226 described herein can be included in the kit.

In another aspect, a kit is provided comprising an agent that modulates the expression and/or activity of CD226, and a package insert comprising instructions for using the agent that modulates the expression and/or activity of CD226 in combination with a PD-1 axis binding antagonist to treat or slow the progression of cancer in an individual. Any of the PD-1 axis binding antagonists and/or agents that modulate the expression and/or activity of CD226 described herein can be included in the kit.

In another aspect, a kit is provided comprising an antagonist that binds to the PD-1 axis and a package insert containing instructions for using the antagonist that binds to the PD-1 axis in combination with an agent that modulates the expression and/or activity of CD226 to enhance immune function in an individual having cancer. Any of the PD-1 axis binding antagonists and/or agents that modulate CD226 expression and/or activity described herein may be included in the kit.

In another aspect, a kit is provided comprising an antagonist that binds to the PD-1 axis and an agent that modulates the expression and/or activity of CD226, and a package insert containing instructions for using the antagonist that binds to the PD-1 axis and the agent that modulates the expression and/or activity of CD226 to enhance immune function in an individual having cancer. Any of the PD-1 axis binding antagonists and/or agents that modulate CD226 expression and/or activity described herein may be included in the kit.

In another aspect, a kit is provided comprising an agent that modulates the expression and/or activity of CD226, and a package insert comprising instructions for using the agent that modulates the expression and/or activity of CD226 in combination with a PD-1 axis binding antagonist to enhance immune function in an individual having cancer. Any of the PD-1 axis binding antagonists and/or agents that modulate the expression and/or activity of CD226 described herein can be included in the kit.

In another aspect, a kit is provided comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with an agent that reduces or inhibits one or more immune coinhibitory receptors, to treat or slow the progression of cancer in an individual or enhance immune function in an individual having cancer. Any of the agents that reduce or inhibit the expression and/or activity of TIGIT and/or the agents that reduce or inhibit one or more immune coinhibitory receptors described herein may be included in the kit.

In another aspect, a kit is provided comprising an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that reduces or inhibits one or more additional immune coinhibitory receptors, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT and the agent that reduces or inhibits one or more additional immune coinhibitory receptors to treat or slow the progression of cancer in an individual or enhance immune function in an individual having cancer. Any of the agents that reduce or inhibit the expression and/or activity of TIGIT and/or the agents that reduce or inhibit one or more immune coinhibitory receptors described herein may be included in the kit.

In another aspect, a kit is provided comprising an agent that reduces or inhibits one or more additional immune coinhibitory receptors, and a package insert comprising instructions for using the agent that reduces or inhibits one or more additional immune coinhibitory receptors, in combination with an agent that reduces or inhibits TIGIT expression and/or activity, to treat or slow the progression of cancer in an individual or enhance immune function in an individual having cancer. Any of the agents that reduce or inhibit TIGIT expression and/or activity and/or the agents that reduce or inhibit one or more immune coinhibitory receptors described herein may be included in the kit.

In another aspect, a kit is provided comprising an agent that reduces or inhibits the expression and/or activity of TIGIT, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT, in combination with an agent that increases or activates one or more additional immune costimulatory receptors, to treat or slow the progression of cancer in an individual or enhance immune function in an individual having cancer. Any of the agents that reduce or inhibit the expression and/or activity of TIGIT and/or the agents that increase or activate one or more immune costimulatory receptors described herein may be included in the kit.

In another aspect, a kit is provided comprising an agent that reduces or inhibits the expression and/or activity of TIGIT and an agent that increases or activates one or more additional immune costimulatory receptors, and a package insert comprising instructions for using the agent that reduces or inhibits the expression and/or activity of TIGIT and the agent that increases or activates one or more additional immune costimulatory receptors to treat or slow the progression of cancer in an individual or enhance immune function in an individual having cancer. Any of the agents that reduce or inhibit the expression and/or activity of TIGIT and/or the agents that increase or activate one or more immune costimulatory receptors described herein may be included in the kit.

In another aspect, a kit is provided comprising an agent that increases or activates one or more additional immune costimulatory receptors, and a package insert comprising instructions for using the agent that increases or activates one or more additional immune costimulatory receptors, in combination with an agent that decreases or inhibits the expression and/or activity of TIGIT, to treat or slow the progression of cancer in an individual or enhance immune function in an individual having cancer. Any of the agents that decrease or inhibit the expression and/or activity of TIGIT and/or the agents that increase or activate one or more immune costimulatory receptors described herein may be included in the kit.

In some embodiments, the kit comprises a container comprising one or more antagonists that bind to the PD-1 axis and agents that reduce or inhibit the expression and/or activity of TIGIT described herein. In some embodiments, the kit comprises a container comprising one or more antagonists that bind to the PD-1 axis and agents that modulate the expression and/or activity of CD226 described herein. In some embodiments, the kit comprises a container comprising one or more agents that reduce or inhibit the expression and/or activity of TIGIT and agents that reduce or inhibit one or more additional immune co-inhibitory receptors described herein. In some embodiments, the kit comprises a container comprising agents that reduce or inhibit the expression and/or activity of TIGIT and agents that increase or activate one or more additional immune costimulatory receptors described herein. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., single-chamber or dual-chamber syringes), and tubes. The container may be made of various materials, such as glass or plastic. In some embodiments, the kit may have a label (e.g., on or associated with the container) or a package insert. The label or package insert may indicate that the compound contained therein may be suitable and intended for treating or slowing the progression of cancer in an individual, or for enhancing immune function in an individual with cancer. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

Examples

The present invention may be better understood by reference to the following examples, which are given by way of illustration and are not intended to limit the scope of the invention.

Example 1: TIGIT is expressed at high levels on depleted CD8 ⁺ and CD4 ⁺ T cells, and its expression correlates with PD-1 expression

To confirm that CD8⁺T cells are competent to express TIGIT following in vitro stimulation with CD8⁺MACS-enriched C57BL6/J mouse splenic T cells were stimulated with plate-immobilized anti-CD3 and anti-CD28 antibodies for 24–48 h in vitro. Flow cytometry was used to determine TIGIT expression. Consistent with previous data on TIGIT expression in CD4⁺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)), mouse CD8⁺T cells expressed TIGIT within 48 h of in vitro stimulation (Fig. 1A).

To assess TIGIT expression by activated CD8 ⁺ T cells in vivo, C57BL6/J mice were infected with the Armstrong strain of lymphoblastic choriomeningitis virus (LCMV), and splenocytes were analyzed 7 days post-infection. Briefly, to recapitulate acute infection, mice were infected intravenously with 2 × ¹⁰⁶ plaque-forming units (PFU) of the Armstrong strain of LCMV. Flow cytometry was used to determine TIGIT expression by naïve (CD44 ^low CD62L ^high ) and CD8 ⁺ and CD4 ⁺ effector memory T cells (CD44 ^high CD62L ^low ). At the peak of the T cell response to LCMV, a subset of CD4 ⁺ effector memory T cells and almost all CD8 ⁺ _T cells expressed high levels of TIGIT (Fig. _1B ). Flow cytometry was used to determine TIGIT expression by PD- ^1high and PD- ^1low effector memory CD8 ⁺ T cells. Interestingly, TIGIT expression correlated almost perfectly with PD-1 expression (Figure 1C).

Because PD-1 is associated with T cell exhaustion, TIGIT expression on chronically stimulated T cells was determined. Briefly, to generate chronic infection, mice were infected intravenously with 2 × 10 ⁶ PFU of LCMV clone 13 and treated with 500 μg and 250 μg of depleting anti-CD4 antibody (clone GK1.5) for 3 days before and 4 days after infection, respectively. Where indicated, mice infected with LCMV clone 13 were injected intraperitoneally with 200 μg of isotype control antibody, 200 μg of anti-PD-L1 antibody, and/or 500 μg of anti-TIGIT antibody 3 times weekly from day 28 to day 42 after infection. Splenocytes were analyzed at 42 days post infection. Flow cytometry was used to determine TIGIT expression by naïve (CD44 ^low CD62L ^high ), central memory CD8 ⁺ and CD4 ⁺ T cells (CD44 ^high CD62L ^low ), and effector memory CD8 ⁺ and CD4 ⁺ T cells (CD44 ^high CD62L ^low ). In fact, in mice chronically infected with LCMV clone 13, TIGIT was expressed at high levels predominantly on PD-1 ^high T cells but not on naïve PD-1 ^low T _cells or central memory T cells (Fig. 1D).

Example 2: Role of TIGIT in T cell depletion in TIGIT-deficient mice

To characterize the role of TIGIT in T cell depletion, mice in which TIGIT was conditionally deleted in T cells were generated (TIGIT ^fl/fl CD4-cre ⁺ (CKO), Fig. 2). Briefly, CD4-cre mice and TIGIT ^LoxP/LoxP mice were generated from C57BL/6J mice using standard methods and cross-breeding. A quality-tested ES cell line (Art B6/3.6 (genetic background: C57BL/6 NTAC) was cultured on a mitotically inactivated mouse embryonic fibroblast feeder layer in ES cell culture medium containing leukemia inhibitory factor and fetal bovine serum. Cells were electroporated with a linearized targeting DNA vector according to Taconic Artemis' Standard Operating Procedures. Selective medium containing G418 and ganciclovir were used as mechanisms to enrich for homologously recombinant clones. Resistant ES cell colonies (ES clones) with distinguishable morphology were isolated at day 8 post-transfection and analyzed by Southern blotting and/or PCR in a primary screen. Homologously recombinant ES cell clones were expanded and were frozen in liquid nitrogen after extensive molecular validation. The neo cassette was removed using flpE recombinase prior to microinjection into female BL/6 albino mice used as donors. Chimeric progeny were generated and tail material was tested by PCR for germline transmission. TIGIT expression was abolished with 96% efficiency from T cells in TIGIT ^fl/FL CD4 ^cre mice.

Mice lacking TIGIT expression on T cells were induced to respond to acute infection with the Armstrong LCMV ^strain in a similar manner to wild ^- type mice (Fig. 3).

To assess the effect in a chronic infection format, TIGIT ^fl/fl CD4-cre ^- (WT) and TIGIT ^fl/fl CD4-cre ⁺ (CKO) mice were rapidly depleted of CD4 ⁺ T cells and infected with LCMV clone 13. Viral titers in splenocytes and liver were determined 42 days after infection. During chronic infection with LCMV clone 13, significantly more CD8 ⁺ and CD4 ⁺ T cells in TIGIT ^fl/fl CD4-cre ⁺ (CKO) mice were competent for interferon-gamma (IFN-γ) production compared with T cells from littermates of wild-type (TIGIT ^fl/fl CD4-cre ^- (WT)) mice (82-86% increase, P<0.01, Figs. 4A-4D). Furthermore, viral load was significantly reduced in chronically infected TIGIT ^fl/fl CD4-cre ⁺ (CKO) mice (68% reduction, P < 0.0001, Fig. 4E).

These results suggest that TIGIT plays an important role in regulating T cell activity and response during chronic immune responses, such as chronic viral infection, and that TIGIT may regulate effector function, in particular the competence to produce effector cytokines such as IFNγ and TNFα, by chronically stimulated or exhausted CD8 ⁺ and CD4 ⁺ T cells.

Example 3: TIGIT and PD-1 synergistically regulate exhausted T cell effector function in vivo

Because TIGIT expression was closely correlated with PD-1 expression, particularly on CD8 ⁺ T cells during acute and chronic viral infection (Fig. 1), blocking TIGIT and PD-1 in combination may restore T cell effector function to a higher level than could be achieved by blocking each coreceptor individually.

To test this hypothesis, C57BL6/J mice were rapidly depleted of CD4 ⁺ T cells and infected with LCMV clone 13. To establish chronic infection, mice were infected intravenously with 2 × ¹⁰⁶ PFU of LCMV clone 13 and treated with 500 μg and 250 μg of depleting anti-CD4 antibody (clone GK1.5) for 3 days before and 4 days after infection, respectively. Where indicated, mice infected with LCMV clone 13 were injected intraperitoneally with 200 μg of isotype-matched control antibody, 200 μg of anti-PD-L1 antibody, and/or 500 μg of anti-TIGIT antibody 3 times weekly from days 28 to 42 post-infection. Treatment was initiated at 28 days post-infection because the T cell response was predominantly 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 Oct;27(4):670-84). Viral titers in splenocytes and liver were determined 42 days post-infection.

In these mice, treatment with anti-PD-L1 antibody induced stronger CD8 ⁺ T cell activation compared with treatment with isotype-matched control antibodies (88% increase, P < 0.0001, Fig. 4F), as reported previously (Barber, DL , et al., Restoring function in exhausted CD8 T cells during chronic viral infection. Nature, 439, 682–687 (2006)). Treatment with anti-TIGIT antibodies alone or in combination with anti-PD-L1 antibodies had no detectable effect on CD8 ⁺ T cell activation (Fig. 4F). Similarly, blocking PD-1 alone resulted in a modest increase in CD8 ⁺ T cell competence to produce cytokines, whereas blocking TIGIT alone had no effect (Fig. 4G). However, the frequency of IFNγ-producing CD8 ⁺ T cells was dramatically increased in mice treated with both anti-TIGIT and anti-PD-L1 antibodies, and to a significantly greater extent than that observed in mice treated with anti-PD-L1 antibodies alone (Fig. 4G, 93% increase, P = 0.0050). A similar effect was observed for CD4 ⁺ T cells (Fig. 5). As also shown in Fig. 3I, co-blockade of TIGIT/PD-L1 significantly enhanced CD8 ⁺ T cell effector function but not CD4 ⁺ T cell effector function in mice compared with mice treated with anti-PD-L1 antibody alone. Similar effects were also observed on T cell expansion and effector function of LCMV gp33 antigen-specific T cells (Fig. 31). These results demonstrate a strong synergy between PD-1 and TIGIT on exhausted CD8 ⁺ T cells and indicate that TIGIT specifically regulates CD8 ⁺ T cell competence for cytokine production and effector function.

Consistent with these results, LCMV viral load was modestly reduced in mice treated with anti-PD-L1 antibody alone, was not reduced in mice treated with anti-TIGIT antibody alone, and was significantly reduced in mice treated with both anti-TIGIT and anti-PD-L1 antibodies (68% reduction in viral titer with anti-PD-L1 antibody treatment, P = 0.0004; 92% reduction in viral titer with anti-TIGIT antibody + anti-PD-L1 antibody treatment, P < 0.0001, Fig. 4H). These data demonstrate a high synergy between the inhibitory effects of PD-1 and TIGIT, suggesting that, unlike PD-1, TIGIT is not a broad inhibitor of effector T cell activation but plays a minor role in limiting T cell competence for cytokine production and effector function.

Example 4: TIGIT expression is elevated in human breast cancer and its expression correlates with the expression of CD8 and inhibitory co-receptors

T cell exhaustion is also a major immunological hallmark of cancer, with large numbers of tumor-infiltrating lymphocytes (TILs), high levels of inhibitory co-receptors, and a lack of ability to produce effector cytokines (Wherry EJ, T cell exhaustion. Nature immunology , 12, 492–499 (2011); Rabinovich GA, Gabrilovich D. & Sotomayor EM, Immunosuppressive strategies that are mediated by tumor cells, Annual review of immunology, 25, 267–296 (2007)).

To determine the extent to which TIGIT inhibits TIL effector function, breast cancer gene expression data from the Cancer Genome Atlas (CGAN) project (Network, C.G.A. Comprehensive molecular portraits of human breast tumors, Nature, 490, 61–70 (2012)) were analyzed using microarray analysis.

TIGIT expression was significantly increased in malignant breast tumors overall (135% increase compared with normal breast samples, P = 6× ^10-12 , Fig. 6A) and in the four main molecular subtypes of breast cancer (Fig. 6A) (Perou CM , et al., Molecular portraits of human breast tumors, 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)). TIGIT expression was highly correlated with CD3ε expression, respectively, with TIL expression ( ^R2 = 0.61, Fig. 6B). Interestingly, TIGIT expression was highly correlated with CD8α but not with CD4, or only moderately correlated with CD4, suggesting that TIGIT primarily regulates CD8 ⁺ TIL function (CD8α, R ² = 0.80; CD4, R ² = 0.42; Fig. 6C).

Given the co-expression of TIGIT and PD-1 in chronic viral infection, the correlation of PD-1 and other inhibitory co-receptors with TIGIT in breast cancer was also assessed. The correlation between TIGIT and PD-1, CTLA4, and LAG3 was very high (PD-1, R ² = 0.87; CTLA4, R ² = 0.76; LAG3, R ² = 0.80; Fig. 6D). Collectively, these data suggest that TIGIT was expressed by TILs, particularly CD8 ⁺ T cells, and that it can suppress their function.

Example 5: TIGIT and PD-1 inhibit antitumor T cell responses

To better characterize TIGIT from TIL cells in mice, BALB/c mice were inoculated with CT26 colorectal carcinoma cells. Briefly, BALB/c mice were inoculated subcutaneously with 1 x 10 ⁵ CT26 colon carcinoma cells suspended in Matrigel (BD Biosciences) in the right side of the thorax. Two weeks later, mice bearing approximately 200 mm ³ tumors were randomly assigned to treatment groups and injected intraperitoneally with 35 mg/kg of isotype-matched control antibody, anti-PD-L1 antibody, and/or anti-TIGIT antibody 3 times per week for 3 weeks. Tumor size was determined twice per week using calipers. Animals with tumors that ulcerated/necrotized or were larger than 2000 mm ³ were euthanized. Splenocytes and tumor-infiltrating lymphocytes (TILs) were analyzed 14 days after inoculation, when tumors had reached approximately 200 mm ³ in size.

Consistent with the TIGIT expression data in human tumors (Fig. 6), CD8 ⁺ and CD4 ⁺ CT26 TILs expressed TIGIT at high levels (Figs. 7A–7B). Furthermore, consistent with the chronic viral infection data, TIGIT expression by TILs closely correlated with the expression of other inhibitory co-receptors, including PD-1 (Figs. 7A–7B) and Tim-3 (Fig. 8). A similar TIGIT expression pattern was found in MC38 colon carcinoma (Fig. 9).

To test the physiological significance of TIGIT expression in the context of the antitumor immune response, BALB/c mice bearing CT26 tumors (approximately 200 ^mm3 in size) were treated with 200 μg of isotype-matched control antibodies, 200 μg of anti-PD-L1 antibodies, 500 μg of anti-TIGIT antibodies, or 200 μg of anti-PD1 antibodies + 500 anti-TIGIT antibodies for 3 weeks.

CT26 tumor growth was slightly inhibited by treatment with either anti-TIGIT or anti-PD-L1 antibodies alone, both of which showed a nonsignificant increase in median survival at day 3 (Figs. 7C–7D). However, combination therapy with both anti-PD-L1 and anti-TIGIT antibodies resulted in a dramatic reduction in tumor growth (75% reduction in median tumor volume at day 16, P<0.0001, Fig. 7C and Fig. 10). Furthermore, 70% of mice treated with the combination of anti-TIGIT and anti-PD-L1 antibodies experienced complete and durable tumor remission and remained alive throughout the study period, even in the absence of additional antibody treatment (Figs. 7C–7D). These effects were also observed in mice bearing tumors treated with a combination of blocking antibodies against TIGIT and PD-1.

To investigate immunity to CT26 tumor cells in these surviving mice, approximately 60 days after the initial inoculation, mice in complete remission (CR) that had received anti-TIGIT + anti-PD-L1 antibodies and naïve BALB/c mice were re-inoculated with CT26 cells in the left side of the thorax (where they had not previously been inoculated). These mice were also inoculated with 1× ¹⁰⁵ EMT6 mammary carcinoma cells in Matrigel into the mammary fat pad. Tumor size was assessed twice weekly. Animals with tumors that ulcerated/necrotized or were larger than 2000 ^mm3 were euthanized.

As shown in Figure 7E, both tumor types grew vigorously in naïve control mice, but EMT6 tumors grew only in mice that had previously cleared CT26 tumors. These results indicate that co-blocking TIGIT and PD-1 during tumorigenesis conferred a state of specific immunity to CT26 tumor cells.

To determine whether the efficacy of TIGIT/PD-L1 co-blockade was mediated by CD8 ⁺ T cells, mice bearing CT26 tumors were depleted of CD8 ⁺ T cells using depleting antibodies at the start of treatment with anti-TIGIT antibody and anti-PDL1 antibody. Mice treated with anti-TIGIT antibody and anti-PD-L1 antibody failed to reject CT26 tumor cells when CD8 ⁺ T cells were depleted at the start of treatment (1532% increase in mean tumor volume at 17 days post-treatment, P = 0.0004, Figs. 32A–32B). Furthermore, CD8 ⁺ T cell depletion impaired the ability of previously treated CR mice to control CT26 tumor re-inoculation (Fig. 32C). Taken together, these results demonstrate that anti-TIGIT antibody and anti-PD-L1 antibody functioned through CD8 ⁺ T cells to induce effective primary and secondary antitumor immune responses.

To determine whether tumor cell expression of PVR is dispensable for the efficacy of TIGIT/PD-L1 co-blockade, wild-type BALB/c mice were inoculated with wild-type CT26 tumors (which express PVR) or PVR-deficient CT26 tumors. Briefly, wild-type CT26 tumors were transiently transfected with a nucleic acid that reduced PVR expression. Approximately two weeks after transfection, CT26 cells were subcloned based on loss of PVR expression using flow cytometry and qPCR. When tumors reached 150–200 mm ³ in size, mice were treated with anti-TIGIT antibody and anti-PD-L1 antibody, or isotype-matched control antibodies. Mice treated with anti-TIGIT antibody and anti-PD-L1 antibody were able to reject wild-type and PVR-deficient tumors, in contrast to tumor-bearing mice treated with control antibodies (Fig. 33). These results showed that anti-TIGIT antibody and anti-PD-L1 antibody function independently of PVR expressed in the tumor.

The efficacy of TIGIT/PD-L1 co-blockade was also tested and confirmed in the MC38 tumor model. Wild-type C57BL6/J mice were inoculated subcutaneously with syngeneic MC38 colorectal carcinoma cells and the established tumors were treated with a combination of anti-TIGIT and PD-L1 blocking antibodies as described above. In contrast to the CT26 model, treatment with anti-PD-L1 antibody alone was sufficient to induce a complete response in some mice (Fig. 25). However, as in the CT26 model, treatment of mice bearing MC38 tumors with both anti-TIGIT and anti-PD-L1 antibodies synergistically prevented tumor growth and induced tumor clearance in the majority of mice (Fig. 26). These effects were also observed in mice inoculated with syngeneic EMT6 mammary carcinoma cells (Fig. 34).

The results showed that combined blockade of TIGIT and PD-1 could induce a robust and antigen-specific antitumor immune response. These results also suggest that adaptive antitumor responses were fully functional and reactivated in treated mice.

To assess the functional effects of TIGIT and PD-1 blockade on tumor-infiltrating lymphocytes themselves, mice were inoculated with CT26 tumor cells and treated with anti-TIGIT antibody and/or anti-PD-L1 antibody as described above. Seven days after the start of treatment, tumors and tumor-draining lymph nodes were collected for flow cytometric analysis.

Tumor-infiltrating and resident CD4 ⁺ T cells in tumor-draining lymph nodes produced low levels of IFNγ and did not produce this cytokine when TIGIT/PD-1 was blocked (Fig. 11). However, tumor-infiltrating CD8 ⁺ T cells from mice treated with both anti-TIGIT and anti-PD-L1 antibodies, but not cells from mice treated with either anti-TIGIT or anti-PD-L1 antibodies, were significantly more competent in IFNγ production when stimulated in vitro (174% increase compared with controls, P = 0.0001, Fig. 13D). Similar results were obtained for CD8 ⁺ TILs in terms of TNFγ production (Fig. 12).

Interestingly, mice treated with anti-TIGIT antibody alone or with anti-PD-L1 antibody or with both antibodies had increased cytokine production competence of resident CD8 ⁺ T cells in tumor-draining lymph nodes (75-113% increase, P<0.001, Fig. 13), suggesting that resident CD8 ⁺ T cells in tumor-draining lymph nodes were inhibited to a lesser extent than their tumor-infiltrating counterparts. The accumulation and phenotypic activation of tumor-infiltrating and resident CD8 ⁺ T cells and CD4 ⁺ T cells in tumor-draining lymph nodes were unaffected and slightly increased after single-antibody treatment and dual-antibody treatment, respectively (Figs. 12-13). The frequencies of CD8 ⁺ T cells co-producing IFNγ/TNFα in tumors and tumor-draining lymph nodes showed similar patterns (Fig. 35A-35B).

Therefore, while blockade of either TIGIT or PD-L1 alone was sufficient to enhance CD8 ⁺ T cell effector function in tumor-draining lymph nodes, blockade of both receptors was required to restore the function of exhausted CD8 ⁺ T cells within the tumor itself, consistent with the notion that the tumor microenvironment is highly immunosuppressive.

Example 6: Coexpression of TIGIT with CD226 CD8 ⁺ T cells infiltrating the tumor

TIGIT competes with the costimulatory receptor CD226 for binding to the 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 in 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 through its effect on CD226 activity.

To assess whether there is a relationship between CD226 and TIGIT in inhibiting T cell responses, TIGIT and CD226 expression was determined on tumor infiltrating CD8⁺T cells.

As shown in Figure 14, C57BL6/J mice were inoculated with MC38 colorectal carcinoma cells. Splenocytes and tumor-infiltrating lymphocytes (TILs) were analyzed using FACS analysis approximately 14 days after inoculation, when tumors reached a size of approximately 200 mm ³ . Representative histogram of CD226 expression by splenic B cells (gray), splenic CD8 ⁺ T cells (blue), and tumor-infiltrating TIGIT ⁺ CD8 ⁺ T cells (red). Data represent the results of two independent experiments; n = 5. Figure 14 shows that splenic CD8 ⁺ T cells expressed high levels of CD226 and tumor-infiltrating TIGIT ⁺ CD8 ⁺ T cells also expressed high levels of CD226. Data show that TIGIT and CD226 are coordinately expressed on murine tumor-infiltrating CD8 ⁺ T cells and can regulate each other's function on CD8 ⁺ T cells. This observation is similar to that seen in activated CD8 ⁺ T cells and NK cells, which also coexpress TIGIT and CD226.

Example 7: Coimmunoprecipitation of TIGIT with CD226 on transfected cells

To determine the extent to which TIGIT interacts with CD226 on the cell surface, cells were cotransfected with human TIGIT and human CD226 and immunoprecipitated. Briefly, COS 7 cells in 15 cm dishes were cotransfected with expression plasmids containing cDNAs of the tagged TIGIT-HA (5 ng) or CD226-Flag (10 ng) proteins, or a control plasmid (pRK). At 23 h posttransfection, cells were washed with PBS and collected in 4 ml ice-cold PBS, centrifuged at 300×g for 5 min, and the cell pellets were resuspended in 2 ml lysis buffer at 4°C. Cells were lysed for 50 min with vortexing every 15 min and then centrifuged at 10,000×g for 15 min at 4°C. The resulting supernatant was preclarified with 160 μl of CL6B Sepharose suspension by rotation for 30 min at 4°C and centrifuged for 2 min at 3,000×g. The supernatant was divided equally into two tubes and immunoprecipitated with anti-HA or anti-flag using standard procedures. Immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting. Western blots were probed with anti-Flag-HRP or anti-HA-HRP.

As shown in Figure 15, the anti-TIGIT antibody precipitated CD226 and the anti-CD226 antibody precipitated TIGIT, demonstrating that TIGIT and CD226 are in physical contact on the cell surface.

Example 8: TIGIT and CD226 interact on CD8 ⁺ T cells

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 assessed. Briefly, MACS-enriched splenic CD8 ⁺ T cells from C57BL6/J mice were stimulated with plate-fixed anti-CD3 and anti-CD28 antibodies and recombinant IL-2 for 48 h and lysed. Cell lysates were immunoprecipitated with anti-TIGIT antibody and probed with anti-CD226 antibody. Figure 16 shows that TIGIT and CD226 interact in activated primary CD8 ⁺ cells, as both were detected in the coimmunoprecipitate. These data indicate 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 FRET) analysis fluorescent inductive-resonant transfer energy)

TR-FRET methodology was used to assess whether there is a molecular interaction between TIGIT and CD226. FRET (fluorescence inductive resonance energy transfer) is based on the transfer of energy between two fluorophores, a donor and an acceptor, when they are in close proximity to each other. Molecular interactions between biomolecules can be assessed by binding each partner to a fluorescent label and determining the level of energy transfer. When the two compounds come close enough to each other, excitation of the donor by the energy source "triggers" the transfer of energy to the acceptor, which in turn emits specific fluorescence at a specific wavelength. Due to these spectral properties, the donor-acceptor complex can be detected without the need for physical separation from unbound partners. The combination of time-resolved FRET (TR) measurement allows for signal purification from background fluorescence. This is usually accomplished by introducing a time delay between excitation of the system and fluorescence measurement to allow the signal to be cleared of all non-specific short-lived emissions.

Using TR-FRET, it was shown that TIGIT and CD226 induced FRET when expressed in the same cell, indicating a molecular interaction between 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. After 24 h, cells were labeled with 100 nM benzylguanine donor-conjugated SNAP-Lumi-4Tb (Cisbio) and 1 μM benzylguanine donor-conjugated SNAP-A647 (New England Biolabs) diluted in DMEM with 10% FCS for 1 h at 37°C under 5% CO ₂ . After three washes with PBS, the FRET signal was recorded at 665 nm for 400 μs after a 60 μs delay after 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 15 min of incubation. The FRET ratio was calculated as the FRET intensity divided by the donor emission at 620 nm. FRET intensity was represented by: (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).

As shown in Figure 17, TIGIT was able to directly disrupt and induce dissociation of CD226 homodimers. As shown in Figure 17A, dissociation of Flag-ST-CD226 homodimers was observed with increasing concentrations of HA-TIGIT, as demonstrated by the decrease in the Flag-ST-CD226 ratio measured in COS-7 cells expressing a constant amount of Flag-ST-CD226 and increasing concentrations of HA-TIGIT. However, as shown in Figure 17b, when anti-TIGIT antibody was added to the cell culture, it blocked the ability of TIGIT and CD226 to associate. This is illustrated by the lack of decrease in the 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 (Figures 17A and 17B).

Using TR-FRET methodology, the ability of TIGIT to associate with CD226 was also demonstrated and is shown in Fig. 17C and 17D. Briefly, after SNAP-labeling with 1 μM donor-conjugated Flag-ST-A647 (see above), cells were washed three times with PBS and incubated with 2 nM donor-conjugated anti-HA Lumi-4Tb (Cisbio) diluted in PBS + 0.2% BSA for 2 h at room temperature. The FRET signal was recorded, the FRET intensity was represented by: (signal at 665 nm from cells labeled with SNAP acceptor and anti-HA donor) - (signal at 665 nm from the same batch of transfected cells labeled with SNAP acceptor and anti-HA donor only).

As shown in Figure 17C, Flag-ST-CD226 was observed to associate with HA-TIGIT, as evidenced by the increasing FRET intensity between Flag-ST-CD226 and HA-TIGIT measured in COS-7 cells expressing a constant amount of Flag-ST-CD226 and increasing concentrations of HA-TIGIT. Upon addition of the anti-TIGIT antibody, the FRET intensity between Flag-ST-CD226 and HA-TIGIT was decreased, as shown in Figure 17D, suggesting that the interaction of TIGIT with CD226 may be blocked by the blocking anti-TIGIT antibody.

To confirm cell surface expression of Flag-ST-CD226 and HA-TIGIT in FRET experiments, ELISAs with anti-Flag and anti-HA antibodies were performed on intact COS-7 cells expressing the indicated tagged constructs. Briefly, COS7 cells were fixed in 4% paraformaldehyde, washed twice, and blocked with phosphate-buffered saline + 1% fetal calf serum (FCS). Cells were then incubated with monoclonal anti-HA antibody (clone 3F10, Roche Applied Science) or monoclonal anti-Flag M2 antibody (Sigma), and conjugated with horseradish peroxidase. After washing, the cells were incubated with SuperSignal ELISA substrate (Pierce) and chemiluminescence was measured on a Safire2 plate reader (Tecan). The specific signal was calculated by subtracting the signal recorded for control transfected cells. As shown in Figure 18, cell surface expression of CD226 and TIGIT was confirmed in the ELISA assay.

To confirm that the TIGIT:CD226 interaction was not due to binding to PVR, Flag-ST-CD226 and HA-TIGIT (WT) or HA-TIGIT Q56R were prepared as described in Stengel et al., (2012) PNAS, 109(14):5399–5904, and FRET ratios were determined as described. As shown in Figure 24, WT TIGIT and Q56R TIGIT bound CD226 with similar efficiency.

These data not only demonstrate that CD226 and TIGIT are associated as complexes, but also that anti-TIGIT antibody can disrupt these interactions and that the TIGIT:CD226 interaction is not mediated by binding to PVR. The data support a role for TIGIT in limiting CD226-mediated T cell activation and suggest that effects on CD226 activity may be an important mechanism by which TIGIT inhibits T cell responses and activity.

Example 10: CD226 blockade abolishes the effects of TIGIT/PD-L1 blockade in vivo

To investigate the physiological significance of the interaction between CD226 and TIGIT, mice were chronically infected with LCMV clone 13 and then treated with anti-TIGIT + anti-PD-L1 antibodies in the absence or presence of blocking anti-CD226 antibodies. Briefly, C57BL6/J mice were acutely depleted of CD4 ⁺ T cells and infected with LCMV clone 13. For chronic infection, mice were infected intravenously with 2 × 10 ⁶ PFU of LCMV clone 13 and treated with 500 μg and 250 μg of depleting anti-CD4 antibody (clone GK1.5) for 3 days before and 4 days after infection, respectively. Where indicated, mice infected with LCMV clone 13 were injected intraperitoneally with 200 μg of isotype-matched control antibody, 500 μg of anti-CD226 antibody, 200 μg of anti-PD-L1 antibody+500 μg of anti-TIGIT antibody, or 500 μg of anti-CD226 antibody+200 μg of anti-PD-L1 antibody+500 μg of anti-TIGIT antibody 3 times weekly starting on days 28–42 post-infection. Treatment was initiated at 28 days post-infection because the T cell response was predominantly 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 Oct;27(4):670-84). Viral titers in splenocytes and liver were determined 42 days post-infection.

In these mice, treatment with anti-CD226 antibody alone had limited effect on CD8 ⁺ T cell incidence, activation, or cytokine competence (Figs. 19A–19C). However, treatment with anti-CD226 antibody significantly abolished the enhancement of CD8 ⁺ T cell activation and IFNg production observed in mice treated with anti-PD-L1 antibody+anti-TIGIT antibody (59% and 58% reduction, respectively, P <0.001; Figs. 19B–19D).

Consistent with these results, LCMV viral load was significantly higher in mice treated with anti-CD226 antibody+anti-PD-L1 antibody+anti-TIGIT antibody than in mice treated with anti-PD-L1 antibody+anti-TIGIT antibody alone (272% increase, P < 0.001, Fig. 19D).

These data suggest that the primary mechanism by which TIGIT limits chronic T cell responses is through interference with CD226-mediated costimulation. The data reveal a previously unknown role for TIGIT in interacting with CD226 and disrupting CD226 dimerization, leading to a reduction or loss of a key costimulatory signal on CD8⁺T cells. The data indicate that interference with CD226-mediated T cell costimulation may be a major mechanism by which TIGIT limits chronic T cell responses, such as those seen in cancer or chronic viral infection. The findings also provide a key parameter for anti-TIGIT antibodies designed to restore effector function to chronically stimulated or depleted CD8⁺or CD4⁺T cells by influencing the ability of TIGIT to interact with CD226 and/or the ability of TIGIT to disrupt CD226 dimerization.

Materials and methods

Mice. C57BL/6J and BALB/c mice were obtained from the Jackson Laboratory and Charles River Laboratories. CD4 ^cre mice and TIGIT ^loxP/LoxP mice were generated from C57BL/6J mice using standard methods and cross-breeding. TIGIT expression by T cells in TIGIT ^loxP/loxP CD4 ^cre mice was abolished with 96% efficiency.

Flow cytometry. Single cell suspensions of spleen, lymph nodes, and tumor were obtained by gentle mechanical disruption. Surface staining was performed using commercially available antibodies to CD4, CD8, CD44, CD62L, PD-1 (eBiosciences), and CD226 (BioLegend). Anti-TIGIT antibodies were obtained from Genentech as described previously (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 instructions (Molecular Probes).

For intracellular cytokine staining (ICS), cells were stimulated for 4 h with 20 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 μM ionomycin (Sigma) in the presence of 3 μg/ml brefeldin A (eBiosciences). Following stimulation, cells were stained for surface markers as described and fixed and permeabilized with FoxP3 fixation buffer (eBioscience) according to the manufacturer's directions. Fixed cells were stained with antibodies to TNFγ and IFNα (eBiosciences).

Blocking antibodies . Blocking monoclonal IgG2a anti-TIGIT antibody (clone 10A7, reactive against murine and human TIGIT) was generated as described previously and cloned into the murine IgG2a backbone. Blocking monoclonal IgG2a anti-PD-L1 antibody (clone 25A1) was generated by immunization of PDL1 ^-/- mice with the PD-1-Fc fusion protein and cloned into the murine IgG2a backbone. Clone 25A1 was modified with previously described mutations abolishing binding to Fcγ receptors. Blocking monoclonal IgG2a anti-CD226 antibody (clone 37F6) was generated by immunization of hamsters with recombinant murine CD226 and cloned into the murine IgG2a backbone. These antibodies were also used in the tests described in other examples provided here.

Viral infections . For acute infection, mice were infected intravenously with 2 × 10 ⁶ plaque-forming units (PFU) of the Armstrong LCMV strain. For chronic infection, mice were infected intravenously with 2 × 10 ⁶ PFU of LCMV clone 13 and treated with 500 μg and 250 μg of depleting anti-CD4 antibodies (clone GK1.5) for 3 days before and 4 days after infection, respectively. Where indicated, mice infected with LCMV clone 13 were injected intraperitoneally with 200 μg of isotype-matched control antibodies, 200 μg of anti-PD-L1 antibodies, and/or 500 μg of anti-TIGIT antibodies 3 times weekly from days 28 to 42 after infection.

Virus titer assay . MC57 cell monolayers were cultured overlaid with 1% methylcellulose and infected with serial dilutions of liver homogenate from LCMV-infected mice. At 72 h post-infection, cells were fixed in 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 o-phenylenediamine (OPD, Sigma).

Bioinformatics. Breast cancer gene expression data using microarray analysis were obtained from The Cancer Genome Atlas (CGAN) project (Network, CGA Comprehensive molecular portraits of human breast tumors, Nature, 490, 61–70 (2012)). Microarray data were processed and normalized using the R programming language (http://r-project.org) and the limma Bioconductor package (http://bioconductor.org). Biological microarray intensity values from each channel were pre-processed using the normal + exponential distribution correction method as described previously in ref. 22. The corrected intensity values were then normalized using quantile normalization as described previously in ref. 23. Normalized log-values were calculated by subtracting the reference channel from the test channel for each microarray. The data were further filtered using a non-specific filter as described previously in reference 24, removing probes that did not map to known genes, and reducing the database to one probe per gene. For differential expression analysis, moderated t-statistics were calculated using the limma package as described previously (Smyth GK, Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology, 3, Article 3 (2004)). Pearson correlation coefficients were used to assess correlation.

CT26 Colon Carcinoma . BALB/c mice were inoculated subcutaneously with 1× ¹⁰⁵ CT26 colon carcinoma cells suspended in Matrigel (BD Biosciences) in the right thorax. Two weeks later, mice bearing approximately 200 ^mm3 tumors were randomly assigned to treatment groups and injected intraperitoneally with 35 mg/kg of isotype-matched control antibody, anti-PD-L1 antibody, and/or anti-TIGIT antibody 3 times per week for 3 weeks. Tumor size was assessed twice per week using calipers. Animals with tumors that ulcerated/necrotized or were larger than 2000 ^mm3 were euthanized.

EMT6 mammary carcinoma . BALB/c mice were inoculated subcutaneously into the fat pad of the fourth mammary gland with 1× ¹⁰⁵ syngeneic EMT6 mammary carcinoma cells in Matrigel (BD Biosciences). Two weeks later, mice bearing 150–200 ^mm3 tumors were randomly assigned to treatment groups and injected intraperitoneally with 35 mg/kg isotype-matched control antibody, anti-PD-L1 antibody, and/or anti-TIGIT antibody 3 times per week for 3 weeks. Tumor size was determined twice per week using calipers, and tumor volume was calculated using a modified ellipsoid formula, ½ × (length × width ² ). Animals whose tumors decreased in size to 32 mm ³ or less were considered complete responders (CR). Animals whose tumors increased in size to more than 2000 mm ³ were considered disease progression and were euthanized. Animals whose tumors ulcerated/necrotized before disease progression or CR were euthanized and excluded from the study.

CT26 booster injection . Where indicated, BALB/c mice previously inoculated with CT26 colon carcinoma cells as described above were boosted with CT26 cells in the left side of the thorax (where they had not previously been inoculated). These mice were also inoculated with 1 x 10 ⁵ EMT6 mammary carcinoma cells in Matrigel in the fat pad of the fourth mammary gland. Tumor size was measured twice weekly. Animals in which tumors ulcerated/necrotized or were larger than 2000 mm ³ were euthanized.

Statistics . Statistical tests were performed using unpaired (paired where indicated) two-tailed Student's t-test. Vertical lines show standard deviation from the mean.

Animal Study Oversight : All animal studies were approved by Genentech's Institutional Animal Care and Use Committee.

Example 11: TIGIT expression is increased in human cancer and its expression correlates with CD8 and PD-1 and CD8 expression ⁺ T cell infiltration

Materials and methods

Bioinformatics . Processing and analysis of RNA sequencing data was performed using the R programming language (http://www.r-project.org) together with several packages from the Bioconductor project (http://www.bioconductor.org). RNA-seq data for malignant tumors and corresponding normal tissue samples were obtained from TCGA for five different indications: breast cancer (Network, CGA Comprehensive molecular portraits of human breast tumors, Nature, 490, 61–70 (2012)), colon adenocarcinoma (Network, TCGA Comprehensive molecular characterization of human colon and rectal cancer, Nature, 487, 330–337 (2012)), clear cell renal cell carcinoma (Network, CGA Comprehensive molecular characterization of clear cell renal cell carcinoma, Nature, 499, 43–49 (2013)), squamous cell lung carcinoma (Network, TCGA Comprehensive genomic characterization of squamous cell lung cancers, Nature, 489, 519–525 (2012)) and carcinoma endometrium (Network, TCGA Integrated genomic characterization of endometrial carcinoma, Nature, 497, 67-73 (2012)).

Raw RNA-seq data were processed using the HTSeqGenie Bioconductor package. Briefly, data 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 data pairs that fell within exons were calculated to estimate gene expression levels for individual genes. Library size values 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)) were used to normalize different samples to their respective sequencing depths.

To obtain a T cell-specific gene signature, the T cell genes identified by the IRIS project were manually curated by removing genes associated with cell cycle processes, genes highly expressed in other tissues, and known coactivating and coinhibitory receptors. This yielded a signature of 15 T cell-specific genes. To calculate the T cell gene expression signature score in the lung squamous cell carcinoma data, we first performed a variance-stabilizing transformation on the raw estimated data using the voom function from the limma Bioconductor package. We then calculated a first eigenvector of the centered and scaled variance-stabilized data from the 15-gene T cell signature. This approach provides a robust estimate of relative abundance in T cells. A linear model incorporating the T cell signature score was then fitted to each gene, again using the limma package. The genes were then ranked by their relationship with the T cell signature in the resulting linear model, selecting only genes that were positively correlated with the T cell signature. To visualize the T cell-associated genes as a heat map, the variance-stabilized data were centered and scaled to unit variance, allowing comparison of genes with different mean expression levels.

To determine the correlation between the expression of TIGIT and other genes, we normalized the estimated RNA-seq data to account for differences in library size using the edgeR method in the 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)). Spearman's rank correlation coefficient was then calculated from the normalized values. Rpo > 0.75 was considered to indicate high correlation, Rpo ≤ 0.75 but > 0.5 was considered to indicate moderate correlation, and Rpo ≤ 0.5 but > 0.25 was considered to indicate low correlation.

To calculate TIGIT/CD3ε ratios for each readout, variance-stabilized data were first calculated for each RNA-seq data set. Then, the log2 ratio of variance-stabilized data for TIGIT and CD3ε was calculated. Standard linear analysis using standard R functions was performed to calculate the difference between tumor and normal samples. A p-value <0.01 was accepted as evidence of a statistically significant difference between tumor and normal tissue.

To identify genes associated with tumor-infiltrating T cells, a gene signature-based approach was used, querying gene expression data from The Cancer Genome Atlas (TCGA) project on the lung squamous cell carcinoma collection (LUSC) (Network, T.C.G.A. Comprehensive genomic characterization of squamous cell lung cancers, Nature 489, 519-525 (2012)). Using a set of immune cell-specific genes identified in 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, a highly specific signature of 15 genes was developed. In examining the genes most associated with the T cell signature, several coinhibitory receptors previously associated with T cell dysfunction in tumors were identified, in particular PD-1 (Fig. 21). In LUSC, TIGIT and CD3ε expression were highly correlated with a Spearman rank correlation coefficient (ρ of 0.82 (Fig. 20A)). In fact, TIGIT and CD3ε expression were also highly correlated in many additional TCGA tumor gene expression databases, including colon adenocarcinoma (COAD), uterine corpus endometrioid carcinoma (UCEC), breast carcinoma (BRCA), and clear cell renal cell carcinoma (KIRC), with ρ values ranging from 0.83 to 0.94 (Figs. 20B-20E). In addition, TIGIT expression was elevated relative to CD3ε expression in many tumor samples, with increased TIGIT/CD3ε ratios in LUSC, COAC, UCEC, and BRCA tumor samples compared with corresponding normal tissues (116%–419% increased, Figs. 20A–20D). The ratio of TIGIT to CD3ε expression was not altered in KIRC samples, although TIGIT and CD3ε expression were significantly higher in KIRC samples than in normal tissue samples (Fig. 20E). These data indicate that TIGIT expression was elevated in tumor infiltrating lymphocytes (TILs) in a wide range of solid tumors.

TIGIT has been previously described as an inhibitor of CD4 ⁺ T cell priming, and its function on CD8 ⁺ T cells remains unknown. However, TIGIT expression in LUSC samples was highly correlated with CD8A and weakly correlated with CD4 (ρ = 0.77 and 0.48, respectively; Fig. 20F). TIGIT expression was also correlated with the expression of its additional costimulatory receptor, CD226, as well as with the expression of PD-1, a key mediator of T cell suppression in tumors and other chronic immune responses (ρ = 0.64 and 0.82, respectively; Figs. 20G–20H). Although these genes are expressed on some cell types other than lymphocytes, our data strongly suggest that tumor-infiltrating T cells, particularly exhausted CD8 ⁺ T cells, expressed TIGIT at high levels.

Example 12: TIGIT and PD-1 are coordinately expressed by human and murine tumor-infiltrating lymphocytes

Materials and methods

Human Tumor Samples and PBMCs . Matched whole blood and freshly surgically resected tumor tissues were obtained from Conversant Biosciences or Foundation Bio. All samples were obtained with written informed consent and were collected using a protocol approved by the Hartford Hospital Institutional Review Board (IRB) (NSCLC patient 1, data shown in Figure 22) or the Western Institutional Review Board (NSCLC patient 2 and CRC patient 1, data shown in Figure 23 and Figure 37). Healthy adult whole blood was obtained from a healthy volunteer. PBMCs were isolated from whole blood by Ficoll gradient centrifugation. Tumor tissues were cut into small pieces and incubated with collagenase and DNase (Roche), and cells were separated using a GentleMACS Disassociator (Miltenyi).

Flow cytometry . Single cell suspensions of mouse spleen, lymph nodes, and tumor were prepared by gentle mechanical disruption. Surface staining was performed using commercially available antibodies to CD4, CD8, CD44, CD62L, PD-1 (eBiosciences), and CD226 (BioLegend). Anti-TIGIT antibodies were obtained from Genentech and conjugated to Alexa Fluor 647 according to the manufacturer's instructions (Molecular Probes).

For intracellular cytokine staining (ICS), cells were stimulated for 4 h with 20 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 μM ionomycin (Sigma) in the presence of 3 μg/ml brefeldin A (eBiosciences). Following stimulation, cells were stained for surface markers as described and fixed and permeabilized with FoxP3 fixation buffer (eBioscience) according to the manufacturer's directions. Fixed cells were stained with antibodies to TNFγ and IFNα (eBiosciences).

Human tumor samples and PBMCs were prepared as described above. Surface staining was performed with vital dye (Molecular Probes), commercial antibodies to CD45 (eBiosciences), CD3, CD4, CD8, PD-1 (BD Biosciences), and anti-TIGIT antibodies prepared as described above.

All samples were run on LSR-II or LSR-Fortessa instruments (BD Biosciences) and analyzed using FlowJo software (Treestar).

To confirm the upregulation of TIGIT in tumor-infiltrating T cells, TIGIT protein expression was assessed on T cells infiltrating human non-small cell lung carcinoma, matched peripheral T cells, and healthy donor peripheral T cells. Cell surface TIGIT was expressed by subsets of NSCLC-infiltrating CD8 ⁺ and CD4 ⁺ T cells (51% and 39%, respectively, Figures 22A-22B). Figure 36 also shows that cell surface TIGIT was expressed by a higher percentage of NSCLC-infiltrating CD8 ⁺ and CD4 ⁺ T cells (TIGIT ⁺ cell counts were 58% and 28%, respectively, Figures 36A-36B). Interestingly, peripheral CD8 ⁺ and CD4 ⁺ T cells from the donor with NSCLC tumor also expressed TIGIT at higher levels compared to cells from healthy donors (Figs. 22A-22B and Figs. 36A-36B). Similar results were obtained with a second collection of matched NSCLC and PBMC samples and a collection of matched colorectal carcinoma (CRC) and PBMC samples (Fig. 23 and Fig. 37). Almost all tumor-infiltrating T cells expressing high levels of TIGIT co-expressed PD-1, consistent with the correlation between TIGIT and PD-1 expression shown in Figure 1 (Fig. 22C).

To extend the observations to preclinical models of human cancer, we characterized TIGIT expression by T cells infiltrating subcutaneous CT26 and MC38 colorectal tumors in wild-type BALB/c mice and C57BL6/J mice, respectively. Two weeks after inoculation, when CT26 and MC38 tumors had grown to 150-200 ^mm3 in size, TIGIT was expressed by approximately 50% of tumor-infiltrating CD8 ⁺ T cells and 25% of tumor-infiltrating CD4 ⁺ T cells, at levels similar to those of primary CD8 ⁺ T cells stimulated in vitro (Figs. 22D-22E and Fig. 2B). On both murine CD8 ⁺ and CD4 ⁺ TILs, CD226 was expressed constitutively, and TIGIT and PD-1 expression were again closely correlated (Figs. 22F–22G).

The results confirmed that TIGIT was expressed at high levels by tumor-infiltrating T cells and that TIGIT expression occurred in parallel with the expression of other coinhibitory receptors, most notably PD-1.

Example 13: CD8 suppression by TIGIT ⁺ T cell responses are dependent on CD226

Unlike PD-1 or CTLA-4, there is no direct biochemical evidence for a T cell inhibitory signaling cascade initiated by TIGIT in cis. However, coinhibitory receptors may also function by downregulating complement co-stimulatory receptor activity, such as by CTLA-4-mediated downregulation of CD28 signaling. Having established that TIGIT is a negative regulator of tumor-infiltrating and antiviral CD8 ⁺ T cells, we asked whether TIGIT induces T cell exhaustion indirectly through downregulation of the complement co-stimulatory receptor, CD226, which is highly expressed by peripheral and tumor-infiltrating CD8 ⁺ T cells (Figure 27).

Wild-type BALB/c mice bearing CT26 tumors measuring 150–200 mm ³ were treated with a combination of anti-PD-L1 and anti-TIGIT antibodies in the presence or absence of a blocking anti-CD226 antibody or with anti-CD226 antibody alone. Treatment with anti-CD226 antibody alone slightly accelerated tumor growth compared with control mice, resulting in a 2-day reduction in median survival (anti-CD226 antibody alone vs. control, P = 0.0118, Figs. 28A–28B). Remarkably, additional administration of anti-CD226 blocking antibodies to mice treated with anti-TIGIT and anti-PD-L1 co-blocking antibodies significantly enhanced tumor growth and completely restored the efficacy of TIGIT/PD-L1 co-blocking 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 antibodies (Fig. 19D). These data indicated that CD226 promoted antitumor and other chronic T cell responses and that TIGIT suppressed these responses, at least in part, by suppressing CD226.

To further understand how TIGIT and CD226 activity influences antitumor T cell responses, we tested whether CD226 alone and in combination with TIGIT influences T cell activation, tumor infiltration, and effector function. Tumors and tumor-draining lymph nodes from mice bearing CT26 tumors treated as described above for seven days were analyzed. As previously shown, combined blockade of PD-L1 and TIGIT increased IFNγ production by both tumor-infiltrating CD8 ⁺ T cells and tumor-draining lymph node CD8 ⁺ T cells (130% and 99% increases, respectively, P < 0.001, Figs. 28C–28D). Blockade of CD226 alone had no effect on IFNγ production by tumor-infiltrating CD8 ⁺ T cells and tumor-draining lymph node resident CD8 ⁺ T cells, suggesting that the costimulatory effects of CD226 are already inhibited in exhausted T cells (Figs. 28C–28E). However, blockade of CD226 reduced the frequency and effector function of tumor-infiltrating CD8 ⁺ T cells in mice treated with the combination of anti-TIGIT and anti-PD-L1 antibodies (57% reduction, P = 0.0015, Fig. 28D). Treatment with anti-CD226 antibody had no such effect on tumor-draining lymph node-resident CD8 ⁺ T cells, whereas anti-PD-L1 antibody alone enhanced CD8 ⁺ T cell effector function, suggesting that blocking PD-L1 was sufficient to enhance CD8 ⁺ T cell effector function even in the absence of CD226. Blocking CD226 also reduced the frequency of tumor-infiltrating CD8 ⁺ T cells (53% reduction, P = 0.0044, Figs. 28E–28F). Taken together, these data suggest that CD226 functions to maintain both the accumulation and effector function of tumor-infiltrating CD8 ⁺ T cells and that TIGIT interferes with the latter.

Example 14: TIGIT impairs CD226 function by directly disrupting CD226 homodimerization

To test the extent to which TIGIT can suppress CD226 activity in cis, the effect of TIGIT on CD226 costimulation in vitro was examined. TIGIT-deficient CD8 ⁺ T cells stimulated with suboptimal concentrations of anti-CD3 antibody responded more strongly to PVR costimulation than did similar wild-type CD8 ⁺ T cells, and this enhanced response was dependent on CD226 (46% increase in proliferation, P = 0.0061; Fig. 29A). Consistent with these data, wild-type CD8 ⁺ T cells stimulated with suboptimal concentrations of anti-CD3 antibody and PVR proliferated to a greater extent in the presence of anti-TIGIT antibody than in the presence of isopypic control antibodies, and this effect was dependent on CD226 (105% increase in proliferation, P = 0.0010, Fig. 29B).

To investigate the role of TIGIT on primary human CD8 ⁺ T cells, CD8 ⁺ T cells were isolated from healthy donors and stimulated with suboptimal concentrations of plate-immobilized anti-CD3 antibody and recombinant human PVR-Fc fusion protein. In the presence of isopypic control antibodies, PVR costimulation modestly enhanced T cell stimulation and proliferation. Furthermore, addition of blocking anti-TIGIT antibodies significantly enhanced the effects of PVR costimulation, consistent with the effects of TIGIT on primary murine CD8 ⁺ T cells (69% increase in proliferation, P = 0.0071, Fig. 29C). These data demonstrate an intracellular role for TIGIT in the inhibition of CD226 function on primary murine and human CD8 ⁺ T cells.

TR-FRET (time-resolved fluorescence inductive resonance energy transfer) technology was used to determine the molecular mechanism by which TIGIT disrupted CD226 activity. Human ST-CD226 was first expressed and labeled with transient donor and acceptor fluorophores. These cells produced a strong FRET signal, confirming the ability of CD226 to homodimerize (Fig. 29D). To examine the interactions of CD226 and TIGIT on the cell surface, ST-CD226 was expressed in the absence or presence of human HA-TIGIT labeled with SNAP-tag substrate and anti-HA antibody, respectively. Surprisingly, coexpression of increasing amounts of TIGIT (monitored by ELISA) attenuated the CD226/CD226 FRET signal, indicating that TIGIT could disrupt CD226 homodimerization (Fig. 29E). Indeed, the acceptor CD226 and donor TIGIT also resulted in a high FRET signal, indicating a direct interaction between these two proteins (Fig. 29F). This interaction was further confirmed by coimmunoprecipitation (Fig. 29G). These data showed that TIGIT and CD226 directly interact on the cell surface, and this interaction can disrupt CD226 homodimerization.

To investigate the effects of TIGIT antibody blocking the TIGIT-CD226 interaction, human ST-CD226 and HA-TIGIT were again coexpressed, this time in the presence or absence of blocking antibodies to human TIGIT. Addition of anti-TIGIT antibodies to cell cultures significantly reduced the ability of TIGIT and CD226 to associate (Fig. 29H). These data suggest that anti-TIGIT antibody treatment can limit the interaction of TIGIT with CD226, a finding consistent with the observation that downregulation of CD226 is a key mechanism by which TIGIT leads to CD8 ⁺ T cell depletion. This is also consistent with the ability of anti-TIGIT antibodies to enhance CD226 costimulation.

The ability of endogenous TIGIT and CD226 to interact was then confirmed (Fig. 30). Primary human T cells were stimulated in vitro with anti-CD3 and anti-CD28 antibodies sorted based on TIGIT expression, maintained, restimulated, 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 antibodies and acceptor-conjugated anti-CD226 antibodies gave a strong FRET signal (Fig. 30). In contrast, only a low FRET signal was detected on T cells that did not express TIGIT or that were labeled with donor-conjugated anti-TIGIT antibodies and acceptor-conjugated anti-HVEM antibodies (Fig. 30), confirming the specific nature of the observed interaction between endogenous TIGIT and CD226.

These results indicate that endogenous TIGIT and CD226 can directly interact on the cell surface and that this interaction disrupts CD226 homodimerization. Given the role of CD226 as a costimulator 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 causes CD8 ⁺ T cell depletion in chronic viral infection and cancer.

Materials and methods

Time-resolved fluorescence inductive resonance energy transfer with transfected cell lines . CHO cells were transfected with CD226 N-terminal SNAP-tag (ST) and N-terminal HA-TIGIT using Lipofectamine 2000 (Life Technologies) and seeded in a white 96-well plate (Costar) at 100,000 cells per well. After 24 h, cells were labeled to measure TR-FRET between SNAP donor/SNAP acceptor or between SNAP acceptor/anti-HA donor. 1) SNAP donor/SNAP acceptor labeling: cells were incubated with 100 nM benzylguanine donor-conjugated SNAP-Lumi-4Tb (Cisbio) and 1 μM benzylguanine acceptor-conjugated SNAP-A647 (New England Biolabs) diluted in DMEM with 10% FCS for 1 h at 37°C under 5% CO ₂ . Cells were then washed three times with PBS before measuring the FRET signal. 2) SNAP acceptor/anti-HA donor: cells were incubated with 1 μM benzylguanine acceptor-conjugated SNAP-A647 diluted in DMEM with 10% FCS for 1 h at 37°C under 5% CO ₂ . After three washes with PBS, the cells were incubated for 2 h with 2 nM Lumi-4Tb anti-HA (Cisbio) in PBS + 0.2% BSA at room temperature. The FRET signal was recorded at 665 nm for 400 μs after a 60 μs delay after laser excitation at 343 nm using a Safire2 plate reader (Tecan). When the anti-TIGIT antibody was tested at a final concentration of 10 μg/ml, the FRET signal was also recorded after 15 min of 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 CD226 expression. FRET intensity was: (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). In the study of Flag-ST-CD226/HA-TIGIT interaction, the FRET ratio was the FRET intensity divided by Flag-ST-CD226 expression, as determined by ELISA with anti-Flag antibody. In this case, FRET intensity was: (signal at 665 nm from cells labeled with SNAP donor and anti-HA donor) - (signal at 665 nm from control transfected cells labeled with SNAP acceptor and anti-HA donor).

Time-resolved fluorescence inductive resonance energy transfer with human T cells . Human anti-TIGIT antibodies (Genentech clone 1F4), anti-CD226 antibodies (Santa Cruz Biotechnology), and anti-HVEM antibodies (eBioscience) were conjugated to TR-FRET-compatible fluorophores (Cisbio). Primary human T cells isolated from blood using MACS were stimulated in vitro with plate-fixed anti-CD3 and anti-CD28 antibodies for 72 h. TIGIT-expressing and non-TIGIT-expressing T cells (all expressing CD226) were then sorted, starved for 72 h, and restimulated for 48 h. Each population was then washed once with Tris-buffered saline (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 in triplicate under the following conditions: 1) anti-TIGIT Ab-Lumi4-Tb (5 2) anti-TIGIT Ab-Lumi4-Tb (5 μg/ml) + anti-HVEM-d2 (10 μg/ml), 3) anti-TIGIT Ab-Lumi4-Tb (5 μg/ml) + anti-CD226 (10 μg/ml), 4) anti-TIGIT Ab-Lumi4-Tb (5 μg/ml) + anti-CD226 (10 μg/ml) + unlabeled anti-TIGIT Ab (clone 1F4) (50 μg/ml). The concentrations indicated were optimized to ensure the highest FRET signal. Cells were incubated for 2 h at room temperature on a rotator and then washed three times with Tris-buffered saline. T cells were then seeded at 400,000 cells/well in a white 96-well plate (Costar) and the TR-FRET signal was recorded at 665 nm for 400 μs after a 60 μs delay after laser excitation at 343 nm using a PHERAstar plate reader (BMG Labtech). FRET intensity was expressed as the 665 nm signal from cells labeled with Ab-Lumi4-Tb + Ab-d2 minus the 665 nm signal from the same batch of cells labeled with Ab-Lumi4-Tb alone. A nonspecific FRET signal was given by T cells incubated with Lumi4Tb + d2 + excess unlabeled Ab.

Coimmunoprecipitation . Briefly, COS 7 cells in 15 cm dishes were cotransfected with expression plasmids containing cDNAs for the tagged TIGIT-HA (5 ng) or CD226-Flag (10 ng) proteins, or a control plasmid (pRK). At 23 h posttransfection, cells were washed with PBS and collected in 4 ml ice-cold PBS, centrifuged at 300 × g for 5 min, and the cell pellets were resuspended in 2 ml lysis buffer at 4°C. Cells were lysed for 50 min with vortexing every 15 min and then centrifuged at 10,000 × g for 15 min at 4°C. The resulting supernatant was pre-clarified with 160 μl of CL6B Sepharose suspension by rotation for 30 min at 4°C and centrifuged for 2 min at 3000×g. The supernatant was divided equally into two tubes and immunoprecipitated with anti-HA or anti-flag using standard procedures. Immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting. Western blots were probed with anti-Flag-HRP or anti-HA-HRP.

All patents, patent applications, documents and articles cited in this specification are incorporated herein by reference in their entirety.

--->

SEQUENCE LIST

<110> GENENTECH, INC.

F. HOFFMANN-LA ROCHE AG

GROGAN, Jane

JOHNSTON, Robert J.

IRVING, Bryan

HACKNEY, Jason

YU, Xin

EATON, Dan

BOWLES, Kristin

COMPS-AGRAR, Laetitia

<120> CANCER TREATMENT METHODS USING ANTAGONISTS,

PD-1 AXIS BINDERS AND TIGIT INHIBITORS

<130> 14639-2025940

<140> Not yet assigned

<141> Concurrently Herewith

<150> US 61/846,941

<151> 2013-07-16

<150> US 61/865,582

<151> 2013-08-13

<150> US 61/950,754

<151> 2014-03-10

<150> US 61/985,884

<151> 2014-04-29

<150> US 61/992,109

<151> 2014-05-12

<160> 49

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Lys Ser Ser Gln Ser Leu Tyr Tyr Ser Gly Val Lys Glu Asn Leu Leu

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Ala Ser Ile Arg Phe Thr

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Gln Gln Gly Ile Asn Asn Pro Leu Thr

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Gly Phe Thr Phe Ser Ser Phe Thr Met His

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Phe Ile Arg Ser Gly Ser Gly Ile Val Phe Tyr Ala Asp Ala Val Arg

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Arg Pro Leu Gly His Asn Thr Phe Asp Ser

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Gly Ile Ser Asn Arg Phe Ser

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Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Pro Gly

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Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val Ser Phe Gly

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Thr Gln Pro Gly Lys

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Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe

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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr

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Gly Phe Thr Phe Ser Asp Ser Trp Ile His

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Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

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Arg His Trp Pro Gly Gly Phe Asp Tyr

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Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

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Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

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Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

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Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

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Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

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Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Leu Val Thr Val Ser Ser Ala Ser Thr Lys

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

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Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ser

115

<210> 42

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<212> Protein

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

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Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

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Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

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<212> Protein

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

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Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

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Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 49

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic construction

<400> 49

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<---

Claims (31)

1. A method of treating or slowing the progression of cancer in an individual, comprising administering to the individual an effective amount of an anti-PD-1 antagonist antibody and an effective amount of an anti-TIGIT antagonist antibody, wherein the anti-TIGIT antagonist antibody inhibits and/or blocks the interaction of CD226 with TIGIT, wherein the anti-TIGIT antagonist antibody inhibits the interaction of PVR with TIGIT; and wherein the anti-PD-1 antagonist antibody inhibits the binding of PD-1 to PD-L1. 2. The method of claim 1, further comprising administering at least one chemotherapeutic agent. 3. The method of claim 1, wherein the cancer has elevated levels of T cell infiltration. 4. The method of claim 1, wherein the anti-TIGIT antagonist antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, and a heteroconjugate antibody. 5. The method of claim 1, wherein the anti-TIGIT antagonist antibody is a full-length antibody. 6. The method of claim 1, wherein the anti-TIGIT antagonist antibody is an antigen-binding fragment of an antibody. 7. The method of claim 1, wherein the PD-1 antagonist antibody is selected from the group consisting of MDX-1106, MK3475, CT-011, and AMP-224. 8. The method of claim 1, wherein the PD-1 antagonist antibody is a full-length antibody. 9. The method of claim 1, wherein the PD-1 antagonist antibody is an antigen-binding fragment of an antibody. 10. The method of claim 1, wherein 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, thymus carcinoma, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell cancer, and malignant blood disease. 11. The method of claim 1, wherein the anti-TIGIT antibody antagonist is administered continuously. 12. The method according to claim 1, wherein the anti-TIGIT antibody antagonist is administered intermittently. 13. The method of claim 1, wherein the anti-TIGIT antagonist antibody is administered prior to the anti-PD-1 antagonist antibody. 14. The method of claim 1, wherein the anti-TIGIT antagonist antibody is administered simultaneously with the anti-PD-1 antagonist antibody. 15. The method of claim 1, wherein the anti-TIGIT antagonist antibody is administered after the anti-PD-1 antagonist antibody. 16. The method of claim 1, wherein the method comprises an effective amount of an anti-TIGIT antibody antagonist. 17. The method of claim 16, wherein the anti-TIGIT antagonist antibody comprises an Fc region that is characterized by human IgG-1, IgG-2, IgG-2A, IgG-2B, IgG-3, or IgG-4 subtypes. 18. The method of claim 1, wherein the method comprises an effective amount of an anti-PD-1 antagonist antibody. 19. The method of claim 18, wherein the PD-1 antagonist antibody comprises an Fc region that is characterized by human IgG-1, IgG-2, IgG-2A, IgG-2B, IgG-3, or IgG-4 subtypes. 20. The method according to claim 1, wherein the individual is a human patient. 21. A kit for treating or slowing the progression of cancer in an individual, comprising an anti-PD-1 antagonist antibody and a package insert containing instructions for using the anti-PD-1 antagonist antibody in combination with an anti-TIGIT antagonist antibody, wherein the anti-TIGIT antagonist antibody inhibits and/or blocks the interaction of CD226 with TIGIT, wherein the anti-TIGIT antagonist antibody inhibits the interaction of PVR with TIGIT; and wherein the anti-PD-1 antagonist antibody inhibits the binding of PD-1 to PD-L1. 22. A kit for treating or slowing the progression of cancer in an individual, comprising an anti-PD-1 antagonist antibody and an anti-TIGIT antagonist antibody, and a package insert containing instructions for use of the anti-PD-1 antagonist antibody and the anti-TIGIT antagonist antibody, wherein the anti-TIGIT antagonist antibody inhibits and/or blocks the interaction of CD226 with TIGIT, wherein the anti-TIGIT antagonist antibody inhibits the interaction of PVR with TIGIT; and wherein the anti-PD-1 antagonist antibody inhibits the binding of PD-1 to PD-L1. 23. A kit for treating or slowing the progression of cancer in an individual, comprising an anti-TIGIT antagonist antibody and a package insert containing instructions for using the anti-TIGIT antagonist antibody in combination with an anti-PD-1 antagonist antibody, wherein the anti-TIGIT antagonist antibody inhibits and/or blocks the interaction of CD226 with TIGIT, wherein the anti-TIGIT antagonist antibody inhibits the interaction of PVR with TIGIT; and wherein the anti-PD-1 antagonist antibody inhibits the binding of PD-1 to PD-L1. 24. The kit according to any one of claims 21-23, wherein the anti-PD-1 antagonist antibody is MDX-1106, MK-3475, CT-011 or AMP-224. 25. The kit according to any one of claims 21-23, wherein the anti-TIGIT antagonist antibody is a full-length antibody. 26. The kit according to any one of claims 21-23, wherein the anti-TIGIT antagonist antibody is an antigen-binding fragment of an antibody. 27. The kit according to any one of claims 21-23, wherein the anti-TIGIT antagonist antibody comprises an Fc region that is characterized by human IgG1, IgG2, IgG2A, IgG2B, IgG3 or IgG4 subtypes. 28. The kit according to any one of claims 21-23, wherein the PD-1 antagonist antibody is a full-length antibody. 29. The kit according to any one of claims 21-23, wherein the PD-1 antagonist antibody is an antigen-binding fragment of an antibody. 30. The kit according to any one of claims 21-23, wherein the PD-1 antagonist antibody comprises an Fc region that is characterized by human IgG1, IgG2, IgG2A, IgG2B, IgG3 or IgG4 subtypes. 31. The kit according to any one of paragraphs 21-23, wherein the individual is a human patient.

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