Methods of treating cancer using PD-1 axis binding antagonists and TIGIT inhibitors

Abstract

The present invention describes combination therapies comprising a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity and methods of use thereof, including methods of treating conditions where enhanced immunogenicity is desired, such as increasing tumor immunogenicity to treat cancer or chronic infection.

Description

[0001] This application is a divisional application of Chinese patent application No. 201480047388.9, filed on July 16, 2014, entitled "Method for treating cancer using a PD-1 axis binding antagonist and a TIGIT inhibitor".

[0002] Cross-citation of related applications

[0003] 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.

[0004] Submission of sequence lists for ASCII text files

[0005] The following submission on an ASCII text file is included in this document in its entirety by reference: Sequence List in Computer-Readable Form (CRF) (filename: 146392025940SEQLISTING.TXT, record date: July 16, 2014, size: 25KB). Technical Field

[0006] This invention describes combination therapy comprising a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity, and methods of using thereof, including methods for treating conditions where enhanced immunogenicity is desired (such as enhancing tumor immunogenicity to treat cancer or chronic infections). Background Technology

[0007] The presentation of two distinct signals to T cells is a widely accepted model of lymphocyte activation by antigen-presenting cells (APCs) in response to resting T lymphocytes. Lafferty et al., Aust. J. Exp. Biol. Med. ScL 53:27-42 (1975). This model further provides for the differentiation between self and non-self and immune tolerance. Bretscher et al., Science 169:1042-1049 (1970); Bretscher, PA, PNASUSA 96:185-190 (1999); Jenkins et al., J. Exp. Med. 165:302-319 (1987). Following recognition of foreign antigenic peptides presented in the context of the major histocompatibility complex (MHC), the first signal, or antigen-specific signal, is transduced via the T cell receptor (TCR). Secondary or co-stimulatory signals are delivered to T cells via co-stimulatory molecules expressed on antigen-presenting cells (APCs) and induce T cells to promote clonal expansion, cytokine secretion, and effector function. (Lenschow et al., Ann. Rev. Immunol. 14:233 (1996)). In the absence of co-stimulation, T cells can become unresponsive to antigenic stimulation, leading to a tolerogenic response to exogenous or endogenous antigens.

[0008] In the two-signal model, T cells receive both positive co-stimulatory and negative co-inhibitory signals. The regulation of these positive and negative signals is crucial for maximizing the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative signals appear to be necessary for inducing T cell tolerance, while positive signals promote T cell activation.

[0009] Both co-stimulatory and co-inhibitory signals are provided to antigen-exposed T cells, and the interaction between these signals is crucial for controlling the magnitude of the immune response. Furthermore, the signals provided to T cells vary with the clearance, deterioration, or persistence of infection or immune stimulation, and these changes powerfully influence responding T cells and remodel the immune response.

[0010] The mechanisms of co-stimulation are of therapeutic interest because it has been shown that manipulation of co-stimulatory signaling provides a means to enhance or terminate cell-based immune responses. Recently, it has been discovered that T cell dysfunction or unresponsiveness occurs in parallel with the induction and sustained expression of the inhibitory receptor (programmed death 1 peptide (PD-1)). Therefore, therapeutic targeting of PD-1 and other molecules that signal via their interaction with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), is an area of ​​great interest.

[0011] PD-L1 is overexpressed in many cancers and is frequently 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, in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes, most tumor-infiltrating T lymphocytes predominantly express PD-1, suggesting that upregulation of PD-1 on tumor-reactive T cells can contribute to impaired antitumor immune responses (Blood 2009 114(8):1537). This could be due to PD-L1 signaling mediated by PD-L1-expressing tumor cells interacting with PD-1-expressing T cells, leading to reduced T cell activation and evasion of immune surveillance (Sharpe et al., NatRev 2002) (Keir ME et al., 2008 Annu.Rev. Immunol. 26:677). Therefore, inhibition of PD-L1 / PD-1 interaction can enhance CD8. + T-cell-mediated tumor killing.

[0012] Inhibition of PD-1 axis signaling via its direct ligands (e.g., PD-L1, PD-L2) has been proposed as a means to enhance T-cell immunity for cancer treatment (e.g., tumor immunity). Furthermore, similar enhancement of T-cell immunity has been observed by inhibiting the binding of PD-L1 to its binding partner B7-1. In addition, combined inhibition of PD-1 signaling with other deregulated signaling pathways in tumor cells can further enhance therapeutic efficacy. Such optimal therapies remain needed for treating, stabilizing, preventing, and / or delaying the development of various cancers.

[0013] All references, publications, and patent applications disclosed herein are hereby fully cited. Summary of the Invention

[0014] This invention describes a combination therapy comprising a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity.

[0015] This article provides methods for treating cancer or delaying cancer progression in an individual, which include administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity.

[0016] This article also provides methods for reducing or inhibiting cancer recurrence or progression in an individual, which include administering an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity to the individual.

[0017] This article also provides methods for treating immune-related diseases or delaying the progression of immune-related diseases in an individual, which include administering an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity to the individual.

[0018] This article also provides methods for reducing or inhibiting the progression of immune-related diseases in an individual, which include administering an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity to the individual.

[0019] In some embodiments, the immune-related disease is associated with T-cell dysfunction. In some embodiments, the T-cell dysfunction is characterized by reduced responsiveness to antigenic stimulation. In some embodiments, the T-cell dysfunction is characterized by T-cell unresponsiveness or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments, the T-cell dysfunction is characterized by T-cell depletion. In some embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments, the immune-related disease is selected from the group consisting of unresolved acute infections, chronic infections, and tumor immunity.

[0020] This article also provides methods for enhancing, strengthening, or stimulating immune responses or functions in individuals by administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity.

[0021] This article also provides methods for treating cancer or delaying cancer progression in an individual, which include administering an effective amount of a PD-1 axis binding antagonist and an agent that modulates CD226 expression and / or activity to the individual.

[0022] This article also provides methods for reducing or inhibiting cancer recurrence or progression in an individual, which include administering an effective amount of a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity to the individual.

[0023] This article also provides methods for treating immune-related diseases or delaying the progression of immune-related diseases in an individual, which include administering an effective amount of a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity to the individual.

[0024] This article also provides methods for reducing or inhibiting the progression of immune-related diseases in individuals, which include administering an effective amount of a PD-1 axis binding antagonist and an agent that modulates CD226 expression and / or activity to the individual.

[0025] In some embodiments, the immune-related disease is associated with T-cell dysfunction. In some embodiments, the T-cell dysfunction is characterized by reduced responsiveness to antigenic stimulation. In some embodiments, the T-cell dysfunction is characterized by T-cell unresponsiveness, or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments, the T-cell dysfunction is characterized by T-cell depletion. In some embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments, the immune-related disease is selected from the group consisting of unresolved acute infections, chronic infections, and tumor immunity.

[0026] This article also provides methods for enhancing, strengthening, or stimulating immune responses or functions in individuals by administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity.

[0027] In some embodiments, the agents that regulate CD226 expression and / or activity can enhance and / or stimulate CD226 expression and / or activity.

[0028] In some embodiments, the agent regulating CD226 expression and / or activity is selected from: agents that inhibit and / or block the interaction between CD226 and TIGIT, antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, and agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR.

[0029] In some embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is an anti-TIGIT antibody or its antigen-binding fragment.

[0030] In some embodiments, the antagonist of TIGIT expression and / or activity is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment.

[0031] In some embodiments, the antagonist expressing and / or activating the PVR is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide.

[0032] In some embodiments, the agents that inhibit and / or block the interaction between TIGIT and PVR are small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0033] In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR are small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0034] The present invention also describes combination therapy comprising agents that reduce or inhibit TIGIT expression and / or activity and agents that reduce or inhibit one or more other immune co-suppressive receptors.

[0035] This article provides a method for enhancing, strengthening, or stimulating an immune response or 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 other immunosuppressive receptors.

[0036] In some embodiments, the one or more other immunosuppressive receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, TIM3, BTLA, and VISTA.

[0037] This invention also describes combination therapy comprising agents that reduce or inhibit TIGIT expression and / or activity and agents that increase or activate one or more other immune co-stimulatory receptors.

[0038] This article provides a method for enhancing, strengthening, or stimulating an immune response or 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 enhances or activates one or more other immune co-stimulatory receptors.

[0039] In some embodiments, the one or more other immune co-stimulatory receptors are selected from the group consisting of CD226, OX-40, CD28, CD27, CD137, HVEM, and GITR. In some embodiments, the one or more other immune co-stimulatory receptors are selected from the group consisting of CD226, OX-40, CD27, CD137, HVEM, and GITR. In some embodiments, the one or more other immune co-stimulatory receptors are selected from the group consisting of OX-40 and CD27.

[0040] In some implementations, any of the above methods further includes the administration of at least one chemotherapeutic agent.

[0041] In some implementations, the individual in any of the above methods has cancer. In some implementations, the individual in any of the above methods is a human being.

[0042] In some embodiments, the CD4 and / or CD8 T cells in the individual have increased or enhanced initiation, activation, proliferation, cytokine release, and / or cell lysis activity relative to prior to administration of the combination.

[0043] In some embodiments, the number of CD4 and / or CD8 T cells increases relative to before administration of the combination. In some embodiments, the number of activated CD4 and / or CD8 T cells increases relative to before administration of the combination. In some embodiments, the activated CD4 and / or CD8 T cells are characterized by γ-IFN. + Generates CD4 and / or CD8 T cells and / or exhibits enhanced cytolytic activity relative to prior to administration of the combination. In some embodiments, the CD4 and / or CD8 T cells exhibit increased release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukins.

[0044] In some embodiments, the CD4 and / or CD8 T cells are effector memory T cells. In some embodiments, the CD4 and / or CD8 effector memory T cells are characterized by γ-IFN. + Generates CD4 and / or CD8 T cells and / or enhances cytolytic activity. In some embodiments, the CD4 and / or CD8 effector memory T cells are characterized by having CD44... 高 CD62L 低 The expression.

[0045] In some implementations, the cancer in any of the above methods has an elevated level of T-cell infiltration.

[0046] In some embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is selected from the group consisting of: antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, and agents that inhibit intracellular signaling and / or interactions mediated by TIGIT bound to PVR.

[0047] In some embodiments, the antagonist of TIGIT expression and / or activity is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0048] In some embodiments, the antagonist for PVR expression and / or activity is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0049] In some embodiments, the agent that inhibits intracellular signaling mediated by TIGIT bound to PVR is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0050] In some embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment.

[0051] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises at least one HVR, which comprises an amino acid sequence selected from the following 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).

[0052] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises a light chain, the light chain containing...

[0053] (SEQ ID NO:13) or

[0054] The amino acid sequence shown in (SEQ ID NO:14) is as follows.

[0055] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises a heavy chain, the heavy chain containing

[0056] (SEQ ID NO:15) or

[0057] The amino acid sequence shown in (SEQ ID NO:16) is as follows.

[0058] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises a light chain and a heavy chain, the light chain comprising...

[0059] (SEQ ID NO:13) or

[0060] (SEQ ID NO:14)

[0061] The shown amino acid sequence indicates that the heavy chain contains...

[0062] (SEQ ID NO:15) or

[0063] The amino acid sequence shown in (SEQ ID NO:16) is as follows.

[0064] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment is selected from humanized antibodies, chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, and immunotoxins.

[0065] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises at least one HVR that is at least 90% identical to any of the following HVRs: 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).

[0066] In some embodiments, the anti-TIGIT antibody or a fragment thereof comprises a light chain and / or a heavy chain, the light chain and the heavy chain respectively comprising, with

[0067] (SEQ ID NO:13) or

[0068] The amino acid sequence shown in (SEQ ID NO:14) or related to

[0069] (SEQ ID NO:15) or

[0070] The amino acid sequence shown in (SEQ ID NO:16) is at least 90% identical to the amino acid sequence shown in the SEQ ID NO:16.

[0071] In some embodiments, the PD-1 axis binding antagonist is selected from the group consisting of PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0072] In some embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 binding antagonist is MDX-1106 (nivolumab). In some embodiments, the PD-1 binding antagonist is Merck 3475 (lambrolizumab). In some embodiments, the PD-1 binding antagonist is CT-011 (pidilizumab). In some embodiments, the PD-1 binding antagonist is AMP-224.

[0073] In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an antibody.

[0074] In some embodiments, the PD-L1 binding antagonist is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105 and MEDI 4736.

[0075] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain and a light chain, the 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 the 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).

[0076] In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence.

[0077] (SEQ ID NO:23), the light chain variable region contains an amino acid sequence.

[0078] (SEQ ID NO:24).

[0079] 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 immunoadhesive.

[0080] In some implementations, the cancers treated are selected from the group consisting of: non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycoses fungoids, Merkel cell carcinoma, and other hematologic malignancies.

[0081] In some embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered continuously. In some embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered intermittently. In some embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered before the PD-1 axis binding antagonist. In some embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered concurrently with the PD-1 axis binding antagonist. In some embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered after the PD-1 axis binding antagonist.

[0082] This document also provides a kit containing a PD-1 axis binding antagonist and a packaging insert containing instructions for the use of the PD-1 axis binding antagonist in an individual to treat cancer or delay cancer progression, in combination with agents that reduce or inhibit TIGIT expression and / or activity.

[0083] This document also provides a kit containing a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions for treating cancer or delaying cancer progression in an individual using the PD-1 axis binding antagonist and the agent that reduces or inhibits TIGIT expression and / or activity.

[0084] This document also provides a kit containing an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions for the use of the agent, which reduces or inhibits TIGIT expression and / or activity, in combination with a PD-1 axis binding antagonist to treat cancer or delay cancer progression in an individual.

[0085] This document also provides a kit containing a PD-1 axis binding antagonist and a packaging insert containing instructions on using the PD-1 axis binding antagonist in combination with agents that reduce or inhibit TIGIT expression and / or activity to enhance the immune function of individuals with cancer.

[0086] This document also provides a kit containing a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions on enhancing the immune function of individuals with cancer using the PD-1 axis binding antagonist and the agent that reduces or inhibits TIGIT expression and / or activity.

[0087] This document also provides a kit containing an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions on enhancing the immune function of individuals with cancer by using the agent that reduces or inhibits TIGIT expression and / or activity in combination with a PD-1 axis binding antagonist.

[0088] This document also provides a kit containing a PD-1 axis binding antagonist and a packaging insert containing instructions on the use of the PD-1 axis binding antagonist in combination with agents that regulate CD226 expression and / or activity to treat cancer or delay cancer progression in an individual.

[0089] This document also provides a kit containing a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity, and a packaging insert containing instructions on the use of the PD-1 axis binding antagonist and the agent that regulates CD226 expression and / or activity in an individual to treat cancer or delay cancer progression.

[0090] This document also provides a kit containing an agent that regulates CD226 expression and / or activity, and a packaging insert containing instructions for the use of the agent that regulates CD226 expression and / or activity in combination with a PD-1 axis binding antagonist to treat cancer or delay cancer progression in an individual.

[0091] This document also provides a kit containing a PD-1 axis binding antagonist and a packaging insert containing instructions on using the PD-1 axis binding antagonist in combination with agents that regulate CD226 expression and / or activity to enhance the immune function of individuals with cancer.

[0092] This document also provides a kit containing a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity, and a packaging insert containing instructions on enhancing the immune function of individuals with cancer using the PD-1 axis binding antagonist and the agent that regulates CD226 expression and / or activity.

[0093] This document also provides a kit containing an agent that regulates CD226 expression and / or activity, and a packaging insert containing instructions on enhancing the immune function of individuals with cancer by using the agent that regulates CD226 expression and / or activity in combination with a PD-1 axis binding antagonist.

[0094] In some embodiments, the PD-1 axis binding antagonist included in the kit is an anti-PD-L1 antibody. In some embodiments, the PD-1 axis binding antagonist included in the kit is an anti-PD-1 antibody. In some embodiments, the agent included in the kit that reduces or inhibits TIGIT expression and / or activity is selected from the group consisting of: antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, and agents that inhibit intracellular signaling and / or interactions mediated by TIGIT bound to PVR. In some embodiments, the antagonist of TIGIT expression and / or activity included in the kit is an anti-TIGIT antibody or its antigen-binding fragment.

[0095] In some embodiments, the kit includes an agent that regulates CD226 expression and / or activity, capable of enhancing and / or stimulating CD226 expression and / or activity. In some embodiments, the agent in the kit that regulates CD226 expression and / or activity is selected from agents that inhibit and / or block the interaction between CD226 and TIGIT, antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, and agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR. In some embodiments, the agent in the kit that inhibits and / or blocks the interaction between CD226 and TIGIT and / or the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment.

[0096] In some aspects, this disclosure provides a method for treating cancer or delaying cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity in the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the agent that reduces or inhibits TIGIT expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in combination with an agent that reduces or inhibits TIGIT expression and / or activity to treat cancer or delay cancer progression. In other respects, this disclosure provides pharmaceutical compositions comprising agents that reduce or inhibit TIGIT expression and / or activity for use in combination with PD-1 axis binding antagonists to treat cancer or delay cancer progression.

[0097] In other aspects, this disclosure provides a method for reducing or inhibiting cancer recurrence or cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for reducing or inhibiting cancer recurrence or cancer progression in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity in the preparation of a medicament for reducing or inhibiting cancer recurrence or cancer progression in an individual, wherein the agent that reduces or inhibits TIGIT expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in combination with an agent that reduces or inhibits TIGIT expression and / or activity to reduce or inhibit cancer recurrence or cancer progression. In other respects, this disclosure provides pharmaceutical compositions comprising agents that reduce or inhibit TIGIT expression and / or activity, which are used in combination with PD-1 axis binding antagonists to reduce or inhibit cancer recurrence or cancer progression.

[0098] In other aspects, this disclosure provides a method for treating an immune-related disease or delaying the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for treating an immune-related disease or delaying the progression of an immune-related disease in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity in the preparation of a medicament for treating an immune-related disease or delaying the progression of an immune-related disease in an individual, wherein the agent that reduces or inhibits TIGIT expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in combination with an agent that reduces or inhibits TIGIT expression and / or activity to treat an immune-related disease or delay the progression of an immune-related disease. In other respects, this disclosure provides pharmaceutical compositions comprising agents that reduce or inhibit TIGIT expression and / or activity for use in combination with PD-1 axis binding antagonists to treat immune-related diseases or delay the progression of immune-related diseases.

[0099] In other respects, this disclosure provides a combination comprising an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity.

[0100] In other aspects, this disclosure provides a method for reducing or inhibiting the progression of immune-related diseases in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for reducing or inhibiting the progression of immune-related diseases in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity in the preparation of a medicament for reducing or inhibiting the progression of immune-related diseases in an individual, wherein the agent that reduces or inhibits TIGIT expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for reducing or inhibiting the progression of immune-related diseases in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other respects, this disclosure provides pharmaceutical compositions comprising agents that reduce or inhibit TIGIT expression and / or activity, which are used in combination with PD-1 axis binding antagonists to reduce or inhibit the progression of immune-related diseases.

[0101] In some embodiments that can be combined with any of the foregoing embodiments, the immune-related disease is associated with T-cell dysfunction. In some embodiments that can be combined with any of the foregoing embodiments, the immune-related disease is a viral infection. In some embodiments that can be combined with any of the foregoing embodiments, the viral infection is a chronic viral infection. In some embodiments that can be combined with any of the foregoing embodiments, the T-cell dysfunction is characterized by reduced responsiveness to antigenic stimulation. In some embodiments that can be combined with any of the foregoing embodiments, the T-cell dysfunction is characterized by T-cell unresponsiveness or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments that can be combined with any of the foregoing embodiments, the T-cell dysfunction is characterized by T-cell depletion. In some embodiments that can be combined with any of the foregoing embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments that can be combined with any of the foregoing embodiments, the immune-related disease is selected from the group consisting of unresolved acute infections, chronic infections, and tumor immunity.

[0102] In other aspects, this disclosure provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for enhancing or stimulating an immune response or function in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity in the preparation of a medicament for enhancing or stimulating an immune response or function in an individual, wherein the agent that reduces or inhibits TIGIT expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for enhancing or stimulating an immune response or function in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides pharmaceutical compositions comprising agents that reduce or inhibit TIGIT expression and / or activity, which are used in combination with PD-1 axis binding antagonists to enhance or stimulate immune responses or function. In other aspects, this disclosure provides a combination comprising an effective amount of a PD-1 axis binding antagonist and agents that reduce or inhibit TIGIT expression and / or activity.

[0103] In other aspects, this disclosure provides a method for treating cancer or delaying cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent regulating CD226 expression and / or activity in the preparation of a medicament for treating cancer or delaying cancer progression in an individual, wherein the agent regulating CD226 expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in combination with an agent regulating CD226 expression and / or activity to treat cancer or delay cancer progression. In other respects, this disclosure provides pharmaceutical compositions comprising agents that regulate CD226 expression and / or activity for use in combination with PD-1 axis binding antagonists to treat cancer or delay cancer progression.

[0104] In other aspects, this disclosure provides a method for reducing or inhibiting cancer recurrence or cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist and an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis-binding antagonist in the preparation of a medicament for reducing or inhibiting cancer recurrence or cancer progression in an individual, wherein the PD-1 axis-binding antagonist is used in combination with an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent regulating CD226 expression and / or activity in the preparation of a medicament for reducing or inhibiting cancer recurrence or cancer progression in an individual, wherein the agent regulating CD226 expression and / or activity is used in combination with a PD-1 axis-binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis-binding antagonist for use in combination with an agent regulating CD226 expression and / or activity to reduce or inhibit cancer recurrence or cancer progression. In other respects, this disclosure provides pharmaceutical compositions comprising agents that regulate CD226 expression and / or activity, which are used in combination with PD-1 axis binding antagonists to reduce or inhibit cancer recurrence or cancer progression.

[0105] In other aspects, this disclosure provides a method for treating an immune-related disease or delaying the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for treating an immune-related disease or delaying the progression of an immune-related disease in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent regulating CD226 expression and / or activity in the preparation of a medicament for treating an immune-related disease or delaying the progression of an immune-related disease in an individual, wherein the agent regulating CD226 expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for use in combination with an agent regulating CD226 expression and / or activity to treat an immune-related disease or delay the progression of an immune-related disease. In other respects, this disclosure provides pharmaceutical compositions comprising agents that regulate CD226 expression and / or activity for use in combination with PD-1 axis binding antagonists to treat immune-related diseases or delay the progression of immune-related diseases.

[0106] In other respects, this disclosure provides a combination comprising an effective amount of a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity.

[0107] In other aspects, this disclosure provides a method for reducing or inhibiting the progression of immune-related diseases in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for reducing or inhibiting the progression of immune-related diseases in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent regulating CD226 expression and / or activity in the preparation of a medicament for reducing or inhibiting the progression of immune-related diseases in an individual, wherein the agent regulating CD226 expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for reducing or inhibiting the progression of immune-related diseases in combination with an agent regulating CD226 expression and / or activity. In other respects, this disclosure provides pharmaceutical compositions comprising agents that regulate CD226 expression and / or activity, which are used in combination with PD-1 axis binding antagonists to reduce or inhibit the progression of immune-related diseases.

[0108] In some embodiments that can be combined with any of the foregoing embodiments, the immune-related disease is associated with a T-cell dysfunction disorder. In some embodiments that can be combined with any of the foregoing embodiments, the immune-related disease is a viral infection. In some embodiments that can be combined with any of the foregoing embodiments, the viral infection is a chronic viral infection. In some embodiments that can be combined with any of the foregoing embodiments, the T-cell dysfunction disorder is characterized by reduced responsiveness to antigenic stimulation. In some embodiments that can be combined with any of the foregoing embodiments, the T-cell dysfunction disorder is characterized by T-cell unresponsiveness, or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments that can be combined with any of the foregoing embodiments, the T-cell dysfunction disorder is characterized by T-cell depletion. In some embodiments that can be combined with any of the foregoing embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments that can be combined with any of the foregoing embodiments, the immune-related disease is selected from the group consisting of unresolved acute infections, chronic infections, and tumor immunity.

[0109] In other aspects, this disclosure provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of a PD-1 axis binding antagonist in the preparation of a medicament for enhancing or stimulating an immune response or function in an individual, wherein the PD-1 axis binding antagonist is used in combination with an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides the use of an effective amount of an agent regulating CD226 expression and / or activity in the preparation of a medicament for enhancing or stimulating an immune response or function in an individual, wherein the agent regulating CD226 expression and / or activity is used in combination with a PD-1 axis binding antagonist. In other aspects, this disclosure provides a pharmaceutical composition comprising a PD-1 axis binding antagonist for enhancing or stimulating an immune response or function in combination with an agent regulating CD226 expression and / or activity. In other aspects, this disclosure provides pharmaceutical compositions comprising agents that regulate CD226 expression and / or activity, which are used in combination with PD-1 axis binding antagonists to enhance or stimulate immune responses or functions. In other aspects, this disclosure provides a combination comprising an effective amount of a PD-1 axis binding antagonist and agents that regulate CD226 expression and / or activity.

[0110] In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an agent that enhances and / or stimulates CD226 expression and / or activity. In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an agent that enhances and / or stimulates the interaction between CD226 and PVR. In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an agent that enhances and / or stimulates intracellular signaling mediated by CD226 binding to PVR. In certain embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is selected from the group consisting of: agents that inhibit and / or block the interaction between CD226 and TIGIT, antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, agents that inhibit and / or block the interaction between TIGIT and PVR L2, agents that inhibit and / or block the interaction between TIGIT and PVR L3, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3, and combinations thereof. In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an agent that inhibits and / or blocks the interaction between CD226 and TIGIT. In some embodiments that can be combined with any of the foregoing embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, or an inhibitory peptide. In some embodiments that can be combined with any of the foregoing embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments that can be combined with any of the foregoing embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is an inhibitory nucleic acid selected from the group consisting of antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras. In some embodiments that can be combined with any of the foregoing embodiments, the antisense polynucleotide targets TIGIT. In some embodiments that can be combined with any of the foregoing embodiments, the interfering RNA targets TIGIT. In some embodiments that can be combined with any of the foregoing embodiments, the catalytic RNA targets TIGIT. In some embodiments that can be combined with any of the foregoing embodiments, the RNA-DNA chimera targets TIGIT.In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an antagonist of TIGIT expression and / or activity. In some embodiments that can be combined with any of the foregoing embodiments, the antagonist of TIGIT expression and / or activity is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments that can be combined with any of the foregoing embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments that can be combined with any of the foregoing embodiments, the antagonist of TIGIT expression and / or activity is an inhibitory nucleic acid selected from the group consisting of antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras. In some embodiments that can be combined with any of the foregoing embodiments, the antagonist of PVR expression and / or activity is selected from the group consisting of small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent for inhibiting and / or blocking the interaction between TIGIT and PVR is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent for inhibiting and / or blocking the interaction between TIGIT and PVR L2 is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent for inhibiting and / or blocking the interaction between TIGIT and PVR L3 is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent for inhibiting and / or blocking intracellular signaling mediated by TIGIT bound to PVR is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In some embodiments that can be combined with any of the foregoing embodiments, the agent inhibiting and / or blocking the interaction between TIGIT and PVR L2 is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In some embodiments that can be combined with any of the foregoing embodiments, the agent inhibiting and / or blocking the interaction between TIGIT and PVR L3 is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0111] In other aspects, this disclosure provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, comprising 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 other co-suppressive immune receptors. In other aspects, this disclosure provides the use of an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity in the preparation of a medicament for enhancing or stimulating an immune response or function in an individual, wherein the agent that reduces or inhibits TIGIT expression and / or activity is used in combination with an agent that reduces or inhibits one or more other co-suppressive immune receptors. In other aspects, this disclosure provides the use of an effective amount of an agent that reduces or inhibits one or more other co-suppressive immune receptors in the preparation of a medicament for enhancing or stimulating an immune response or function in an individual, wherein the agent that reduces or inhibits one or more other co-suppressive immune receptors is used in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides pharmaceutical compositions comprising an agent that reduces or inhibits TIGIT expression and / or activity, which are used in combination with an agent that reduces or inhibits one or more other immunosuppressive receptors to enhance or stimulate an immune response or function. In other aspects, this disclosure provides a combination comprising 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 other immunosuppressive receptors. In some embodiments that can be combined with any of the foregoing embodiments, the one or more other immunosuppressive 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 foregoing embodiments, the one or more other immunosuppressive receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, and TIM3.

[0112] In other aspects, this disclosure provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, comprising administering to the individual an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity and an agent that enhances or activates one or more other immune co-stimulatory receptors. In other aspects, this disclosure provides the use of an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity in the preparation of a medicament for enhancing or stimulating an immune response or function in an individual, wherein the agent that reduces or inhibits TIGIT expression and / or activity is used in combination with an agent that enhances or activates one or more other immune co-stimulatory receptors. In other aspects, this disclosure provides the use of an effective amount of an agent that enhances or activates one or more other immune co-stimulatory receptors in the preparation of a medicament for enhancing or stimulating an immune response or function in an individual, wherein the agent that enhances or activates one or more other immune co-stimulatory receptors is used in combination with an agent that reduces or inhibits TIGIT expression and / or activity. In other aspects, this disclosure provides pharmaceutical compositions comprising agents that reduce or inhibit TIGIT expression and / or activity, which are used in combination with agents that enhance or stimulate immune responses or function by increasing or activating one or more other immune co-stimulatory receptors. In other aspects, this disclosure provides a combination comprising an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity and an agent that enhances or activates one or more other immune co-stimulatory receptors. In some embodiments that can be combined with any of the foregoing embodiments, the one or more other immune co-stimulatory 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 foregoing embodiments, the one or more additional immune co-stimulatory 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 foregoing embodiments, the one or more additional immune co-stimulatory receptors are selected from the group consisting of OX-40 and CD27.

[0113] In some embodiments that can be combined with any of the foregoing embodiments, the method further includes administering at least one chemotherapeutic agent. In some embodiments that can be combined with any of the foregoing embodiments, the individual has cancer. In some embodiments that can be combined with any of the foregoing embodiments, the individual is human. In some embodiments that can be combined with any of the foregoing embodiments, the CD4 and / or CD8 T cells in the individual have increased or enhanced initiation, activation, proliferation, cytokine release, and / or cytolysis activity relative to before the administration of the combination. In some embodiments that can be combined with any of the foregoing embodiments, the number of CD4 and / or CD8 T cells is increased relative to before the administration of the combination. In some embodiments that can be combined with any of the foregoing embodiments, the number of activated CD4 and / or CD8 T cells is increased relative to before the administration of the combination. In some embodiments that can be combined with any of the foregoing embodiments, the activated CD4 and / or CD8 T cells are characterized by γ-IFN. + Generative CD4 and / or CD8 T cells and / or enhanced cytolytic activity relative to prior to administration of the combination. In some embodiments that can be combined with any of the foregoing 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 that can be combined with any of the foregoing embodiments, the CD4 and / or CD8 T cells are effector memory T cells. In some embodiments that can be combined with any of the foregoing embodiments, the CD4 and / or CD8 effector memory T cells are characterized by γ-IFN... + Generative D4 and / or CD8 T cells and / or enhanced cytolytic activity. In certain embodiments that can be combined with any of the foregoing embodiments, the CD4 and / or CD8 effector memory T cells are characterized by having CD44 高 CD62L 低The expression of TIGIT. In certain embodiments that can be combined with any of the foregoing embodiments, the cancer has an elevated level of T cell infiltration. In certain embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is selected from the group consisting of: antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, agents that inhibit and / or block the interaction between TIGIT and PVR L2, agents that inhibit and / or block the interaction between TIGIT and PVR L3, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3, and combinations thereof. In certain embodiments that can be combined with any of the foregoing embodiments, the antagonist of TIGIT expression and / or activity is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the antagonist of PVR expression and / or activity is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent that inhibits and / or blocks the interaction between TIGIT and PVR is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent that inhibits and / or blocks the interaction between TIGIT and PVR L2 is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent for inhibiting and / or blocking the interaction between TIGIT and PVR L3 is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent for inhibiting and / or blocking intracellular signaling mediated by TIGIT bound to PVR is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In certain embodiments that can be combined with any of the foregoing embodiments, the agent for inhibiting and / or blocking intracellular signaling mediated by TIGIT bound to PVR L2 is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides.In some embodiments that can be combined with any of the foregoing embodiments, the agent that inhibits and / or blocks intracellular signaling mediated by TIGIT bound to PVR L3 is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In some embodiments that can be combined with any of the foregoing embodiments, the antagonist of TIGIT expression and / or activity is an inhibitory nucleic acid selected from the group consisting of: antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras. In some embodiments that can be combined with any of the foregoing embodiments, the antisense polynucleotide targets TIGIT.

[0114] In some embodiments that can be combined with any of the foregoing embodiments, the interfering RNA targets TIGIT. In some embodiments that can be combined with any of the foregoing embodiments, the catalytic RNA targets TIGIT. In some embodiments that can be combined with any of the foregoing embodiments, the RNA-DNA chimera targets TIGIT. In some embodiments that can be combined with any of the foregoing embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises at least one HVR, which comprises an amino acid sequence selected from the following amino acid sequences: (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ ID NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4), FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6); or (2) RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and GLRGFYAMDY (SEQ ID NO:12). In certain embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody light chain, the antibody light chain comprising...

[0115] (SEQ ID NO:13) or

[0116]

[0117] The amino acid sequence shown in SEQ ID NO:14. In certain embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody heavy chain containing...

[0118] (SEQ ID NO:15) or

[0119] The amino acid sequence shown in SEQ ID NO:16. In certain embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody light chain and an antibody heavy chain, the antibody light chain comprising...

[0120]

[0121] (SEQ ID NO:13) or

[0122]

[0123] The antibody heavy chain contains the amino acid sequence shown in SEQ ID NO:14.

[0124] (SEQ ID NO:15) or

[0125] The amino acid sequence shown in SEQ ID NO:16. In certain embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment, wherein the antibody is selected from the group consisting of: humanized antibodies, chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, and immunotoxins. In some embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises at least one HVR that is at least 90% identical to any of the HVRs shown below: (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 that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or fragment comprises a light chain and / or a heavy chain, the light chain comprising […].

[0126]

[0127] (SEQ ID NO:13) or

[0128]

[0129] The heavy chain contains at least 90% identical amino acid sequences to those shown in (SEQ ID NO:14), and contains amino acid sequences that are identical to those shown in the heavy chain. (SEQ ID NO:15) or

[0130] The amino acid sequence shown in (SEQ ID NO:16) is at least 90% identical. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is selected from the group consisting of PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partner. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 binding antagonist is an antibody. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 binding antagonist is MDX-1106. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 binding antagonist is MK-3475. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 binding antagonist is CT-011. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments that can be combined with any of the foregoing embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments that can be combined with any of the foregoing 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 foregoing embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments that can be combined with any of the foregoing embodiments, the PD-L1 binding antagonist is selected from the group consisting of: YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736.In certain embodiments that can be combined with any of the foregoing embodiments, the anti-PD-L1 antibody comprises a heavy chain and a light chain, the 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 the 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 certain embodiments that can be combined with any of the foregoing embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence.

[0131] (SEQ ID NO:23),

[0132] (SEQ ID NO:40), or

[0133] (SEQ ID NO:41), the light chain variable region contains an amino acid sequence.

[0134] (SEQ ID NO:24). In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments that can be combined with any of the foregoing embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments that can be combined with any of the foregoing embodiments, the PD-L2 binding antagonist is an immunoadhesin. In some embodiments that can be combined with any of the foregoing embodiments, the cancer is selected from the group consisting of: non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphoma, myeloma, mycoses fungoids, Merkel cell carcinoma, and other hematologic malignancies. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered continuously. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered intermittently. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered before the PD-1 axis binding antagonist. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered concurrently with the PD-1 axis binding antagonist. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered after the PD-1 axis binding antagonist. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is administered before the agent that regulates CD226 expression and / or activity. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is administered concurrently with the agent regulating CD226 expression and / or activity. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is administered after the agent regulating CD226 expression and / or activity. In some embodiments that can be combined with any of the foregoing embodiments, the agent reducing or inhibiting TIGIT expression and / or activity is administered before the agent reducing or inhibiting one or more other immunosuppressive receptors. In some embodiments that can be combined with any of the foregoing embodiments, the agent reducing or inhibiting TIGIT expression and / or activity is administered concurrently with the agent reducing or inhibiting one or more other immunosuppressive receptors.In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered after the agent that reduces or inhibits one or more other immunosuppressive receptors. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered before the agent that increases or activates one or more other immunostimulatory receptors. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered simultaneously with the agent that increases or activates one or more other immunosuppressive receptors. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is administered after the agent that increases or activates one or more other immunosuppressive receptors.

[0135] In other respects, this disclosure provides a kit comprising a PD-1 axis binding antagonist and a packaging insert containing instructions for the use of the PD-1 axis binding antagonist in an individual for the treatment of cancer or for delaying cancer progression, in combination with agents that reduce or inhibit TIGIT expression and / or activity.

[0136] In other respects, this disclosure provides a kit comprising a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions for treating cancer or delaying cancer progression in an individual using the PD-1 axis binding antagonist and the agent that reduces or inhibits TIGIT expression and / or activity.

[0137] In other respects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions for the use of the agent that reduces or inhibits TIGIT expression and / or activity in combination with a PD-1 axis binding antagonist to treat cancer or delay cancer progression in an individual.

[0138] In other respects, this disclosure provides a kit comprising a PD-1 axis binding antagonist and a packaging insert containing instructions on using the PD-1 axis binding antagonist in combination with agents that reduce or inhibit TIGIT expression and / or activity to enhance the immune function of individuals with cancer.

[0139] In other respects, this disclosure provides a kit comprising a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions on enhancing the immune function of individuals with cancer using the PD-1 axis binding antagonist and the agent that reduces or inhibits TIGIT expression and / or activity.

[0140] In other respects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions on enhancing the immune function of individuals with cancer by using the agent that reduces or inhibits TIGIT expression and / or activity in combination with a PD-1 axis binding antagonist.

[0141] In other respects, this disclosure provides a kit comprising a PD-1 axis binding antagonist and a packaging insert containing instructions for the use of the PD-1 axis binding antagonist in combination with agents that regulate CD226 expression and / or activity to treat cancer or delay cancer progression in an individual.

[0142] In other respects, this disclosure provides a kit comprising a PD-1 axis binding antagonist and an agent regulating CD226 expression and / or activity, and a packaging insert containing instructions for treating cancer or delaying cancer progression in an individual using the PD-1 axis binding antagonist and the agent regulating CD226 expression and / or activity.

[0143] In other respects, this disclosure provides a kit comprising an agent that regulates CD226 expression and / or activity, and a packaging insert containing instructions for the use of the agent that regulates CD226 expression and / or activity in combination with a PD-1 axis binding antagonist to treat cancer or delay cancer progression in an individual.

[0144] In other respects, this disclosure provides a kit comprising a PD-1 axis binding antagonist and a packaging insert containing instructions on using the PD-1 axis binding antagonist in combination with agents that regulate CD226 expression and / or activity to enhance the immune function of individuals with cancer.

[0145] In other respects, this disclosure provides a kit comprising a PD-1 axis binding antagonist and an agent regulating CD226 expression and / or activity, and a packaging insert containing instructions on enhancing the immune function of individuals with cancer using the PD-1 axis binding antagonist and the agent regulating CD226 expression and / or activity.

[0146] In other respects, this disclosure provides a kit comprising an agent that regulates CD226 expression and / or activity, and a packaging insert containing instructions on enhancing the immune function of an individual with cancer by using the agent that regulates CD226 expression and / or activity in combination with a PD-1 axis binding antagonist.

[0147] In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody. In some embodiments that can be combined with any of the foregoing 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 can be combined with any of the foregoing embodiments, the anti-PD-L1 antibody comprises a heavy chain and a light chain, the 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 the 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 that can be combined with any of the foregoing embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence.

[0148] (SEQ ID NO:23),

[0149] (SEQ ID NO:40), or

[0150] (SEQ ID NO:41), the light chain variable region contains an amino acid sequence.

[0151] (SEQ ID NO:24). In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is an anti-PD-1 antibody. In some embodiments that can be combined with any of the foregoing embodiments, the anti-PD-1 antibody is MDX-1106, MK-3475, or CT-011. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is AMP-224. In some embodiments that can be combined with any of the foregoing embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments that can be combined with any of the foregoing embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments that can be combined with any of the foregoing embodiments, the PD-L2 binding antagonist is an immunoadhesive.

[0152] In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing 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 other immunosuppressive receptors to treat cancer or delay cancer progression in an individual. In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity and an agent that reduces or inhibits one or more other immunosuppressive receptors, and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity and the agent that reduces or inhibits one or more other immunosuppressive receptors to treat cancer or delay cancer progression in an individual. In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits one or more other immunosuppressive receptors and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity in combination with the agent that reduces or inhibits one or more other immunosuppressive receptors to treat cancer or delay cancer progression in an individual. In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity in combination with the agent that reduces or inhibits one or more other immunosuppressive receptors to enhance the immune function of an individual with cancer. In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity and an agent that reduces or inhibits one or more other immunosuppressive receptors, and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity and the agent that reduces or inhibits one or more other immunosuppressive receptors to enhance the immune function of an individual with cancer. In other respects, this disclosure provides a kit comprising an agent that reduces or inhibits one or more other immunosuppressive receptors and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity to enhance the immune function of an individual with cancer. In some embodiments that can be combined with any of the foregoing embodiments, the one or more other immunosuppressive 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 foregoing embodiments, the one or more other immunosuppressive receptors are selected from the group consisting of PD-1, CTLA-4, LAG3, and TIM3.

[0153] In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity in combination with an agent that increases or activates one or more other immune co-stimulatory receptors to treat cancer or delay cancer progression in an individual. In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity and an agent that increases or activates one or more other immune co-stimulatory receptors, and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity and the agent that increases or activates one or more other immune co-stimulatory receptors to treat cancer or delay cancer progression in an individual. In other aspects, this disclosure provides a kit comprising an agent that enhances or activates one or more other immune co-stimulatory receptors and a packaging insert containing instructions for using the agent that enhances or activates one or more other immune co-stimulatory receptors in combination with an agent that reduces or inhibits TIGIT expression and / or activity to treat cancer or delay cancer progression in an individual. In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity in combination with an agent that enhances or activates one or more other immune co-stimulatory receptors to enhance the immune function of an individual with cancer. In other aspects, this disclosure provides a kit comprising an agent that reduces or inhibits TIGIT expression and / or activity and an agent that enhances or activates one or more other immune co-stimulatory receptors, and a packaging insert containing instructions for using the agent that reduces or inhibits TIGIT expression and / or activity and the agent that enhances or activates one or more other immune co-stimulatory receptors to enhance the immune function of an individual with cancer. In other respects, this disclosure provides a kit comprising an agent that enhances or activates one or more other immune co-stimulatory receptors and a packaging insert containing instructions for using the agent that enhances or activates one or more other immune co-stimulatory receptors in combination with an agent that reduces or inhibits TIGIT expression and / or activity to enhance the immune function of an individual with cancer. In some embodiments that can be combined with any of the foregoing embodiments, the one or more other immune co-stimulatory 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 foregoing embodiments, the one or more other immune co-stimulatory 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 foregoing embodiments, the one or more other immune co-stimulatory receptors are selected from the group consisting of OX-40 and CD27.

[0154] In some embodiments that can be combined with any of the foregoing embodiments, the individual is a person. In some embodiments that can be combined with any of the foregoing embodiments, the agent that reduces or inhibits TIGIT expression and / or activity is selected from the group consisting of: antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, agents that inhibit and / or block the interaction between TIGIT and PVR L2, agents that inhibit and / or block the interaction between TIGIT and PVR L3, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2, and agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3. In some embodiments that can be combined with any of the foregoing embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an agent that enhances and / or stimulates CD226 expression and / or activity. In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an agent that enhances and / or stimulates the interaction between CD226 and PVR. In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an agent that enhances and / or stimulates intracellular signaling mediated by CD226 binding to PVR. In certain embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is selected from the group consisting of: agents that inhibit and / or block the interaction between CD226 and TIGIT, antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, agents that inhibit and / or block the interaction between TIGIT and PVR L2, agents that inhibit and / or block the interaction between TIGIT and PVR L3, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2, and agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3. In some embodiments that can be combined with any of the foregoing embodiments, the agent regulating CD226 expression and / or activity is an agent that inhibits and / or blocks the interaction between CD226 and TIGIT.In some embodiments that can be combined with any of the foregoing embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, or an inhibitory peptide. In some embodiments that can be combined with any of the foregoing embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises at least one HVR, which comprises an amino acid sequence selected from the following amino acid sequences: (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ ID NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4), FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6); or (2) RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and GLRGFYAMDY (SEQ ID NO:12). In some embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody light chain containing... (SEQ ID NO:13) or

[0155] The amino acid sequence shown in SEQ ID NO:14. In certain embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody heavy chain containing...

[0156] (SEQ ID NO:15) or

[0157] The amino acid sequence shown in SEQ ID NO:16. In certain embodiments that can be combined with any of the foregoing embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody light chain and an antibody heavy chain, the antibody light chain comprising...

[0158] (SEQ ID NO:13) or

[0159] The antibody heavy chain contains the amino acid sequence shown in SEQ ID NO:14.

[0160] (SEQ ID NO:15) or

[0161] The amino acid sequence shown in (SEQ ID NO:16) is as follows. Attached Figure Description

[0162] Figures 1A-1D TIGIT was shown to be depleted on CD8. + and CD4 + Highly expressed on T cells. Figure 1A Depicting MACS-enriched C57BL6 / J spleen CD8 cells after 24–48 hours of plate-bound anti-CD3 and anti-CD28 stimulation. + T cells. Flow cytometry bar graphs represent TIGIT expression (red) relative to isotype staining (gray). Quantification of TIGIT MFI is also shown. ***, P < 0.001. Data represent 2 independent experiments; n = 3. Figure 1B-1C In this study, C57BL6 / J mice were infected with Armstrong LCMV strain, and spleen cells were analyzed 7 days post-infection. Data represent two independent experiments; n=5. Figure 1B The flow cytometry bar graph shown represents immature (CD44) cells. 低 CD62L 高 ) and effect memory (CD44) 高 CD62L 低 CD4 + and CD8 + TIGIT expression on T cells. Quantification of TIGIT MFI was also shown. ***, P<0.001. Figure 1C The flow cytometry bar graph shown represents PD-1. 高 and PD-1 低 Effect memory CD8 + TIGIT expression on T cells. TIGIT MFI quantification was also shown. ***, P < 0.001. Figure 1D It showed that transient CD4 reduction was achieved in C57BL6 / J mice. +T cells were infected with 13 clones of LCMV. Splenic cells were analyzed 42 days post-infection. Flow cytometry bar graphs represent naïve (CD44) cells. 低 CD62L 高 ), Central Memory (CD44) 高 CD62L 高 ), and effect memory (CD44) 高 CD62L 低 CD8 + TIGIT expression on T cells. Quantification of TIGIT MFI is also shown. ***, P < 0.001. Data represent two independent experiments; n = 5. Error bars depict the standard error of the mean.

[0163] Figure 2 Display TIGIT loxP / loxP Mouse design. The loxP site is flanked by exon 1 of TIGIT, and standard techniques were used.

[0164] Figures 3A-3D TIGIT defect type CD8 was displayed + and CD4 + T cells respond normally to acute viral infection. TIGIT was infected with Armstrong strain LCMV. fl / fl CD4 cre (CKO) and TIGIT fl / fl Litterctic pups (WT). Spleen cells were analyzed 7 days post-infection. Data represent two independent experiments; n = 5. Figure 3A Displayed on CD8 + A representative FACS image of T cell-gated cells, with the box indicating activated (CD44) cells. 高 Cells. Activated CD8 + T cells as part of total CD8 + Quantification of the percentage of T cells. Figure 3B The results showed that after in vitro stimulation, CD8 + A representative FACS image of T cell-gated cells, with IFNγ-generating cells highlighted in boxes. IFNγ-generating cells are represented by total CD8+. + Quantification of the percentage of T cells. Figure 3C Displayed on CD4 + A representative FACS image of T cell-gated cells, with the box indicating activated (CD44) cells. 高 Cells. Activated CD4 + T cells as total CD4 + Quantification of the percentage of T cells. Figure 3D The results showed that after in vitro stimulation, CD4 +A representative FACS image of T cell-gated cells, with IFNg-producing cells highlighted in boxes. IFNg-producing cells are a representative component of total CD4+. + Quantification of the percentage of T cells. Error bars depict the standard error of the mean.

[0165] Figures 4A-4H This study demonstrated that TIGIT and PD-1 synergistically regulate effector function in depleted T cells in vivo. Figures 4A-4E In China, regarding TIGIT fl / fl CD4-cre - (WT) and TIGIT fl / fl CD4-cre + (CKO) mice transiently reduced CD4 + T cells were infected with 13 clones of LCMV. Splenic cells and hepatitis virus titers were analyzed 42 days post-infection. Data represent two independent experiments, with n = 6–9 per group. Figure 4A Depicted on CD8 + A representative FACS image of T cell-gated cells, with activated cells (CD44) indicated by boxes. 高 CD62L 低 Activated cells as total CD8 + Quantification of the percentage of T cells. Figure 4B Depicting CD8 after in vitro stimulation + A representative FACS image of T cell-gated IFNγ, with the box indicating IFNγ. + Cells. IFNγ-producing cells as CD8 + Quantification of the percentage of T cells. Figure 4C Depicted on CD4 + A representative FACS image of T cell-gated cells, with activated cells (CD44) indicated by boxes. 高 CD62L 低 Activated cells as total CD4 + Quantification of the percentage of T cells. Figure 4D Depicting CD4 after in vitro stimulation + A representative FACS image of T cell-gated IFNγ, with the box indicating IFNγ. + Cells. IFNγ-producing cells act as CD4+ cells. + Quantification of the percentage of T cells. Figure 4E Quantification of liver LCMV titer. ***, P < 0.0001. Figure 4F-4H In C57BL6 / J mice, CD4 was transiently reduced. +T cells were collected and mice were infected with clonal 13 LCMV strains. Mice were treated with isotype-matched controls, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies starting at day 28 post-infection. Splenic cell and hepatic virus titers were analyzed at day 42 post-infection. Data represent two independent experiments; n = 10. Figure 4F Depicted on CD8 + A representative FACS image of T cell-gated cells, with activated cells (CD44) indicated by boxes. 高 CD62L 低 Activated cells as total CD8 + Quantification of the percentage of T cells. ***, P<0.0001. Figure 4G Depicting the activation of CD8 after in vitro stimulation + A representative FACS image of T cell-gated IFNγ, with the box indicating IFNγ. + Cells. IFNγ-producing cells act as activated CD8 cells. + Quantification of the percentage of T cells. *, P = 0.0352. **, P = 0.0047. Figure 4H Describes the quantification of liver LCMV titer. *, P = 0.0106. **, P = 0.0047. Error bars depict the standard error of the mean.

[0166] Figures 5A-5B It showed that TIGIT / PD-L1 co-blocked enhanced CD4 during chronic viral infection. + T cell effector function. CD4 reduction in C57BL6 / J mice. + T cells were collected and mice were infected with clonal LCMV strain 13. Mice were treated with isotype control, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies starting 28 days post-infection. Splenic cell and hepatic virus titers were analyzed at 42 days post-infection. Data represent two independent experiments; n = 10. Figure 5A Depicted on CD4 + A representative FACS image of T cell-gated cells, with activated cells (CD44) indicated by boxes. 高 CD62L 低 Activated CD4 + T cells as total CD4 + Quantification of the percentage of T cells. Figure 5B Depicting CD4 after in vitro stimulation + A representative FACS image of T cell-gated cells, with IFNγ-producing cells highlighted in boxes. IFNγ-producing cells are a representative component of total CD4+. + Quantification of the percentage of T cells. *, P = 0.019. Error bars depict the standard error of the mean.

[0167] Figures 6A-6D The study showed that TIGIT expression is elevated in human breast cancer and is associated with the expression of CD8 and inhibitory co-receptors. Microarray data on breast cancer gene expression generated by The Cancer Gene Atlas Network were analyzed. Gene expression data were normalized and expressed as log2. Figure 6A TIGIT expression was depicted in normal and all breast tumor samples (left) and in breast tumor subtypes (right). ***, P = 6 x 10⁻⁶ -12 The diagram shows the boxes and whiskers. Figure 6B Describe the correlation between TIGIT and CD3ε expression. R 2 =0.61. Figure 6C Depicting TIGIT and CD8α (left, R) 2 =0.80) or CD4 (right, R) 2 The correlation was 0.42. Figure 6D Depicting TIGIT and PD-1 (left, R) 2 =0.87), LAG3 (in, R 2 =0.80), and CTLA4 (right, R 2 The correlation was 0.76.

[0168] Figures 7A-7F TIGIT and PD-1 were shown to inhibit anti-tumor T cell responses. Figures 7A-7B In this study, BALB / c mice were inoculated with CT26 colorectal cancer cells. Fourteen days post-inoculation, when the tumor size reached approximately 200 mm... 3 At the same time, spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed. Data represent one experiment; n=6. Figure 7A The flow cytometry bar graphs depict the spleen and tumor-invasive CD8 cells. + TIGIT expression on T cells. Quantification of TIGIT MFI was also shown. **, P = 0.0023. Figure 7B The flow cytometry bar graph depicts the spleen and tumor-invasive CD4 cells. + TIGIT expression on T cells. Quantification of TIGITMFI was also shown. ***, P = 0.0002. Figures 7C-7E In this study, BALB / c mice were inoculated with CT26 colorectal cancer cells. When the tumor size reached approximately 200 mm... 3 Mice were treated for 3 weeks with isotype control, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibody. Data represent two independent experiments; n = 10-20 ( Figures 7C-7D ) or 7-10 ( Figure 7E ). Figure 7C Describe the median CT26 tumor volume over time. Figure 7D Describe mouse survival. Figure 7E This shows that approximately 60 days after initial inoculation, mice in complete regression (CR) receiving anti-TIGIT+anti-PD-L1 and untreated BALB / c mice were inoculated with CT26 cells in their left thoracic flanks and EMT6 breast cancer cells in their mammary fat pads. Median (left) and individual (right) tumor volumes of CT26 (square) and EMT6 (triangle) tumors are shown in CR mice (purple and green) and untreated mice (black and orange). Figure 7F Display as shown Figure 7C Mice were inoculated with CT26 tumors and treated as described in the previous section. Tumor invasiveness and T cells residing in tumor-draining lymph nodes were analyzed by flow cytometry. CD8+ cells were detected after in vitro stimulation. + A representative FACS image of TILs, with IFNγ-producing cells highlighted within boxes. IFNγ-producing CD8 cells. + TIL as the total CD8 + Quantification of the percentage of TIL. ***, P = 0.0003. Data represent 2 independent experiments; n = 5. Error bars depict the standard error of the mean.

[0169] Figures 8A-8B The study showed that TIGIT expression in CT26 tumor-infiltrating lymphocytes was correlated with Tim-3 expression. BALB / c mice were inoculated with CT26 colorectal cancer cells. Approximately 14 days post-inoculation, when the tumor size reached approximately 200 mm... 3 At the same time, spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed. Data represent one experiment; n=6. Figure 8A Depicting representative bar graphs of spleen and tumor-invasive CD8 + TIGIT expression on T cells. Quantification of TIGIT MFI. **, P = 0.0026. Figure 8B Describing spleen and tumor-invasive CD4 + A representative bar chart of TIGIT expression on T cells. Quantification of TIGIT MFI. ***, P < 0.0001. Error bars depict the standard error of the mean.

[0170] Figures 9A-9B The study showed that TIGIT expression in MC38 tumor-infiltrating lymphocytes was associated with PD-1 and Tim-3 expression. C57BL6 / J mice were inoculated with MC38 colorectal cancer cells. Approximately 14 days post-inoculation, when the tumor size reached approximately 200 mm... 3 At the same time, spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed. Data represent one experiment; n=5. Figure 9A Describing spleen and tumor-invasive CD8 +Representative bar chart of TIGIT expression on T cells. Quantification of TIGIT MFI. ***, P<0.0001. Figure 9B Describing spleen and tumor-invasive CD4 + Representative bar chart of TIGIT expression on T cells. Quantification of TIGIT MFI. *, P = 0.0136. **, P = 0.0029. Error bars depict the standard error of the mean.

[0171] Figure 10 The growth of CT26 tumors in mice treated with anti-PD-L1 and / or anti-TIGIT is shown. Untreated BALB / c mice were inoculated with CT26 tumor cells and treated with anti-PD-L1 and / or anti-TIGIT or allotype-matched control antibodies, such as... Figure 4D-4F As described above. Tumor volume over time is shown for individual mice in each treatment group. Data represent two independent experiments.

[0172] Figures 11A-11F CD4 was displayed + Flow cytometry analysis of TILs and tumor-draining lymph node T cells. BALB / c mice were inoculated with CT26 colorectal cancer cells. When the tumor size reached approximately 200 mm... 3 Mice were treated for 7 days with isotype control, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies. Tumors and tumor-draining lymph nodes were harvested. Data represent two independent experiments; n = 5. CD8+ was observed in tumor-draining lymph nodes after in vitro stimulation. + A representative FACS image of T cell-gated cells, with boxes indicating IFNγ-producing cells. IFNγ + Cells as total CD8 + Quantification of the percentage of T cells. ***, P<0.001. CD8 + Quantification of T cells as a percentage of total TILs. **, P = 0.0065. Activated (CD44) 高 CD62L (low) CD8 + T cells as part of total CD8 + Quantification of TIL percentage. *, P = 0.012. CD8 + Quantification of the percentage of T cells in total tumor-draining lymph nodes. Activated CD8+ cells in tumor-draining lymph nodes. + T cells as part of total CD8 + Quantification of the percentage of T cells. *, P<0.05. Figure 11C Depicting CD4 + Quantification of T cells as a percentage of total TILs. *, P = 0.016. Figure 11D Depicting activated CD4 +T cells as total CD4 + Quantification of the percentage of TIL. Figure 11E Depicting CD4 + Quantification of T cells as a percentage of total tumor-draining lymph node cells. Figure 11F Depicting activated CD4 in tumor-draining lymph nodes + T cells as total CD4 + Quantification of the percentage of T cells. Figure 11A Depicting IFNγ after in vitro stimulation + Cells as CD4 + Quantification of the percentage of TIL. Figure 11B Depicting IFNγ in tumor-draining lymph nodes after in vitro stimulation + Cells as CD4 + Quantification of the percentage of T cells. Error bars depict the standard error of the mean.

[0173] Figures 12A-12C CD8 was displayed + Further flow cytometry analysis of TILs. As shown in Figure 4, BALB / c mice were inoculated with CT26 colorectal cancer cells and treated with isotype controls, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies. Tumors were harvested 7 days after treatment and analyzed by flow cytometry. Data represent two independent experiments; n = 5. Figure 12A Describing TNFα + Cells as total CD8 + Quantification of the percentage of TIL. **, P<0.01. Figure 12B Depicting CD8 + TIL is quantified as a percentage of total TIL. **, P<0.01. Figure 12C Depicting the activated (CD44) 高 CD62L 低 CD8 + TIL as the total CD8 + Quantification of the percentage of TIL. *, P<0.05. Error bars depict the standard error of the mean.

[0174] Figures 13A-13D CD8 cells show tumor-draining lymph node residency. + Flow cytometry analysis of T cells. As shown in Figure 4, BALB / c mice were inoculated with CT26 colorectal cancer cells and treated with isotype controls, anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT antibodies. Tumor draining lymph nodes were harvested 7 days after treatment and analyzed by flow cytometry. Data represent two independent experiments; n = 5. Figure 13A Depicting CD8 receptors residing in tumor-draining lymph nodes after in vitro stimulation+ A representative FACS image of T cell-gated cells, with boxes indicating IFNγ-producing cells. IFNγ + Cells as total CD8 + Quantification of the percentage of T cells. ***, P<0.001. Figure 13B Depicting CD8 + The quantification of T cells as a percentage of total cells in tumor-draining lymph nodes. Figure 13C Depicting the activated (CD44) 高 CD62L 低 CD8 + T cells as part of total CD8 + Quantification of the percentage of T cells. *, P<0.05. Error bars depict the standard error of the mean. Figure 13D Depicting TNFα-producing cells as CD8+ cells in total tumor-draining lymph nodes + Quantification of the percentage of T cells.

[0175] Figure 14 It showed tumor-invasive CD8 + Co-expression of CD226 and TIGIT on T cells. C57BL6 / J mice were inoculated with MC38 colorectal cancer cells. Approximately 14 days post-inoculation, when the tumor reached approximately 200 mm... 3 When analyzing the size of spleen cells and tumor-infiltrating lymphocytes (TILs), spleen B cells (gray) and spleen CD8 cells were also examined. + T cells (blue), and TIGIT + Tumor-invasive CD8 + A representative bar chart of CD226 expression on T cells (red). Data represent two independent experiments; n = 5.

[0176] Figure 15 The image shows CD226 and TIGIT co-precipitate (co-IP) from transfected cells. COS7 cells were co-transfected with an expression plasmid containing either a TIGIT-HA (5 ng) or CD226-Flag (10 ng) labeled protein, or a control plasmid (pRK). After transfection, cells were washed, centrifuged, and cell clumps were lysed. The pre-clarified and centrifuged supernatant was then aliquoted into two tubes and immunoprecipitated using either anti-HA or anti-flag immunoprecipitation according to standard protocol. The immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting. Western blotting was probed with either anti-Flag-HRP or anti-HA-HRP.

[0177] Figure 16 TIGIT and CD226 were displayed on the original CD8. +Interactions in T cells. Stimulation of MACS-enriched spleen C57BL6 / J CD8 cells with plate-bound anti-CD3 and anti-CD28 antibodies and recombinant IL-2. + T cells were lysed after 48 hours. Cell lysates were immunoprecipitated with anti-TIGIT and probed with anti-CD226. Path: Molecular weight ladder (1), input (2), immunoprecipitate flow filtrate (3), and immunoprecipitate. Arrows indicate the expected molecular weight of CD226.

[0178] Figures 17A-17D The detection of TIGIT / CD226 interaction via TR-FRET is shown. Figure 17A Depicting the dissociation of Flag-ST-CD226 homodimer by HA-TIGIT. FRET ratio between Flag-ST-CD226 expressed at constant levels and in COS-7 cells with increased HA-TIGIT concentrations. Figure 17B The FRET ratio between Flag-ST-CD226 was recorded after 15 minutes of incubation with either PBS (white column) or anti-TIGIT antibody (black column). Figure 17C The binding of Flag-ST-CD226 to HA-TIGIT was depicted. FRET intensity between Flag-ST-CD226 and HA-TIGIT was measured in the same batch of transfected COS-7 cells using an anti-Flag ELISA assay. Figure 17D The FRET variation between Flag-ST-CD226 and HA-TIGIT was plotted after 15 minutes of incubation with PBS (white column) or anti-TIGIT antibody (black column). Data in A and C represent four independent experiments, each performed in triplicate. Data in B and D represent two independent experiments, each performed in triplicate.

[0179] Figure 18 Cell surface expression of Flag-ST-CD226 and HA-TIGIT is shown. Anti-Flag and anti-HA ELISA were performed on intact COS-7 cells expressing the tagged constructs. Data represent three independent experiments, each performed in triplicate.

[0180] Figures 19A-19D This study demonstrated that CD226 blockade reversed the enhanced antiviral T-cell response induced by TIGIT / PD-L1 co-blockade. Figures 19A-19D In C57BL6 / J mice, CD4 was transiently reduced. +T cells were collected and mice were infected with clonal LCMV strains. Starting 28 days post-infection, mice were treated with isotype-matched controls, anti-CD226, anti-PD-L1 + anti-TIGIT, or anti-PD-L1 + anti-TIGIT + anti-CD226 antibodies. Splenic cell and hepatic virus titers were analyzed 42 days post-infection. Figure 19A Depicting CD8 + Quantification of the percentage of T cells as spleen cells. Figure 19B Depicting activated CD8 + T cells as part of total CD8 + Quantification of the percentage of T cells. ***, P<0.001. Figure 19C Depicting IFNg-producing cells as activated CD8 + Quantification of the percentage of T cells. ***, P<0.001. Figure 19D Describe the quantification of liver LCMV titer. ***, P < 0.001. Error bars depict the standard error of the mean.

[0181] Figures 20A-20H The results show that TIGIT expression is elevated in human cancers and strongly correlated with CD8 and PD-1. Gene expression analysis in human cancers was performed as described in Example 11. Scatter plots show per-gene counts normalized to library size. Box and whisker plots show the variation in the stable expression ratios of TIGIT and CD3e. Figure 20A The correlation between TIGIT and CD3e RNA expression in LUSC (grey) and normal lung (black) was depicted. ρ = 0.86. The quantification of the TIGIT / CD3e expression ratio was also shown. The LUSC ratio was increased by 372%. ***, P = 1.46 x 10⁻⁶. -46 . Figure 20B The correlation between TIGIT and CD3e RNA expression in COAD (grey) and normal colon (black) was depicted. ρ = 0.83. The quantification of the TIGIT / CD3e expression ratio was also shown. COAD ratio increase = 116%. ***, P = 3.66 x 10⁻⁶ -6 . Figure 20C The correlation between TIGIT and CD3eRNA expression in UCEC (grey) and normal endometrium (black) was depicted. ρ = 0.87. The quantification of the TIGIT / CD3e expression ratio was also shown. The UCEC ratio increased by 419%. ***, P = 7.41 x 10⁻⁶. -5 . Figure 20D The correlation between TIGIT and CD3e RNA expression in BRCA (grey) and normal breast (black) was depicted. ρ = 0.82. The quantification of the TIGIT / CD3e expression ratio was also shown. BRCA ratio increase = 313%. ***, P = 4.6 x 10⁻⁶ -44 . Figure 20EThe correlation between TIGIT and CD3e RNA expression was depicted in clear cell renal cell carcinoma (gray) and normal kidney (black). ρ = 0.94. The quantification of the TIGIT / CD3e expression ratio was also shown. Figure 20F The correlations between TIGIT and CD8A (left) or TIGIT and CD4 (right) are depicted in squamous cell carcinoma of the lung (gray) and normal lung (black). The values ​​are ρ = 0.77 and 0.48, respectively. Figure 20G The correlation between TIGIT and PD-1 (Pdcd1) was depicted in lung squamous cell carcinoma (gray) and normal lung (black). ρ = 0.82. Figure 20H The correlation between TIGIT and CD226 is depicted in squamous cell carcinoma of the lung (red) and normal lung (black). ρ = 0.64.

[0182] Figure 21 Analysis of T-cell-related gene expression in lung squamous cell carcinoma (LUSC) is presented. Gene expression in LUSC and normal tissue samples was analyzed as described in Example 11, and a heatmap of genes with the best correlation to gene signatures in the LUSC samples was generated. Hierarchical clustering was used to cluster both genes and samples using Ward links on the Euclidean distance matrix of the centralized and calibrated expression data.

[0183] Figures 22A-22G The study showed that TIGIT and PD-1 were co-expressed by human and mouse tumor-infiltrating lymphocytes. Figures 22A-22C This diagram shows an analysis of lymphocytes from freshly excised human NSCLC tumors, tumor-matched peripheral blood, and normal donor peripheral blood. The data represent two independently analyzed tumors. Figure 22A The representative FACS map depicts peripheral and tumor-invasive CD8 cells. + TIGIT expression on T cells, with TIGIT highlighted in the box. + cell. Figure 22B The representative FACS map depicts peripheral and tumor-invasive CD4+. + TIGIT expression on T cells, with TIGIT highlighted in the box. + cell. Figure 22C The flow cytometry bar chart depicting PD-1 高 (Red) and PD-1 低 (Blue) NSCLC Infiltrating CD8 + (Left) and CD4 + (Right) TIGIT expression on T cells. Figure 22D-22G In this study, BALB / c mice were inoculated with syngeneic CT26 colorectal cancer cells. Fourteen days post-inoculation, when the tumor reached approximately 200 mm... 3When analyzing the size of spleen cells and tumor-infiltrating lymphocytes (TILs), the data represent two independent experiments; n = 5–6. Figure 22D Describing tumor-invasive CD8 + A representative FACS image of TIGIT expression on T cells, with TIGIT highlighted in boxes. + cell. Figure 22E Describing tumor-invasive CD4 + A representative FACS image of TIGIT expression on T cells, with TIGIT highlighted in boxes. + Cells. TIGIT + The frequency of T cells is quantified as a percentage of all T cells. *, P = 0.0134. ***, P < 0.0001. Figure 22F The flow cytometry bar chart depicting PD-1 高 and PD-1 低 Tumor-invasive CD8 + T cells and spleen CD8 + TIGIT expression on T cells. Quantification of TIGIT MFI was also shown. **, P = 0.0023. Figure 22G The flow cytometry bar chart depicting PD-1 高 and PD-1 低 Tumor-invasive CD4 + T cells and spleen CD4 + TIGIT expression on T cells. Quantification of TIGIT MFI is also shown. ***, P = 0.0002. Error bars depict the standard error of the mean.

[0184] Figures 23A-23D Characterization of TIGIT expression in human tumor-infiltrating T cells was demonstrated. Figures 23A-23B The depicted FACS map shows invasive CD8+ in NSCLC tumors. + and CD4 + T cells ( Figure 23A ) and donor-matched PBMC CD8 + and CD4 + T cells ( Figure 23B The TIGIT expression, where the box indicates TIGIT. + cell. Figures 23C-23D The depicted FACS map shows CRC tumor infiltration of CD8. + and CD4 + T cells ( Figure 23C ) and donor-matched PBMC CD8 + and CD4 + T cells ( Figure 23D The TIGIT expression, where the box indicates TIGIT. +cell.

[0185] Figure 24 The results showed that the TIGIT:CD226 interaction was not driven by PVR TIGIT:CD226, and the TIGIT Q56R:CD226 interaction was detected by TR-FRET. The FRET ratios between Flag-ST-CD226 and HA-TIGIT or HA-TIGIT Q56R showed that WT and Q56R TIGIT bind to CD226 with equal potency. Data represent three independent experiments performed in triplicate.

[0186] Figures 25A-25C The efficacy of TIGIT / PD-L1 antibody co-blockade in mice carrying MC38 tumors was demonstrated. Figures 25A-25C In this study, mice carrying MC38 tumors were generated as described above and treated for 3 weeks with blocking antibodies against PD-L1 (red), TIGIT (blue), TIGIT and PD-L1 (purple), or an isotype-matched control antibody (black). N = 10 (control, anti-PD-L1 alone, anti-TIGIT alone) or 20 (anti-TIGIT + anti-PD-L1). Figure 25A The median (left) and individual (right) MC38 tumor volumes are depicted over time. Figure 25B The tumor volume of MC38 cells was plotted 14 days after antibody treatment. ***, P = 0.0005. **, P = 0.0093. *, P = 0.0433. Figure 25C The mouse survival over time is plotted. Error bars depict the standard error of the mean.

[0187] Figures 26A-26E Further characterization of TIGIT expression in mouse tumor-infiltrating T cells was demonstrated. Figure 26A Depicting spleen C57BL6 / JCD8 enrichment via MACS + T cells were cultured with plate-coated anti-CD3 and anti-CD28 agonist antibodies. Representative bar graphs of TIGIT (red) staining over time and isotype-matched controls (solid gray). Quantification of TIGIT MFI. ***, P < 0.001. Stimulated cells induced PD-1 expression and constitutively expressed CD226 (data not shown). Data represent 2 independent experiments; n = 5. Figure 26B-26E Wild-type C57BL6 / J mice were subcutaneously inoculated with syngeneic MC38 colorectal cancer cells. Tumors were allowed to grow without intervention until they reached a size of 150-200 mm. 3 The data represent two independent experiments; n = 5. Figure 26B Describing tumor-invasive CD8 + A representative FACS image of T cells, with TIGIT highlighted in the box.+ Cells. TIGIT + Cell frequency as a measure of all tumor invasiveness or spleen CD8 + Quantification of the percentage of T cells. ***, P<0.0001. Figure 26C Describing tumor-invasive CD4 + A representative FACS image of T cells, with TIGIT highlighted in the box. + Cells. TIGIT + Cell frequency as a measure of all tumor invasiveness or spleen CD4 + Quantification of the percentage of T cells. ***, P<0.0001. Figure 26D Depicting PD-1 高 and PD-1 低 Tumor-invasive CD8 + T cells (red and blue, respectively) and spleen CD8 + A representative bar chart of TIGIT expression in T cells (gray). Quantification of TIGIT MFI. ***, P<0.0001. Figure 26E Depicting PD-1 高 and PD-1 低 Tumor-invasive CD4 + T cells and spleen CD4 + Representative bar chart of TIGIT expression on T cells. Quantification of TIGIT MFI. *, P = 0.0136. **, P = 0.0029. Error bars depict the standard error of the mean.

[0188] Figures 27A-27B It showed tumor-invasive CD8 + and CD4 + T cells maintained high levels of CD226 expression. Wild-type BALB / c mice were inoculated with CT26 tumor cells as described in this article. Tumors grew to approximately 150-200 mm. 3 After determining the size, the tumor and spleen were analyzed by flow cytometry. Figure 27A Depicting CD226 + CD8 + T cells, CD4 + T cells and non-T cells, respectively, represent all CD8 cells. + T cells, CD4 + Quantification of the percentage of T cells and non-T cells. Figure 27B A representative bar chart depicting CD226 expression in tumors and spleen. Data represent two independent experiments; n = 5. Error bars depict the standard error of the mean.

[0189] Figures 28A-28F CD8 was displayed +TIGIT inhibition of the T-cell response is CD226-dependent. BALB / c mice were subcutaneously inoculated with CT26 colorectal cancer cells in the right lateral thorax. When the tumor reached approximately 200 mm... 3 When measuring the size, mice were treated for 3 weeks with isotype control (black), anti-CD226 (orange), anti-PD-L1 (red), anti-TIGIT + anti-PD-L1 (purple), or anti-TIGIT + anti-PD-L1 + anti-CD226 (green) antibodies. Data represent one experiment; n = 10 (AB) or 5 (CF). Figure 28A Median (left) and individual (right) CT26 tumor volumes over time are depicted. Figure 28B Describe the survival of mice over time. Figures 28C-28F In this study, after 7 days of treatment, tumor-infiltrating lymphocytes and lymphocytes residing in tumor-draining lymph nodes were assessed by flow cytometry. Figure 28C Depicting CD8 generation of IFNγ after in vitro stimulation + TIL as the total CD8 + Quantification of the percentage of TILs. **, P < 0.01. FIG 28D depicts IFNγ-producing cells as total CD8+ after in vitro stimulation. + Quantification of the percentage of T cells. *, P<0.05. Figure 28E Depicting CD8 + TIL as a percentage of total TIL. **, P < 0.01. FIG 28F depicts CD8. + T cells are quantified as the percentage of all tumor-draining lymph nodes residing in the lymph nodes. Error bars depict the standard error of the mean.

[0190] Figures 29A-29H This demonstrates that TIGIT weakens CD226 function by directly disrupting CD226 homodimerization. Figure 29A Depicted from TIGIT fl / fl CD4 cre (CKO) and TIGIT fl / fl CD4 wt (WT) Litters enrich CD8 through MACS + T cells were stimulated, as shown, in the presence of either anti-CD226 or an allotype-matched control antibody. H3-thymidine uptake was indicated by the ratio of cell cultures using anti-CD3+PVR-Fc to those using anti-CD3 alone. **, P = 0.0061. ***, P < 0.0001. Data represent two independent experiments; n = 5. Figure 29B Depicting the enrichment of wild-type C57BL6 / JCD8 by MACS +T cells were stimulated, as shown, in the presence of anti-TIGIT, anti-CD226, and / or allotype-matched control antibodies. The ratio of cell cultures using anti-CD3+PVR-Fc to those using anti-CD3 alone was used to show H... 3 - Thymidine uptake. ***, P < 0.001 in paired t-test. Figure 29C Depicting the enrichment of primary human CD8 from blood via MACS + T cells were stimulated with suboptimal levels of plate-bound anti-CD3 in the presence or absence of human recombinant PVR-Fc. Anti-TIGIT antibody or an allotype-matched control antibody was added as shown. 3 Quantification of H-thymidine uptake. **, P = 0.0071 and 0.0014, respectively. Figure 29D The study depicted transient transfection of CHO cells with elevated concentrations of both recipient and donor FLAG-ST-CD226 as shown. FRET intensity was quantified relative to donor emission. Data represent 3 independent experiments; n=3. Figures 29E-29F In this study, CHO cells were transiently transfected with FLAG-ST-CD226 and elevated concentrations of HA-TIGIT, as shown. Data represent two or more independent experiments; n = 4. Data were normalized to the maximum signal. Figure 29E Quantization of the CD226:CD226 FRET ratio (FRET ratio 1). Figure 29F Quantization of TIGIT:CD226 FRET ratio (FRET ratio 2). Figure 29G This image depicts anti-FLAG (left) and anti-HA (right) immunoblotting results of either anti-FLAG (left) or anti-HA (right) immunoprecipitates prepared from COS-7 cells transfected with either the self-administered empty pRK vector or a combination of Flag-CD226 and HA-TIGIT. Data represent two independent experiments. Figure 29H Plot the quantification of the TIGIT:CD226FRET ratio after incubation with PBS (white) or anti-TIGIT antibody (red). ***, P < 0.001. Data represent 4 independent experiments; n = 3. Error bars plot the standard error of the mean.

[0191] Figure 30 Primary human T cells were enriched from blood via MACS and stimulated with anti-CD3 and anti-CD28. TIGIT sorting was performed. + and TIGIT - Cells were conditioned, rested, restimulated, and labeled with the antibody for FRET as shown. Data represent two independent experiments. ***, P < 0.001. Error bars depict the standard error of the mean.

[0192] Figures 31A-31CThis demonstrates that TIGIT and PD-1 co-blockade does not restore depleted CD4 during chronic viral infection. + T cell effector function. Figure 31A Depicting CD8 + Quantification of T cells as a percentage of all spleen cells. Figure 31B Describing GP33 pentamer + Cells as all spleen CD8 + Quantification of the percentage of T cells. **, P = 0.0040. Figure 31C Depicting gp33 pentamer after in vitro stimulation + CD8 + A representative FACS image of T cell-gated IFNγ, with the box indicating IFNγ. + Cells. IFNγ-producing cells as all gp33 pentamers + CD8 + Quantification of the percentage of T cells. *, P = 0.0319. **, P = 0.0030. Error bars depict the standard error of the mean.

[0193] Figures 32A-32C The efficacy of TIGIT / PD-L1 co-blockade was shown to be CD8-dependent. + T cells. In Figures 32A-32B In Figure 7, wild-type BALB / c mice were inoculated with CT26 tumors. When the tumor reached 100-150 mm... 3 At the size of [the target], CD8 was temporarily reduced in mice. + T cells were treated with anti-TIGIT and anti-PD-L1. Data represent one experiment; n = 10 / group. Figure 32A Median (left) and individual (right) CT26 tumor volumes over time are depicted. Figure 32B The quantification of CT26 tumor volume was depicted 17 days after the start of treatment. ***, P = 0.0004. Figure 32C In this study, wild-type BALB / c mice were inoculated with CT26 tumors and treated with anti-TIGIT + anti-PD-L1, followed by rechallenge with CT26 tumors. During the rechallenge, CD8 was transiently reduced. + T cells. Data represent 2 independent experiments; n=5. Figure 32C The median (left) and individual (right) CT26 tumor volumes are plotted over time. Error bars depict the standard error of the mean.

[0194] Figure 33 This showed that PVR expression on tumor cells was negligible for the co-blockade effect of TIGIT / PD-L1. Wild-type BALB / c mice were inoculated with wild-type or PVR-deficient (PVR.KO) tumors as described. When the tumors reached 150-200 mm...3 When the size was reached, mice were treated with anti-TIGIT + anti-PD-L1 or an isotype-matched control antibody. Data represent one experiment; n = 10 / group. Figure 33 Median (left) and individual (right) CT26 tumor volumes over time are depicted.

[0195] Figure 34 The efficacy of TIGIT / PD-L1 antibody co-blockade in mice carrying EMT6 tumors was demonstrated. Mice carrying EMT6 tumors were generated as described above and treated for 3 weeks with blocking antibodies against PD-L1 (red), TIGIT (blue), TIGIT and PD-L1 (purple), or an isotype-matched control antibody (black). N = 10 (control, anti-PD-L1 alone, anti-TIGIT alone) or 20 (anti-TIGIT + anti-PD-L1). Figure 34 Median (left) and individual (right) EMT6 tumor volumes over time are depicted.

[0196] Figures 35A-35B TIGIT showed modulation of tumor-invasive CD8 + T-cell effector function. As shown in Figure 7, BALB / c mice were subcutaneously inoculated with CT26 colorectal cancer cells in the right lateral thorax and treated with anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT. Tumor-draining lymph node (dLN) resident and tumor-infiltrating T cells were analyzed by flow cytometry at 7 days after treatment initiation. Data represent two independent experiments; n = 5. Figure 35A Depicting CD8+ of IFNγ / TNFα dual-generative dLN residency + and CD4 + T cells, as CD8 cells residing in total dLN, respectively + and CD4 + Quantification of the percentage of T cells. Dual cytokine production in unstimulated T cells was also shown. **, P = 0.002, 0.003, and 0.001, respectively. Figure 35B Describing IFNγ / TNFα dual-genetic tumor-invasive CD8 + and CD4 + T cells were used as a representative of total tumor-infiltrating CD8 cells. + and CD4 + Quantification of the percentage of T cells. Dual cytokine production by unstimulated T cells is also shown. ***, P < 0.0001. Error bars depict the standard error of the mean.

[0197] Figures 36A-36BThis paper presents an analysis of lymphocytes from resected human NSCLC tumors, tumor-matched peripheral blood, and normal donor peripheral blood. Data were pooled from three independently acquired samples. Figure 36A Depicting TIGIT + Cells as all CD8 + Quantification of the percentage of T cells. *, P<0.05. Figure 36B Depicting TIGIT + Cells as all CD4 + Quantification of the percentage of T cells.

[0198] Figures 37A-37B This demonstrates the characterization of TIGIT expression in human tumors. Figure 37A Depicting isotype staining (gray) relative to subset matching, NSCLC tumor-resident lymphocytes (red, CD45) + FSC 低 Myeloid cells (blue, CD45) + FSC 高 ), and non-hematopoietic cells (green, CD45) - Representative flow cytometry bar graph of TIGIT expression. Figure 37B Depicting PD-1 高 and PD-1 低 NSCLC tumor-invasive CD8 + and CD4 + T-cell gating strategies. Detailed Implementation

[0199] I. General Technology

[0200] The techniques and procedures described or mentioned herein are generally well understood and commonly used by those skilled in the art, and are performed using conventional methods, such as those widely used, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (edited by F.M. Usubel et al., (2003)); Methods in Enzymology (Academic Press, Inc.): PCR2: A Practical Approach (edited by M.J. MacPherson, B.D. Hames and G.T. Taylor, (1995)); Harlow and Lane (edited by Harlow and Lane, (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by R.R. Freshney, (1987)); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Manual. Notebook (JECellis, 1998) Academic Press; Animal Cell Culture (RIFreshney), 1987); Introduction to Cell and Tissue Culture (JPMather and PERoberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A.Doyle, JBGriffiths, and DG Newell, 1993-8) J.Wiley and Sons; Handbook of Experimental Immunology (eds. DMWeir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos (ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); CurrentProtocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty. ed., IRL Press, 1988-1989); Monoclonal Antibodies: APractical Approach (edited by P.Shepherd and C.Dean, Oxford University Press, 2000); Using Antibodies: ALaboratory Manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (edited by V.T. DeVita et al., J.L. Bippincott Company, 1993).

[0201] II. Definition

[0202] The term "PD-1 axis binding antagonist" is a molecule that inhibits the interaction between a PD-1 axis binding partner and one or more of its binding partners, thereby eliminating T cell dysfunction originating from signal transduction along the PD-1 signaling axis (resulting in the restoration or enhancement of T cell function, such as proliferation, cytokine production, and target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0203] The term "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction originating from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partners. In one specific aspect, a 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, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction originating from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, a PD-1 binding antagonist reduces negative co-stimulatory signals via PD-1, thereby making dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition), said negative co-stimulatory signals being mediated by or via cell surface proteins expressed on T lymphocytes. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In one specific aspect, the PD-1 binding antagonist is MDX-1106, as described herein. In another specific aspect, the PD-1 binding antagonist is Merck3745, as described herein. In yet another specific aspect, the PD-1 binding antagonist is CT-011, as described herein. In yet another specific aspect, the PD-1 binding antagonist is AMP-224, as described herein.

[0204] The term "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction originating from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In one specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, a PD-L1 binding antagonist includes an anti-PD-L1 antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, an oligopeptide, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction originating from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and B7-1. In one embodiment, the PD-L1 binding antagonist reduces negative co-stimulatory signals via PD-L1, thereby making dysfunctional T cells less susceptible to dysfunction (e.g., enhanced effector responses to antigen recognition), said negative co-stimulatory signals being mediated by or via signal transduction mediated by cell surface proteins expressed on T lymphocytes. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In one specific aspect, the anti-PD-L1 antibody is YW243.55.S70 described herein. In another specific aspect, the anti-PD-L1 antibody is MDX-1105 described herein. In yet another specific aspect, the anti-PD-L1 antibody is MPDL3280A described herein. In yet another specific aspect, the anti-PD-L1 antibody is MEDI 4736 described herein.

[0205] The term "PD-L2 binding antagonist" is a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction originating 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 with its binding partners. In one specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, a PD-L2 antagonist includes an anti-PD-L2 antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, an oligopeptide, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction originating from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2 binding antagonist reduces negative co-stimulatory signals via PD-L2, thereby making dysfunctional T cells less dysfunctional (e.g., enhancing effector responses to antigen recognition), said negative co-stimulatory signals being mediated by or via signal transduction mediated by cell surface proteins expressed on T lymphocytes. In some implementations, the PD-L2 binding antagonist is an immunoadhesive.

[0206] The term "aptamer" refers to a nucleic acid molecule capable of binding to a target molecule, such as a polypeptide. For example, the aptamer of the present invention specifically binds to the TIGIT polypeptide or molecules in signaling pathways that regulate TIGIT expression. The generation and therapeutic uses of aptamers are well established in the art. See, for example, U.S. Patent No. 5,475,096, and... (Eyetech, New York) is effective in treating age-related macular degeneration.

[0207] The term "antagonist" is used in the broadest sense to include any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of the natural polypeptides disclosed herein. Similarly, the term "agonist" is used in the broadest sense to include any molecule that mimics the biological activity of the natural polypeptides disclosed herein. Suitable agonist or antagonist molecules explicitly include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of polypeptides may include contacting the polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities typically associated with said polypeptide.

[0208] The terms "TIGIT antagonist" and "antagonist of TIGIT activity or expression" are used interchangeably to refer to compounds that interfere with the normal functioning of TIGIT by reducing the transcription or translation of TIGIT-encoding nucleic acids, or by inhibiting or blocking the activity of TIGIT peptides, or both. Examples of TIGIT antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, TIGIT-specific aptamers, anti-TIGIT antibodies, TIGIT-binding fragments of anti-TIGIT antibodies, TIGIT-binding small molecules, TIGIT-binding peptides, and other peptides 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 other domains), such that the interaction between the TIGIT antagonist and TIGIT results in a reduction or cessation of TIGIT activity or expression. Those skilled in the art will understand that in some cases, a TIGIT antagonist may antagonize one TIGIT activity without affecting another TIGIT activity. For example, a desired TIGIT antagonist for use in some of the methods described herein is a TIGIT antagonist that antagonizes TIGIT activity in response to, for example, one of the PVR interaction, PVRL3 interaction, or PVRL2 interaction, but does not affect or minimally affects any other TIGIT interaction.

[0209] The terms "PVR antagonist" and "antagonist of PVR activity or expression" are used interchangeably to refer to compounds that interfere with the normal functioning of PVR by reducing the transcription or translation of PVR-encoded nucleic acids, or by inhibiting or blocking the activity of PVR peptides, or both. Examples of PVR antagonists include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, PVR-specific aptamers, anti-PVR antibodies, PVR-binding fragments of anti-PVR antibodies, PVR-binding small molecules, PVR-binding peptides, and other peptides 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 other domains), such that the interaction between the PVR antagonist and PVR results in a reduction or cessation of PVR activity or expression. Those skilled in the art will understand that in some cases, a PVR antagonist may antagonize one PVR activity without affecting another. For example, a desired PVR antagonist for use in some of the methods described herein is a PVR antagonist that antagonizes PVR activity in response to TIGIT interaction but does not affect PVR-CD96 and / or PVR-CD226 interaction.

[0210] The term "functional impairment" in the context of immune dysfunction refers to a reduced immune responsiveness to antigenic stimuli. This term encompasses both the elements of exhaustion and / or non-responsiveness, where antigen recognition may occur, but the subsequent immune response is ineffective in controlling infection or tumor growth.

[0211] As used herein, the term “dysfunction” also includes a lack of sensitivity or response to antigen recognition, and in particular, an impaired ability to translate antigen recognition into downstream T cell effector functions, such as proliferation, cytokine production (e.g., IL-2), and / or target cell killing.

[0212] The term "unresponsiveness" refers to incomplete or insufficient signaling originating from T cell receptors (e.g., intracellular Ca2+ in the absence of ras activation). +2 The unresponsive state to antigen stimulation is increased. T cell unresponsiveness can also occur after antigen stimulation in the absence of co-stimulation, resulting in cells that become insensitive to subsequent antigen activation even in the context of co-stimulation. The unresponsive state can often be overcome by the presence of interleukin-2. Unresponsive T cells do not undergo clonal expansion and / or acquire effector function.

[0213] The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction arising from persistent TCR signaling, which occurs during many chronic infections and cancers. It differs from unresponsiveness in that it occurs not through incomplete or absent signaling, but due to persistent signaling. It is characterized by poor effector function, persistent inhibitory receptor expression, and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Exhaustion can originate from both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) and intrinsic cellular negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

[0214] "Enhancing T cell function" refers to inducing, eliciting, or stimulating T cells to possess sustained or amplified biological functions, or restoring or reactivating exhausted or inactive T cells. Examples of enhancing T cell function include increased levels of CD8+ cells relative to pre-intervention levels. + T cell gamma-interferon secretion, increased proliferation, and increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance). In one embodiment, the enhancement level is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. The manner in which this enhancement is measured is known to those skilled in the art.

[0215] "T-cell dysfunction" is a T-cell disorder or condition characterized by reduced responsiveness to antigenic stimuli. In one specific embodiment, T-cell dysfunction is a disorder specifically associated with inappropriately elevated signaling via PD-1. In another embodiment, T-cell dysfunction is a disorder in which T cells are unresponsive or have reduced capacity to secrete cytokines, proliferate, or perform cytolytic activities. In one specific aspect, reduced responsiveness leads to ineffective control of pathogens or tumors expressing immunogens. Examples of T-cell dysfunction characterized by T-cell dysfunction include undifferentiated acute infections, chronic infections, and tumor immunity.

[0216] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when this evasion weakens, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0217] "Immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps to eliminate tumor cells through an immune response. Examples of enhancing tumor immunogenicity include, but are not limited to, treatment with PD-1 axis binding antagonists (e.g., anti-PD-L1 antibodies) and TIGIT inhibitors (e.g., anti-TIGIT antibodies).

[0218] "Sustained response" refers to the continued effect of reducing tumor growth after treatment has been stopped. For example, the tumor size may remain the same or smaller compared to the size at the start of the application phase. In some embodiments, the sustained response has a duration at least the same as, at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times the duration of treatment.

[0219] The term "antibody" includes monoclonal antibodies (including full-length antibodies having the Fc region of an immunoglobulin), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies, and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0220] A basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites, while IgA antibodies contain 2-5 basic 4-chain units that combine with the J chain to form a multivalent assembly. In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each light chain is linked to the heavy chain by a covalent disulfide bond, and two heavy chains are linked to each other by one or more disulfide bonds, the number of which depends on the homotype of the heavy chains. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at its N-terminus. H ), followed by three (for α and γ chains) or four (for μ and ε isotypes) constant fields (C H Each light chain has a variable domain (V) at its N-terminus. L ), followed by a constant domain (C) at its other end. L V L With V H Arranged together, and C L With the first constant field of the heavy chain (C H 1) Arranged together. It is believed that specific amino acid residues form interfaces between the variable domains of the light and heavy chains. A V H And a V LThey pair together to form an antigen-binding site. For information on the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th ed., Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Based on their constant domain amino acid sequence, L-chains from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ). Based on their heavy chain constant domain (C... H Based on their amino acid sequences, immunoglobulins can be classified into different categories or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each possessing heavy chains called α, δ, ε, γ, and μ, respectively. According to C... H With minor differences in sequence and function, γ and α classes can be further subdivided into subclasses, such as the following subclasses expressed in humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0221] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the antibody's heavy or light chain. The heavy chain variable domain and the light chain variable domain can be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0222] The term "variable" refers to the fact that certain segments within a variable domain exhibit significant sequence differences between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire span of the variable domain. Instead, it is concentrated in three segments within both the light and heavy chain variable domains, called hypervariable regions (HVRs). The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, which mostly adopt a β-sheet conformation and are linked by three HVRs that form a loop and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FRs and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD (1991)). The constant domain does not directly participate in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as the participation of antibodies in the cytotoxicity of antibody-dependent cells.

[0223] The term "monoclonal antibody," as used herein, refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical, except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in very small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Unlike typical polyclonal antibody preparations, which contain different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, a key advantage of monoclonal antibodies is that they are synthesized from hybridoma cultures, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be generated through any particular method. For example, monoclonal antibodies to be used according to the present invention can be generated by a variety of techniques, including, for example, hybridoma methods (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, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display technology (see, for example, 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): 29 9-310(2004); Lee et al., J.Mol.Biol.340(5):1073-1093(2004); Fellouse, Proc.Nat.Acad.Sci.USA 101(34):12467-12472(2004); Lee et al., J. Immunol. Methods 284(1-2):119-132(2004)), and techniques for generating human or human-like antibodies in animals having partial or whole 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); US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). .

[0224] The term "naked antibody" refers to an antibody that is not conjugated with a cytotoxic module or a radiolabel.

[0225] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody in essentially its complete form, distinct from antibody fragments. Specifically, intact antibodies include those containing both heavy and light chains (including the Fc region). The constant domain can be a natural sequence constant domain (e.g., the human natural sequence constant domain) or a variant of its amino acid sequence. In some cases, intact antibodies may possess one or more effector functions.

[0226] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding and / or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Digestion of an antibody with papain produces two identical antigen-binding fragments, termed the "Fab" fragment, and a residual "Fc" fragment, the names reflecting its ease of crystallization. The Fab fragment consists of the complete L chain and the variable region (V) of the H chain. H ) and the first constant field of the heavy chain (C H1) Composition. Each Fab fragment is monovalent for antigen binding, meaning it has only one antigen-binding site. Pepsin treatment of the antibody produces a larger F(ab')2 fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, possessing different antigen-binding activities while still being able to cross-link the antigen. The Fab' fragment, due to its C... H The 1 domain differs from the Fab fragment by the addition of a few residues at its carboxyl terminus, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the terminology used herein for Fab' where the constant-domain cysteine ​​residues carry free thiol groups. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0227] The Fc fragment contains the carboxyl-terminal portions of two H chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0228] "Fv" is the smallest antibody fragment containing both a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently bound heavy chain variable region and a light chain variable region. Six hypervariable rings (three rings each from the heavy and light chains) emanate from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than the complete binding site.

[0229] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a type of Fv containing V molecules linked together to form a single polypeptide chain. H and V L Antibody fragments containing antibody domains. Preferably, the sFv peptide is in V H With V L The domains further contain peptide linkers, which enable sFv to form the desired structure for binding antigens. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315, 1994.

[0230] The "functional fragment" of the antibody of this invention comprises a portion of the complete antibody, generally including the antigen-binding or variable region of the complete antibody, or a retained or modified Fc region of the antibody with FcR binding capability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0231] The term "double antibody" refers to antibodies produced by transvaginal administration of V... H and V L Small antibody fragments are prepared by constructing sFv fragments (see previous paragraph) using short linkers (approximately 5-10 residues) between the domains. Due to the short linkers, the V domains pair interchain rather than intrachain, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific biantibodies are heterodimers of two "crossover" sFv fragments, where the V domains of the two antibodies... H and V L The domains are located on different polypeptide chains. More detailed descriptions of biantibodies can be found in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0232] Monoclonal antibodies explicitly include, as described herein, “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include “primate-derived” antibodies in which the antigen-binding region of the antibody is derived from an antibody generated by immunizing a macaque monkey with, for example, an antigen of interest. As used herein, “humanized antibodies” are a subset of “chimeric antibodies.”

[0233] A “humanized” form of a nonhuman (e.g., mouse) antibody refers to a chimeric antibody that minimally contains a sequence derived from a nonhuman immunoglobulin. In one embodiment, a humanized antibody refers to an immunoglobulin (receptor antibody) in which the HVR residues (defined below) of a human immunoglobulin are replaced with HVR residues of a nonhuman species (donor antibody) (such as a mouse, rat, rabbit, or nonhuman primate) having the desired specificity, affinity, and / or ability. In some cases, the framework (“FR”) residues of the human immunoglobulin are replaced with corresponding nonhuman residues. Furthermore, humanized antibodies may contain residues not found in the receptor antibody or in the donor antibody. These modifications can be made to further improve antibody performance, such as binding affinity. Generally, humanized antibodies will contain at least one, typically two, variable domains that are substantially all of the following: all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulin sequences, and all or substantially all FR regions are FR regions of human immunoglobulin sequences, although the FR regions may contain one or more individual FR residue substitutions that improve antibody performance (such as binding affinity, isomerization, immunogenicity, etc.). The number of these amino acid substitutions in the FRs is typically no more than 6 in the H chain and no more than 3 in the L chain. Humanized antibodies may optionally also contain at least a portion of the immunoglobulin constant region (Fc), which is typically the constant region of human immunoglobulins. For more details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0234] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by humans and / or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J.Mol.Biol.227:381(1991); Marks et al., J.Mol.Biol.222:581(1991)). Methods described in the following literature can also be used to prepare human monoclonal antibodies: Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77(1985); Boerner et al., J.Immunol.147(1):86-95(1991). See also van Dijk and van de Winkel, Curr.Opin.Pharmacol., 5:368-74(2001). Human antibodies can be prepared by administering antigens to transgenic animals, such as immunized xenomic mice, that have been modified to generate human antibodies in response to antigenic stimulation but whose endogenous loci have lost their ability (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, concerning XENOMOUSE). TM (Technology). See also, for example, Li et al., Proc. Natl. Ascad. Sci. USA, 103:3557-3562 (2006), on human antibodies generated by human B-cell hybridoma technology.

[0235] The terms “hypervariant region,” “HVR,” or “HV” as used in this article refer to regions within the variable domain of an antibody that are highly variable in sequence and / or form structurally defined loops. Typically, antibodies 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 among these six HVRs, and H3 is considered to play a unique role, particularly in conferring precise specificity to the antibody. See, for example, 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 camellid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0236] This article uses and covers many descriptions of HVRs. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers to the location of the structural loop (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loops, and is derived from the use of OxfordMolecular's AbM antibody modeling software. "Contact" HVRs are based on the analysis of the available crystal structure of the complex. The residues of each of these HVRs are described below.

[0237]

[0238] HVRs can include the following “extended HVRs”: 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. For each of these definitions, the variable domain residues are as numbered according to Kabat et al., see above.

[0239] The phrases "according to Kabat variable domain residue numbering" or "according to Kabat amino acid position numbering" and their variations refer to Kabat, etc., as described above in the numbering system used for editing antibody heavy chain or light chain variable domains. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to shortening or insertion of variable domains FR or HVR. For example, a heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat residue numbering scheme for a given antibody can be determined by comparing the antibody sequence with homologous regions of a "standard" Kabat numbered sequence.

[0240] "Frame" or "FR" residues refer to those residues in the variable domain other than the HVR residues defined herein.

[0241] The "human common framework" or "receptor human framework" refers to the framework representing the most common amino acid residues in the selected human immunoglobulin VL or VH framework sequence. Typically, the human immunoglobulin VL or VH sequence selection is derived from a variable domain sequence subgroup. Commonly, these subgroups are such as those used by Kabat et al. Sequences of Proteins Immunological InterestSubgroups as defined in the 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include, for VL, subgroups could be subgroups κI, κII, κIII, or κIV, as in Kabat et al., see above. Additionally, for VH, subgroups could be subgroup I, subgroup II, or subgroup III, as in Kabat et al., see above. Alternatively, a human common frame can be derived from the aforementioned frames, where specific residues, such as human frame residues, are selected based on their homology with the donor frame by comparing the donor frame sequence with a set of various human frame sequences. Recipient human frames or human common frames “derived” from human immunoglobulin frames can contain their same amino acid sequence, or they can contain previously existing amino acid sequence variations. In some embodiments, the number of previously existing amino acid variations 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.

[0242] The “VH subgroup III common framework” comprises a common sequence obtained from the amino acid sequences of the variable heavy chain subgroup III described above by Kabat et al. In one embodiment, the VH subgroup III common framework amino acid sequence comprises at least a portion 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).

[0243] The “VLκI common framework” comprises a common sequence obtained from the amino acid sequence of the variable light chain κ subgroup I from Kabat et al., see above. In one embodiment, the VL subgroup I common framework amino acid sequence comprises at least a portion 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).

[0244] An “amino acid modification” at a specified location (e.g., the Fc region) refers to the substitution or deletion of a specified residue, or the insertion of at least one amino acid residue near the specified residue. An insertion “near” the specified residue means an insertion within one or two residues. The insertion can be at the N-terminus or C-terminus of the specified residue. The preferred amino acid modification in this document is substitution.

[0245] "Affinity-matured" antibodies are those with one or more alterations in one or more of their HVR domains, resulting in improved affinity for the antigen compared to parent antibodies without these alterations. In one embodiment, the affinity-matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity-matured antibodies can be generated using procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation via VH and VL domain shuffling. The following literature records the random mutagenesis of HVR and / or framework residues: for example, Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0246] As used herein, the terms "specific binding" or "specific to..." refer to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines 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) binds to that target with greater affinity, strength, ease, and / or duration than it binds to other targets. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the antibody-bound target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody that specifically binds to the 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 a conserved epitope on a protein from different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

[0247] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous protein ("adhesin") with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence having a desired binding specificity (i.e., being "heterologous") different from the antigen recognition and binding sites of an antibody and an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule is typically a continuous amino acid sequence containing at least a receptor or ligand binding site. The immunoglobulin constant domain sequence in an immunoadhesin can be derived from any immunoglobulin, such as IgG-1, IgG-2 (including IgG2A and IgG2B), IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM. Preferably, the Ig fusion comprises a substitution of at least one variable region within the Ig molecule with a domain of a polypeptide or antibody as described herein. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2, and CH3 regions of an IgG1 molecule, or the hinge, CH1, CH2, and CH3 regions. For generating immunoglobulin fusions, see also U.S. Patent No. 5,428,130, published June 27, 1995. For example, useful immunoadhesins as a second agent that can be used in combination therapies described herein include polypeptides comprising an extracellular or PD-1-binding portion of PD-L1 or PD-L2 fused to a constant domain of an immunoglobulin sequence, such as PD-L1 ECD–Fc, PD-L2 ECD–Fc, and PD-1ECD–Fc, respectively. The combination of an immunoadhesin of Ig Fc and the cell surface receptor ECD is sometimes referred to as a soluble receptor.

[0248] "Fusion proteins" and "fusion peptides" refer to peptides having two covalently linked parts, each with different properties. These properties can be biological, such as in vitro or in vivo activity. They can also be simple chemical or physical, such as binding to target molecules, catalysis of reactions, etc. The two parts can be directly linked by a single peptide bond or via a peptide linker, but connected in a reading frame manner.

[0249] "PD-1 oligopeptide," "PD-L1 oligopeptide," or "PD-L2 oligopeptide" are oligopeptides that bind (preferably specifically bind) to PD-1, PD-L1, or PD-L2 negative co-stimulatory peptides, respectively, comprising receptors, ligands, or signal transduction components as described herein. Such oligopeptides can be chemically synthesized using known oligopeptide synthesis techniques or prepared and purified using recombinant techniques. These oligopeptides are typically at least about 5 amino acids in length, or 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, or 50 amino acids in length. 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 known techniques.In this regard, it should be noted that techniques for screening oligopeptide libraries for oligopeptides that specifically bind to polypeptide targets are well known in the art (see, for example, U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publications Nos. WO 84 / 03506 and WO84 / 03564; Geysen et al., Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82:178-182 (1985); Geysen et al., 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, SE et al., Proc. Natl. Acad. Sci. USA, 87: 6378 (1990); Lowman, HB et al., Biochemistry, 30: 10832 (1991); Clackson, T. et al., Nature, 352: 624 (1991); Marks, JD et al., J. Mol. Biol., 222: 581 (1991); Kang, AS et al., Proc. Natl. Acad. Sci. USA, 88: 8363 (1991), and Smith, GP., Current Opin. Biotechnol., 2: 668 (1991).

[0250] "Blocking" antibodies or "antagonist" antibodies are antibodies that inhibit or reduce the biological activity of the antigens they bind to. In some embodiments, blocking or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. The anti-PD-L1 antibody of the present invention blocks signaling via PD-1, thereby restoring the functional responses of T cells (e.g., proliferation, cytokine production, target cell killing) from a dysfunctional state to antigen stimulation.

[0251] "Agonist" or activating antibodies are antibodies that enhance or initiate signal transduction by binding to an antigen. In some embodiments, agonist antibodies induce or activate signal transduction in the absence of a natural ligand.

[0252] The term "Fc region" is used herein to define the C-terminal region of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. While the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as the segment from its Cys226 or Pro230 position to the carboxyl terminus. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region can be eliminated, for example, during antibody production or purification, or through recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, compositions of complete antibodies may include antibody groups with all K447 residues eliminated, antibody groups with none of the K447 residues eliminated, or antibody groups that are a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for use in the antibodies of this invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0253] The term "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. Preferred FcRs are native human FcRs. Furthermore, preferred FcRs are FcRs (γ receptors) that bind IgG antibodies, including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor-based tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor-based tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see M). Annu.Rev.Immunol.15:203-234(1997)). For reviews of FcR, see Ravetch and Kinet, Annu.Rev.Immunol.9:457-492(1991); Capel et al., Immunomethods 4:25-34(1994); de Haas et al., J.Lab.Clin.Med.126:330-41(1995). The term “FcR” in this paper covers other FcRs, including those to be identified in the future.

[0254] The term “Fc receptor” or “FcR” also includes the neonatal receptor, FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Methods for measuring binding to FcRn are known (see, for example, 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); WO2004 / 92219 (Hinton et al.)). It can determine the in vivo binding and serum half-life of human FcRn high-affinity binding peptides to FcRn, for example in transgenic mice expressing human FcRn or in transfected human cell lines, or in primates administered peptides with a variant Fc region. WO2004 / 42072 (Presta) describes antibody variants whose binding to FcR is modified or eliminated. See also, for example, Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0255] The phrase "substantially reduced" or "substantially different," as used herein, refers to a sufficiently high degree of difference between two values ​​(typically one relating to a molecule and the other to a reference / comparison molecule) such that a person skilled in the art would consider the difference between the two values ​​to be statistically significant within the context of the biological characteristic measured using the values ​​(e.g., the Kd value). As a function of the reference / comparison molecule value, the difference between the 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%.

[0256] The phrases “substantially similar” or “substantially identical” as used herein indicate a sufficiently high degree of similarity between two values ​​(e.g., one relating to an antibody of the present invention and the other to a reference / comparison antibody) such that a person skilled in the art would consider the difference between the two values ​​to be small or non-biologically and / or statistically significant within the context of the biological characteristics measured using the values ​​(e.g., Kd values). As a function of the reference / comparison 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%.

[0257] When used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or mammals exposed to them at the doses and concentrations employed. Typically, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; 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 dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming anti-charge ions such as sodium; and / or nonionic surfactants such as TWEEN. TM Polyethylene glycol (PEG) and PLURONICS TM .

[0258] "Packaging inserts" refer to the information typically included in the commercial packaging of a drug, which contains information about the indications, usage, dosage, administration, contraindications, other drugs that may be used in conjunction with the packaged product, and / or warnings.

[0259] As used herein, “treatment / management” refers to a clinical intervention designed to alter the natural course of an individual or cells being treated during a clinicopathological process. The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. For example, an individual is considered successfully “treated” if one or more cancer-related symptoms are reduced or eliminated, including but not limited to reduced proliferation (or destruction) of cancerous cells, reduction of symptoms originating from the disease, improvement of the quality of life of individuals with the disease, reduction of the dosage of other medications required to treat the disease, delay of disease progression, and / or prolongation of individual survival.

[0260] As used herein, “delayed disease progression” means to postpone, hinder, slow, delay, stabilize, and / or slow the formation of a disease (such as cancer). This delay can be of varying lengths depending on the individual’s medical history and / or treatment. As will be apparent to those skilled in the art, a sufficient or significant delay can substantially encompass prevention, as the individual does not develop the disease. For example, the formation of late-stage cancer, such as metastases, can be delayed.

[0261] As used in this article, “reducing or inhibiting cancer recurrence” means reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression.

[0262] As used herein, “cancer” and “cancerous” refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers, 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 this type of cancer include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (hepatic carcinoma), bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (kidney or renal cancer), prostate cancer, vulvar cancer, thyroid cancer, and various types of head and neck cancer, as well as B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small aneuploid NHL, and bulky lymphoma). Diseases including NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs syndrome.

[0263] As used in this article, “metastasis” refers to the spread of cancer from its primary site to other locations in the body. Cancer cells can detach from the primary tumor, infiltrate the lymphatic and blood vessels, circulate via the bloodstream, and grow in distant lesions (metastases) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a continuous process, depending on whether tumor cells detach from the primary tumor, spread via the bloodstream, and stop at a distant site. At the new site, the cell establishes a blood supply and can grow to form a life-threatening mass. Stimulating and inhibitory molecular pathways within tumor cells regulate this behavior, and the interaction between tumor cells and host cells in distant sites is also important.

[0264] An "effective dose" is at least the minimum concentration required to achieve measurable improvement or prevention of a specific condition. The effective dose as used herein can vary with factors such as the patient's disease state, age, sex, weight, and the ability of the antibody to elicit the desired response in the individual. An effective dose is also the amount at which the beneficial effect of treatment outweighs any toxic or adverse effects of treatment. For prophylactic use, beneficial or desired outcomes include results such as elimination or reduction of risk, mitigation of severity, or delay of disease onset, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during disease development. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reduction of one or more symptoms arising from the disease, improvement of the quality of life of those suffering from the disease, reduction of the dosage of other medications required to treat the disease, enhancement of the effect of another medication (e.g., via targeted therapy), delay of disease progression, and / or prolongation of survival. In the case of cancer or tumors, an effective amount of a drug may be effective in reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., to some extent slowing or desiredly stopping) the infiltration of cancer cells into peripheral organs; inhibiting (i.e., to some extent slowing and desiredly stopping) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating one or more symptoms associated with the disease to some extent. An effective amount may be administered in one or more applications. For the purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a preventive or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may be achieved with or without another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if, together with one or more other agents, it achieves or achieves the desired result.

[0265] As used in this article, "in combination with / in combination with / together with" refers to the application of one therapeutic modality in addition to another. Therefore, "in combination with / in combination with / together with" means the application of another therapeutic modality before, during, or after the application of one therapeutic modality to an individual.

[0266] As used herein, “subject” refers to a mammal, including but not limited to humans or non-human mammals such as cows, horses, dogs, sheep, or cats. Preferably, a subject refers to a human. In this document, a patient is also a subject.

[0267] As used in this article, “complete response” or “CR” refers to the disappearance of all target damage; “partial response” or “PR” refers to a reduction of at least 30% in the longest diameter of the target damage (SLD) with the baseline SLD as a reference; and “stable disease” or “SD” refers to a reduction in the minimum SLD at the start of treatment with neither sufficient target damage contraction to meet PR nor sufficient increase to meet PD.

[0268] As used in this article, “progressive disease” or “PD” refers to at least a 20% increase in the SLD of the target lesion or the presence of one or more new lesions, with the minimum SLD recorded from the start of treatment as a reference.

[0269] As used in this article, “progression-free survival” (PFS) refers to the length of time during and after treatment when the treated disease (e.g., cancer) does not worsen. PFS can include the amount of time a patient has experienced a full or partial response, and the amount of time a patient has experienced stable disease.

[0270] As used in this article, “Overall Response Rate” (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0271] As used in this article, “overall survival” refers to the percentage of individuals in a group who are likely to survive after a specific duration.

[0272] "Chemotherapy agents" refer to chemical compounds that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide. Alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedepa, and uredopa; ethylenimines and methylamelamines, including altretamine and triethylene melamine. Leneemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); tetrahydrocannabinol (dronabinol), ); β-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue topotecan) CPT-11 (irinotecan) Acetylcamptothecin, scopoletin, and 9-aminocamptothecin; bryostatin; pemetrexed; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid); teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oral α-4 integrin inhibitor; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide Hydrochlorides, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediynes (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, for example, Nicolaou et al., Angew. Chem. Intl. Ed. Engl.).33:183-186(1994)); anthracycline antibiotics (dynemicins), including dynemicins A; esperamicin; and neocarzinostatin chromophore and related chromogenic chromophores of ethynylene antibiotics), aclacinomysin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin D, daunorubicin, detorubicin, 6-diazol-5-oxo-L-leucine, doxorubicin (including). Morpholin-doxorubicin, cyanomorpholin-doxorubicin, 2-pyrrole-doxorubicin, doxorubicin hydrochloride liposome injection ( And deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin Mycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and gemcitabine. Tegafur Capecitabine Epothilone; and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, and 6-azinon. Glycosides, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine, and imatinib (a 2-phenylaminopyrimidine derivative), as well as other c-Kit inhibitors; anti-adrenergic drugs, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as frolinic acid. acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-Ethylhydrazide; Procarbazine; Polysaccharide complexes (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine Dacarbazine; mannomustine; mitobronitol; mitolactalol; pipebroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, such as pallitaxel. Albumin-modified nanoparticle formulation of palitaxane (ABRAXANE) TM ), and doxetaxel. Chloranbucil; thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine. Platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine Oxaliplatin; leucovovin; vinorelbine Novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; pharmaceutically acceptable salts, acids, or derivatives of any of the above substances; and combinations of two or more of the above substances, such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone) and FOLFOX (oxaliplatin). TM (This is an abbreviation for a treatment regimen combining 5-FU and leucovorin.)

[0273] This definition also includes anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or generalized therapy. They may themselves be hormones. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including...). Tamoxifen, Raloxifene Droloxifen, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene. Antiprogesterone derivatives; estrogen receptor downregulators (ERDs); estrogen receptor antagonists, such as fulvestrant. Drugs that function to inhibit or shut down the ovaries, such as luteinizing hormone-releasing hormone (LHRH) agonists, such as leuprolide acetate. and Goserelin acetate, buserelin acetate, and tripterelin; antiandrogens such as flutamide, nilutamide, and bicalutamide; and aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, and megestrol acetate. Exemestane Formate, Fadrozole, Vorozole Letrozole And anastrozole Additionally, this definition of chemotherapy agents includes bisphosphonates, such as clodronate (e.g.) or etidronate sodium NE-58095, zoledronic acid / zoledronate alendronate Pamidronate tiludronate Or risedronate And troxacitabine (a 1,3-dioxolane cytosine analog); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in aberrant cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as Vaccines and gene therapy vaccines, such as vaccine, Vaccines and Vaccines; topoisomerase 1 inhibitors (e.g.) Anti-estrogens, such as fulvestrant; Kit inhibitors, such as imatinib or EXEL-0862 (a tyrosine kinase inhibitor); EGFR inhibitors, such as erlotinib or cetuximab; anti-VEGF inhibitors, such as bevacizumab; arinotecan; rmRH (e.g.) Lapatinib and lapatinib ditosylate (a small molecule inhibitor of dual tyrosine kinases ErbB-2 and EGFR, also known as GW572016); 17AAG (a geldanamycin derivative, which is a heat shock protein (Hsp) 90 toxin); and any pharmaceutically acceptable salts, acids, or derivatives of the above substances.

[0274] As used herein, the term "cytokine" generally refers to a protein released by a population of cells that acts as an intercellular mediator on another cell or has an autocrine effect on the cell that produces the protein. Examples of such cytokines include lymphokines, monokines; interleukins ("ILs"), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, including... rIL-2; tumor necrosis factors, such as TNF-α or TNF-β, TGF-β1-3; and other polypeptide factors, including leukemia inhibitory factor (“LIF”), ciliary ganglion neurotrophic factor (“CNTF”), CNTF-like cytokines (“CLC”), cardiotrophic protein (“CT”), and kit ligand (“KL”).

[0275] As used herein, the term "chemokine" refers to soluble factors (such as cytokines) that have the ability to selectively induce chemotaxis and activation of leukocytes. They also trigger processes such as angiogenesis, inflammation, wound healing, and tumorigenesis. Examples of chemokines include IL-8, a human homologue of mouse keratinocyte chemoattractant (KC).

[0276] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise.

[0277] In this document, the reference to "about" a value or parameter includes (and describes) changes involving said value or parameter itself. For example, a description of "about X" includes a description of "X".

[0278] As used herein, the phrase “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable organic or inorganic salt of the compounds of the present invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannins, pantothenates, bitartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates (methylsulfonates), ethanesulfonates, benzenesulfonates, p-toluenesulfonates and pyrates (i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthoic acid)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate ions, succinate ions, or other counterions. Counterions can be any organic or inorganic module that stabilizes the charge of the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. In cases where multiple charged atoms are components of a pharmaceutically acceptable salt, multiple counterions may be present. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0279] If the compound of the present invention is a base, then the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, such as treating the free base with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, etc.) or with organic acids (such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid / fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid (such as glucuronic acid or galacturonic acid), α-hydroxy acids (such as citric acid or tartaric acid), amino acids (such as aspartic acid or glutamic acid), aromatic acids (such as benzoic acid or cinnamic acid), sulfonic acids (such as p-toluenesulfonic acid or ethanesulfonic acid), etc.).

[0280] If the compounds of the present invention are acids, then the desired pharmaceutically acceptable salts can be prepared by any suitable method, such as treating the free acid with inorganic or organic bases (such as amines (primary, secondary, or tertiary)), alkali metal hydroxides, or alkaline earth metal hydroxides, etc. Exemplary examples of suitable salts include, but are not limited to, organic salts derived from amino acids (such as glycine and arginine), ammonium, primary / secondary / 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.

[0281] The phrase “pharmaceutically acceptable” indicates that the substance or composition must be chemically and / or toxicologically compatible with the other components constituting the formulation and / or the mammals to which it is used for treatment.

[0282] It should be understood that the aspects and variations of the invention described herein include those that are "composed" of and / or substantially composed of the aspects and variations.

[0283] III. Methods

[0284] In one aspect, this article provides a method for treating cancer or delaying cancer progression in an individual, which includes administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity.

[0285] In another aspect, this article provides a method for reducing or inhibiting cancer recurrence or progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist in combination with an agent that reduces or inhibits TIGIT expression and / or activity. As disclosed herein, cancer recurrence and / or progression includes, but is not limited to, cancer metastasis.

[0286] In another aspect, this article provides a method for treating immune-related diseases or delaying the progression of immune-related diseases in an individual, which includes administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity.

[0287] In another aspect, this article provides a method for reducing or inhibiting the progression of immune-related diseases in an individual, which includes administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity.

[0288] In some embodiments, the immune-related disease is associated with T-cell dysfunction. 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 dysfunction is characterized by reduced responsiveness to antigenic stimulation. In some embodiments, the T-cell dysfunction is characterized by T-cell unresponsiveness or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments, the T-cell dysfunction is characterized by T-cell depletion. In some embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments, the T-cell dysfunction includes unresolved acute infections, chronic infections, and tumor immunity.

[0289] In another aspect, this article provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, which includes administering an effective amount of a PD-1 axis-binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity.

[0290] In another aspect, this article provides a method for treating cancer or delaying cancer progression in an individual, which includes administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity.

[0291] In another aspect, this article provides a method for reducing or inhibiting cancer recurrence or progression in an individual, which includes administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity.

[0292] In another aspect, this article provides a method for treating immune-related diseases or delaying the progression of immune-related diseases in an individual, which includes administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity.

[0293] In another aspect, this article provides a method for reducing or inhibiting the progression of immune-related diseases in an individual, which includes administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that modulates CD226 expression and / or activity.

[0294] In some embodiments, the immune-related disease is associated with T-cell dysfunction. 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 dysfunction is characterized by reduced responsiveness to antigenic stimulation. In some embodiments, the T-cell dysfunction is characterized by T-cell unresponsiveness or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments, the T-cell dysfunction is characterized by T-cell depletion. In some embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments, the immune-related disease is selected from the group consisting of unresolved acute infections, chronic infections, and tumor immunity.

[0295] In another aspect, this article provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual by administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that regulates CD226 expression and / or activity.

[0296] In some embodiments, the agents regulating CD226 expression and / or activity are capable of enhancing and / or stimulating CD226 expression and / or activity; enhancing and / or stimulating the interaction of CD226 with PVR, PVRL2, and / or PVRL3; and enhancing and / or stimulating intracellular signaling mediated by CD226 binding to PVR, PVRL2, and / or PVRL3. As used herein, agents capable of enhancing and / or stimulating CD226 expression and / or activity include, but are not limited to, agents that enhance and / or stimulate CD226 expression and / or activity. As used herein, agents capable of enhancing and / or stimulating the interaction of CD226 with PVR, PVRL2, and / or PVRL3 include, but are not limited to, agents that enhance and / or stimulate the interaction of CD226 with PVR, PVRL2, and / or PVRL3. As used herein, agents capable of enhancing and / or stimulating intracellular signaling mediated by CD226 binding to PVR, PVRL2, and / or PVRL3 include, but are not limited to, agents that enhance and / or stimulate intracellular signaling mediated by CD226 binding to PVR, PVRL2, and / or PVRL3.

[0297] In some embodiments, the agent regulating CD226 expression and / or activity is selected from agents that inhibit and / or block the interaction between CD226 and TIGIT, antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, agents that inhibit and / or block the interaction between TIGIT and PVR L2, agents that inhibit and / or block the interaction between TIGIT and PVR L3, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3, and combinations thereof.

[0298] In some embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is an inhibitory nucleic acid selected from antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras.

[0299] In some embodiments, the antagonist of TIGIT expression and / or activity is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments, the antagonist of TIGIT expression and / or activity is an inhibitory nucleic acid selected from antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras.

[0300] In some embodiments, the antagonist of PVR expression and / or activity is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the antagonist of PVR expression and / or activity is selected from small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0301] In some embodiments, the agent inhibiting and / or blocking the interaction between TIGIT and PVR is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the agent inhibiting and / or blocking the interaction between TIGIT and PVR is selected from small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0302] In some embodiments, the agent that inhibits and / or blocks the interaction between TIGIT and PVR L2 is selected from small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0303] In some embodiments, the agent that inhibits and / or blocks the interaction between TIGIT and PVR L3 is selected from small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0304] In some embodiments, the agent that inhibits and / or blocks intracellular signaling mediated by TIGIT bound to PVR is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the agent that inhibits and / or blocks intracellular signaling mediated by TIGIT bound to PVR is selected from small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0305] In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2 are selected from small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0306] In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3 are selected from small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0307] In another aspect, this document provides a method for enhancing, strengthening, or stimulating an immune response or 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 the expression and / or activity of one or more other immunosuppressive receptors. In some embodiments, the one or more other immunosuppressive receptors are selected from PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and CD96. In some embodiments, the one or more other immunosuppressive receptors are selected from PD-1, CTLA-4, LAG3, and TIM3.

[0308] In another aspect, this document provides a method for enhancing, strengthening, or stimulating an immune response or 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 increases or activates the expression and / or activity of one or more other immune co-stimulatory receptors. In some embodiments, the one or more other immune co-stimulatory receptors are selected from CD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4. In some embodiments, the one or more other immune co-stimulatory receptors are selected from CD226, OX-40, CD28, CD27, CD137, HVEM, and GITR. In some embodiments, the one or more other immune co-stimulatory receptors are selected from OX-40 and CD27.

[0309] The method of the present invention can be applied in situations where enhanced immunogenicity is desired (such as increasing tumor immunogenicity to treat cancer or T-cell dysfunction).

[0310] It can treat a variety of cancers or delay their progression.

[0311] In some embodiments, the individual has non-small cell lung cancer. The non-small cell lung cancer may be in an early stage or in a late stage. In some embodiments, the individual has small cell lung cancer. The small cell lung cancer may be in an early stage or in a late stage. In some embodiments, the individual has renal cell carcinoma. The renal cell carcinoma may be in an early stage or in a late stage. In some embodiments, the individual has colorectal cancer. The colorectal cancer may be in an early stage or in a late stage. In some embodiments, the individual has ovarian cancer. The ovarian cancer may be in an early stage or in a late stage. In some embodiments, the individual has breast cancer. The breast cancer may be in an early stage or in a late stage. In some embodiments, the individual has pancreatic cancer. The pancreatic cancer may be in an early stage or in a late stage. In some embodiments, the individual has gastric cancer. The gastric cancer may be in an early stage or in a late 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 can be in an early or late stage. In some embodiments, the individual has melanoma. The melanoma can be in an early or late stage. In some embodiments, the individual has head and neck cancer. The head and neck cancer can be in an early or late stage. In some embodiments, the individual has thyroid cancer. The thyroid cancer can be in an early or late stage. In some embodiments, the individual has sarcoma. The sarcoma can be in an early or late stage. In some embodiments, the individual has prostate cancer. The prostate cancer can be in an early or late stage. In some embodiments, the individual has glioblastoma. The glioblastoma can be in an early or late stage. In some embodiments, the individual has cervical cancer. The cervical cancer can be in an early or late stage. In some embodiments, the individual has thymic carcinoma. The thymic carcinoma can be in an early or late stage. In some embodiments, the individual has leukemia. The leukemia can be in an early or late stage. In some embodiments, the individual has lymphoma. The lymphoma can be in an early or late stage. In some embodiments, the individual has myeloma. The myeloma can be in an early or late stage. In some embodiments, the individual has mycoses fungoids. The mycoses fungoids can be in an early or late stage. In some embodiments, the individual has Merkel cell carcinoma.The Merkel cell carcinoma can be in an early or late stage. In some embodiments, the individual has hematologic malignancy. The hematologic malignancy can be in an early or late stage. In some embodiments, the individual is a person.

[0312] In some embodiments of the method of the present invention, the CD4 and / or CD8 T cells in the individual have increased or enhanced initiation, activation, proliferation, cytokine release and / or cell lysis activities relative to prior to administration of the combination.

[0313] In some embodiments of the method of the present invention, the number of CD4 and / or CD8 T cells increases relative to before the application of the combination. In some embodiments of the method of the present invention, the number of activated CD4 and / or CD8 T cells increases relative to before the application of the combination.

[0314] In some embodiments of the method of the present invention, activated CD4 and / or CD8 T cells are characterized by γ-IFN. + Generative CD4 and / or CD8 T cells and / or enhanced cell lysis activity relative to prior to application of the combination.

[0315] In some embodiments of the method of the present invention, the CD4 and / or CD8 T cells exhibit increased release of cytokines selected from the group consisting of IFN-γ, TNF-α, and interleukins.

[0316] In some embodiments of the method of the present invention, the CD4 and / or CD8 T cells are effector memory T cells. In some embodiments of the method of the present invention, the CD4 and / or CD8 effector memory T cells are characterized by γ-IFN. + Generates CD4 and / or CD8 T cells and / or enhances cytolytic activity. In some embodiments of the method of the present invention, the CD4 and / or CD8 effector memory T cells are characterized by having CD44 高 CD62L 低 The expression.

[0317] In some embodiments of the method of the present invention, the cancer has an elevated level of T-cell infiltration.

[0318] In some embodiments, the method of the present invention may further include the administration of other therapies. These other therapies may be radiotherapy, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing therapies. These other therapies may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the other therapy is the administration of a side effect limiter (e.g., an agent intended to reduce the occurrence and / or severity of treatment side effects, such as an antinausea agent). In some embodiments, the other therapy is radiotherapy. In some embodiments, the other therapy is surgery. In some embodiments, the other therapy may be one or more chemotherapeutic agents described above.

[0319] Any PD-1 axis binding antagonist and agent that reduces or inhibits TIGIT expression and / or activity can be used in the methods of the present invention.

[0320] In some implementations, 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.) are human proteins.

[0321] PD-1 axis binding antagonists

[0322] This article provides a method for treating cancer or delaying cancer progression in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This article also provides a method for reducing or inhibiting cancer recurrence or progression in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This article further provides a method for treating immune-related diseases or delaying the progression of immune-related diseases in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This article also provides a method for reducing or inhibiting the progression of immune-related diseases in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This article also provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, which includes administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity.

[0323] For example, PD-1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0324] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In one specific aspect, the PD-1 ligand binding partner is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In one specific aspect, the PD-L1 binding partner is PD-1 and / or B7-1. In another embodiment, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In one specific aspect, the PD-L2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0325] In some embodiments, the PD-1 binding antagonist is selected from: MDX-1106 (nivolumab), Merck 3745 (lambrolizumab), CT-011 (pidilizumab), and AMP-224. In some embodiments, the PD-1 binding antagonist is selected from: YW243.55.S70, MPDL3280A, MDX-1105, and MEDI 4736. In some embodiments, the PD-L2 binding antagonist is AMP-224. In some embodiments, the PD-1 binding antagonist is AMP-224. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007 / 005874. Antibody YW243.55.S70 (SEQ ID No. 20) is an anti-PD-L1 antibody described in WO 2010 / 077634 A1 and US 8,217,149 (which are included herein by reference). MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO2006 / 121168. Merck 3745, also known as MK 3475, MK-3475, SCH-900475, or lambolizumab, is an anti-PD-1 antibody described in WO2009 / 114335. CT-011, also known as hBAT, hBAT-1, or pidilizumab, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0326] Examples of anti-PD-L1 antibodies that can be used in the methods of the present invention and methods for generating them are described in PCT patent applications WO2010 / 077634 A1 and US 8,217,149, which are incorporated herein by reference.

[0327] In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is capable of inhibiting binding between PD-L1 and PD-1 and / or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab')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.

[0328] Anti-PD-L1 antibodies (including compositions containing such antibodies) useful in this invention, such as those described in WO 2010 / 077634 A1 and US 8,217,149, can be used in combination with agents that reduce or inhibit TIGIT expression and / or activity, with or without any other therapy (e.g., chemotherapy), to treat cancer or immune-related diseases (e.g., T-cell dysfunction, viral infections, chronic viral infections, etc.).

[0329] In one embodiment, the anti-PD-L1 antibody contains a heavy chain variable region polypeptide comprising HVR-H1, HVR-H2, and HVR-H3 sequences, wherein:

[0330] (a) The HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO:33);

[0331] (b) The HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO:34);

[0332] (c) The HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:19);

[0333] Furthermore, X1 is D or G; X2 is S or L; X3 is T or S.

[0334] In one specific aspect, X1 is D; X2 is S and X3 is T. In another aspect, the polypeptide further comprises a variable region heavy chain framework sequence juxtaposed between HVRs according to the following formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another aspect, the framework sequence is derived from a human shared framework sequence. In still another aspect, the framework sequence is a VH subgroup III shared framework. In yet another aspect, at least one framework sequence is as follows:

[0335] HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:25)

[0336] HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:26)

[0337] HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:27)

[0338] HC-FR4 is WGQGTLVTVSA (SEQ ID NO:28).

[0339] In another aspect, the heavy chain polypeptide is further combined with a variable region light chain, wherein the variable region light chain comprises HVR-L1, HVR-L2, and HVR-L3, wherein:

[0340] (a) The HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO:35);

[0341] (b) The HVR-L2 sequence is SASX9LX 10 S(SEQ ID NO:36);

[0342] (c) The HVR-L3 sequence is QQX 11 X 12 X 13 X 14 PX 15 T(SEQ ID NO:37);

[0343] Furthermore, where: X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; X 10 It is Y or A; X 11 It is Y, G, F, or S; X 12 It is L, Y, F, or W; X 13 It is Y, N, A, T, G, F, or I; X 14 It is H, V, P, T, or I; X 15 It is A, W, R, P, or T.

[0344] In another respect, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 It is Y; X 11 It is Y; X 12 It is L; X 13 It is Y; X 14 It is H; X 15 It is A. In another aspect, the light chain further comprises a variable region light chain frame sequence juxtaposed between HVRs according to the following formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the frame sequence is derived from a human shared frame sequence. In another aspect, the frame sequence is a VLκI shared frame. In yet another aspect, at least one frame sequence is as follows:

[0345] LC-FR1 is DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:29)

[0346] LC-FR2 is WYQQKPGKAPKLLIY(SEQ ID NO:30)

[0347] LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:31)

[0348] LC-FR4 is FGQGTKVEIKR (SEQ ID NO:32).

[0349] In another embodiment, an isolated anti-PD-L1 antibody or antigen-binding fragment is provided, comprising heavy chain and light chain variable region sequences, wherein:

[0350] (a) The heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, wherein:

[0351] (i) The HVR-H1 sequence is GFTFSX1SWIH (SEQ ID NO:33).

[0352] (ii) The HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO:34)

[0353] (iii) The HVR-H3 sequence is RHWPGGFDY (SEQ ID NO:19), and (b) the light chain contains HVR-L1, HVR-L2, and HVR-L3, wherein:

[0354] (i) The HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO:35).

[0355] (ii) The HVR-L2 sequence is SASX9LX 10 S(SEQ ID NO:36), and

[0356] (iii) The HVR-L3 sequence is QQX 11 X 12 X 13 X 14 PX 15 T(SEQ ID NO:37)

[0357] Furthermore, where: X1 is D or G; X2 is S or L; X3 is T or S; X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; X 10 It is Y or A; X 11 It is Y, G, F, or S; X 12It is L, Y, F, or W; X 13 It is Y, N, A, T, G, F, or I; X 14 It is H, V, P, T, or I; X 15 It is A, W, R, P, or T.

[0358] In one specific aspect, X1 is D; X2 is S and X3 is T. In another aspect, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 It is Y; X 11 It is Y; X 12 It is L; X 13 It is Y; X 14 It is H; X 15 It is A. In another aspect, X1 is D; X2 is S and X3 is T, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 It is Y; X 11 It is Y; X 12 It is L; X 13 It is Y; X 14 It is H and X 15 It is A.

[0359] In another aspect, the heavy chain variable region contains one or more frame sequences juxtaposed between HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region contains one or more frame sequences juxtaposed between HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the frame sequences are derived from human shared frame sequences. In yet another aspect, the heavy chain frame sequences are derived from Kabat subgroup I, II, or III sequences. In yet another aspect, the heavy chain frame sequences are VH subgroup III shared frames. In yet another aspect, one or more heavy chain frame sequences are as follows:

[0360] HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:25)

[0361] HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:26)

[0362] HC-FR3RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:27)

[0363] HC-FR4WGQGTLVTVSA (SEQ ID NO:28).

[0364] In another aspect, the light chain framework sequence is derived from the KabatκI, II, III, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VLκI common framework. In yet another aspect, one or more light chain framework sequences are as follows:

[0365] LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:29)

[0366] LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:30)

[0367] LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:31)

[0368] LC-FR4 FGQGTKVEIKR (SEQ ID NO: 32).

[0369] In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another 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, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function arises from "effector-less Fc mutations" or aglycosylation. In yet another embodiment, the effector-less Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0370] In yet another embodiment, an anti-PD-L1 antibody is provided, comprising heavy chain and light chain variable region sequences, wherein:

[0371] (a) The heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity with GFTFSDSWIH (SEQ ID NO:17), AWISPYGGSTYYADSVKG (SEQ ID NO:18), and RHWPGGFDY (SEQ ID NO:19), respectively, or

[0372] (b) The light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences that have at least 85% sequence identity with RASQDVSTAVA (SEQ ID NO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT (SEQ ID NO:22), respectively.

[0373] (c) In one specific aspect, 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 frame sequences juxtaposed between HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more frame sequences juxtaposed between HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the frame sequence is derived from a human common frame sequence. In yet another aspect, the heavy chain frame sequence is derived from a Kabat subgroup I, II, or III sequence. In another aspect, the heavy-chain framework sequence is a common framework of VH subgroup III. In yet another aspect, one or more heavy-chain framework sequences are as follows:

[0374] HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:25)

[0375] HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:26)

[0376] HC-FR3RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:27)

[0377] HC-FR4WGQGTLVTVSA (SEQ ID NO:28).

[0378] In another aspect, the light chain framework sequence is derived from the KabatκI, II, III, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VLκI common framework. In yet another aspect, one or more light chain framework sequences are as follows:

[0379] LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:29)

[0380] LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:30)

[0381] LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:31)

[0382] LC-FR4 FGQGTKVEIKR (SEQ ID NO: 32).

[0383] In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another 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, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function arises from a "smaller effector Fc mutation" or glycosylation-free design. In yet another embodiment, the smaller effector Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0384] In yet another embodiment, an isolated anti-PD-L1 antibody is provided, comprising heavy chain and light chain variable region sequences, wherein:

[0385] (a) The heavy chain sequence has at least 85% sequence identity with the following heavy chain sequences:

[0386] (SEQ ID NO:23),

[0387] (SEQ ID NO:40), or

[0388] (SEQ ID NO:41),

[0389] or

[0390] (b) The light chain sequence has at least 85% sequence identity with the following light chain sequences:

[0391] (SEQ ID NO:24).

[0392] In one specific aspect, 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 contains one or more frame sequences juxtaposed between HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region contains one or more frame sequences juxtaposed between HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the frame sequence is derived from a human common frame sequence. In yet another aspect, the heavy chain frame sequence is derived from a Kabat subgroup I, II, or III sequence. In another aspect, the heavy-chain framework sequence is a common framework of VH subgroup III. In yet another aspect, one or more heavy-chain framework sequences are as follows:

[0393] HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:25)

[0394] HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:26)

[0395] HC-FR3RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:27)

[0396] HC-FR4WGQGTLVTVSA (SEQ ID NO:28).

[0397] In another aspect, the light chain framework sequence is derived from the KabatκI, II, III, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VLκI common framework. In yet another aspect, one or more light chain framework sequences are as follows:

[0398] LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:29)

[0399] LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:30)

[0400] LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:31)

[0401] LC-FR4 FGQGTKVEIKR (SEQ ID NO: 32).

[0402] In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another 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, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function originates from generation in prokaryotic cells. In yet another specific aspect, minimal effector function originates from "smaller effector Fc mutation" or glycosylation-free. In yet another embodiment, the smaller effector Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0403] In yet another embodiment, the present invention provides a composition comprising any of the anti-PD-L1 antibodies described above in combination with at least one pharmaceutically acceptable carrier.

[0404] In yet another implementation, an isolated nucleic acid is provided that encodes a light chain or heavy chain variable region sequence of an anti-PD-L1 antibody, wherein:

[0405] (a) The heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity with GFTFSDSWIH (SEQ ID NO:17), AWISPYGGSTYYADSVKG (SEQ ID NO:18), and RHWPGGFDY (SEQ ID NO:19), respectively.

[0406] (b) The light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences having at least 85% sequence identity with RASQDVSTAVA (SEQ ID NO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT (SEQ ID NO:22), respectively.

[0407] In one specific aspect, sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In each aspect, the heavy chain variable region contains one or more frame sequences juxtaposed between HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region contains one or more frame sequences juxtaposed between HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the frame sequence is derived from a human-shared frame sequence. In still another aspect, the heavy chain frame sequence is derived from a Kabat subgroup I, II, or III sequence. In another aspect, the heavy-chain framework sequence is a common framework of VH subgroup III. In yet another aspect, one or more heavy-chain framework sequences are as follows:

[0408] HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:25)

[0409] HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:26)

[0410] HC-FR3RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:27)

[0411] HC-FR4WGQGTLVTVSA (SEQ ID NO:28).

[0412] In another aspect, the light chain framework sequence is derived from the KabatκI, II, III, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VLκI common framework. In yet another aspect, one or more light chain framework sequences are as follows:

[0413] LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:29)

[0414] LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:30)

[0415] LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:31)

[0416] LC-FR4 FGQGTKVEIKR (SEQ ID NO: 32).

[0417] In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another 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, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function originates from generation in prokaryotic cells. In yet another specific aspect, minimal effector function originates from "smaller effector Fc mutation" or glycosylation-free. In yet another embodiment, the smaller effector Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0418] In another embodiment, an isolated anti-PD-L1 antibody is provided, comprising heavy chain and light chain variable region sequences, wherein:

[0419] (a) The heavy chain sequence has at least 85% sequence identity with the following heavy chain sequences:

[0420] (SEQ ID NO:41), or

[0421] (b) The light chain sequence has at least 85% sequence identity with the following light chain sequences:

[0422] (SEQ ID NO:24).

[0423] In one specific aspect, 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 contains one or more frame sequences juxtaposed between HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region contains one or more frame sequences juxtaposed between HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the frame sequence is derived from a human common frame sequence. In yet another aspect, the heavy chain frame sequence is derived from a Kabat subgroup I, II, or III sequence. In another aspect, the heavy-chain framework sequence is a common framework of VH subgroup III. In yet another aspect, one or more heavy-chain framework sequences are as follows:

[0424] HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:25)

[0425] HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:26)

[0426] HC-FR3RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:27)

[0427] HC-FR4WGQGTLVTVSS (SEQ ID NO:42).

[0428] In another aspect, the light chain framework sequence is derived from the KabatκI, II, III, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VLκI common framework. In yet another aspect, one or more light chain framework sequences are as follows:

[0429] LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:29)

[0430] LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:30)

[0431] LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:31)

[0432] LC-FR4 FGQGTKVEIKR (SEQ ID NO: 32).

[0433] In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another 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, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function originates from generation in prokaryotic cells. In yet another specific aspect, minimal effector function originates from "smaller effector Fc mutation" or glycosylation-free. In yet another embodiment, the smaller effector Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0434] In another aspect, the heavy chain variable region comprises one or more frame sequences juxtaposed between HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region comprises one or more frame sequences juxtaposed between HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In another aspect, the frame sequences are derived from human shared frame sequences. In another aspect, the heavy chain frame sequences are derived from Kabat subgroup I, II, or III sequences. In another aspect, the heavy chain frame sequences are VH subgroup III shared frames. In another aspect, one or more heavy chain frame sequences are as follows:

[0435] HC-FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS(SEQ ID NO:43)

[0436] HC-FR2 WVRQAPGKGLEWVA(SEQ ID NO:44)

[0437] HC-FR3RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:27)

[0438] HC-FR4WGQGTLVTVSS (SEQ ID NO:45).

[0439] In another aspect, the light chain framework sequence is derived from the KabatκI, II, III, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VLκI common framework. In yet another aspect, one or more light chain framework sequences are as follows:

[0440] LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:29)

[0441] LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:30)

[0442] LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:31)

[0443] LC-FR4 FGQGTKVEIK (SEQ ID NO: 46).

[0444] In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another 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, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function arises from a "smaller effector Fc mutation" or glycosylation-free process. In yet another embodiment, the smaller effector Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0445] In yet another embodiment, an anti-PD-L1 antibody is provided, comprising heavy chain and light chain variable region sequences, wherein:

[0446] (d) The heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity with GFTFSDSWIH (SEQ ID NO:17), AWISPYGGSTYYADSVKG (SEQ ID NO:18), and RHWPGGFDY (SEQ ID NO:19), respectively, or

[0447] (e) The light chain further comprises HVR-L1, HVR-L2 and HVR-L3 sequences that have at least 85% sequence identity with RASQDVSTAVA (SEQ ID NO:20), SASFLYS (SEQ ID NO:21) and QQYLYHPAT (SEQ ID NO:22), respectively.

[0448] In one specific aspect, 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 contains one or more frame sequences juxtaposed between HVRs as follows: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region contains one or more frame sequences juxtaposed between HVRs as follows: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the frame sequence is derived from a human common frame sequence. In yet another aspect, the heavy chain frame sequence is derived from a Kabat subgroup I, II, or III sequence. In another aspect, the heavy-chain framework sequence is a common framework of VH subgroup III. In yet another aspect, one or more heavy-chain framework sequences are as follows:

[0449] HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:25)

[0450] HC-FR2 WVRQAPGKGLEWV(SEQ ID NO:26)

[0451] HC-FR3RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR(SEQ ID NO:27)

[0452] HC-FR4WGQGTLVTVSSASTK (SEQ ID NO:47).

[0453] In another aspect, the light chain framework sequence is derived from the KabatκI, II, III, or IV subgroup sequence. In yet another aspect, the light chain framework sequence is a VLκI common framework. In yet another aspect, one or more light chain framework sequences are as follows:

[0454] LC-FR1 DIQMTQSPSSSLSASVGDRVTITC(SEQ ID NO:29)

[0455] LC-FR2 WYQQKPGKAPKLLIY(SEQ ID NO:30)

[0456] LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO:31)

[0457] LC-FR4 FGQGTKVEIKR (SEQ ID NO: 32).

[0458] In yet another specific aspect, the antibody further comprises a human or mouse constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In yet another 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, and IgG3. In yet another aspect, the mouse constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, minimal effector function arises from a "smaller effector Fc mutation" or glycosylation-free design. In yet another embodiment, the smaller effector Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0459] In yet another embodiment, an isolated anti-PD-L1 antibody is provided, comprising heavy chain and light chain variable region sequences, wherein:

[0460] (a) The heavy chain sequence has at least 85% sequence identity with the following heavy chain sequences:

[0461] (SEQ ID NO:40), or

[0462] (b) The light chain sequence has at least 85% sequence identity with the following light chain sequences:

[0463] (SEQ ID NO:24).

[0464] In some embodiments, an isolated anti-PDL1 antibody is provided comprising heavy and light chain variable region sequences, 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 with the amino acid sequence of SEQ ID NO:24. In some embodiments, an isolated anti-PDL1 antibody is provided comprising heavy and light chain variable region sequences, 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 with the amino acid sequence of SEQ ID NO:40. In some embodiments, an isolated anti-PDL1 antibody is provided, comprising heavy chain and light chain variable region sequences, 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 with the amino acid sequence of SEQ ID NO:24, and the heavy chain variable region sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:40.

[0465] In yet another embodiment, an isolated anti-PD-L1 antibody is provided, comprising heavy chain and light chain sequences, wherein:

[0466] (a) The heavy chain sequence has at least 85% sequence identity with the following heavy chain sequences:

[0467] (SEQ ID NO:48), or

[0468] (b) The light chain sequence has at least 85% sequence identity with the following light chain sequences:

[0469] (SEQ ID NO:49).

[0470] In some embodiments, an isolated anti-PDL1 antibody is provided comprising heavy and light chain sequences, 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 with the amino acid sequence of SEQ ID NO:48. In some embodiments, an isolated anti-PDL1 antibody is provided comprising heavy and light chain sequences, 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 with the amino acid sequence of SEQ ID NO:48. In some embodiments, an isolated anti-PDL1 antibody is provided, comprising heavy and light chain sequences, 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 with the amino acid sequence of SEQ ID NO:48, and the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:48.

[0471] In another aspect, the nucleic acid further comprises a vector suitable for expressing the nucleic acid, said nucleic acid encoding any of the previously described anti-PD-L1 antibodies. In yet another specific aspect, the vector further comprises a host cell suitable for expressing the nucleic acid. In yet another specific aspect, the host cell is a eukaryotic or prokaryotic cell. In yet another specific aspect, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO).

[0472] Anti-PD-L1 antibodies or antigen-binding fragments thereof may be generated using methods known in the art, such as by culturing host cells containing nucleic acids under conditions suitable for generating such antibodies or fragments, and recovering the antibodies or fragments, wherein the nucleic acids encode any one of the previously described anti-PD-L1 antibodies or antigen-binding fragments in a form suitable for expression.

[0473] In yet another embodiment, the present invention provides a composition comprising the anti-PD-L1 antibody or its antigen-binding fragment provided above and at least one pharmaceutically acceptable carrier.

[0474] Drugs that reduce or inhibit TIGIT expression and / or activity

[0475] This article provides a method for treating or delaying cancer progression in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This article also provides a method for reducing or inhibiting cancer recurrence or progression in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This article further provides a method for treating or delaying the progression of immune-related diseases in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This article also provides a method for reducing or inhibiting the progression of immune-related diseases in an individual, comprising administering an effective amount of a PD-1 axis binding antagonist to the individual in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This document also provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist in combination with an agent that reduces or inhibits TIGIT expression and / or activity. This document also provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, comprising 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 other immune co-suppressive receptors. This document also provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual, comprising administering to the individual an effective amount of an agent that reduces or inhibits TIGIT expression and / or activity and an agent that increases or activates one or more other immune co-stimulatory receptors. For example, agents that reduce or inhibit TIGIT expression and / or activity include antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, agents that inhibit and / or block the interaction between TIGIT and PVR L2, agents that inhibit and / or block the interaction between TIGIT and PVR L3, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3, and combinations thereof.

[0476] In some embodiments, the antagonist of TIGIT expression and / or activity includes small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0477] In some embodiments, the antagonist of PVR expression and / or activity includes small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0478] In some embodiments, the agents that inhibit and / or block the interaction between TIGIT and PVR include small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0479] In some embodiments, the agents that inhibit and / or block the interaction between TIGIT and PVR L2 include small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0480] In some embodiments, the agents that inhibit and / or block the interaction between TIGIT and PVR L3 include small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0481] In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR include small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0482] In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2 include small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0483] In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3 include small molecule inhibitors, inhibitory antibodies or antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0484] In some embodiments, the antagonist of TIGIT expression and / or activity is an inhibitory nucleic acid selected from antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras.

[0485] In some embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment.

[0486] Anti-TIGIT antibodies (including compositions containing such antibodies) useful in this invention, such as those described in WO 2009 / 126688, can be used in combination with PD-1 axis binding antagonists.

[0487] Anti-TIGIT antibody

[0488] This invention provides anti-TIGIT antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugated antibodies. Those skilled in the art will understand that this invention also provides antibodies against other peptides (i.e., anti-PVR antibodies), and any description herein specifically relating to the creation, generation, modification, use, or other aspects of anti-TIGIT peptides will also apply to antibodies specific to other non-TIGIT peptides.

[0489] Polyclonal antibodies

[0490] Anti-TIGIT antibodies may include polyclonal antibodies. Methods for preparing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be generated in mammals, for example, by single or multiple injections of an immunizing agent and, if necessary, an adjuvant. Typically, the immunizing agent and / or adjuvant will be injected into the mammal via multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a TIGIT peptide or a fusion protein thereof. Conjugating the immunizing agent to a protein known to be immunogenic in the immunized mammal may be useful. Examples of such immunogenic proteins include, but are not limited to, foraminifera hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitors. Examples of adjuvants that may be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization regimen can be selected by those skilled in the art without extensive experimentation.

[0491] Monoclonal antibodies

[0492] Alternatively, anti-TIGIT antibodies can be monoclonal antibodies. Monoclonal antibodies can be prepared using hybridoma methods, as described in Kohler and Milstein, Nature 256:495 (1975). In the hybridoma method, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce the production or generation of lymphocytes that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0493] Immunotherapy agents typically include the TIGIT peptide or its fusion protein. Peripheral blood lymphocytes (“PBLs”) are typically used, if human-derived cells are desired, or spleen cells or lymph node cells are used, if non-human mammalian-derived cells are desired. The lymphocytes are then fused with an immortalized cell line to form hybridoma cells using a suitable fusion agent, such as polyethylene glycol (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103). The immortalized cell line is typically a transformed mammalian cell line, particularly rodent, bovine, and human-derived myeloma cells. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells are cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), then the culture medium used for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (“HAT medium”), which inhibit the growth of HGPRT-deficient cells.

[0494] Preferred immortalized cell lines are those that are efficiently fused, support stable high levels of antibody expression in selected antibody-producing cells, and are sensitive to media such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained from, for example, the Salk Institute Cell Distribution Center (San Diege, California, USA) and the American Type Culture Collection (Manassas, Virginia, USA). Human myeloma and mouse-human heterologous myeloma cell lines for generating human monoclonal antibodies have also been described (Kozbor, J. Immunol. 133: 3001 (1984); Brodeuret al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987) pp. 51-63).

[0495] The presence of monoclonal antibodies against the peptide can then be determined in the culture medium in which the hybridoma cells are cultured. Preferably, the binding specificity of the monoclonal antibodies generated by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be determined by, for example, the Scatchard analysis of Munson and Pollard, Anal. Biochem. 107:220 (1980).

[0496] After identifying the desired hybridoma cells, the clone can be subcloned using a limiting dilution process and cultured using standard methods (Goding, see above). Suitable media for this purpose include, for example, Dulbecco's modified Eagle's medium or RPMI-1640 medium. Alternatively, hybridoma cells can be cultured in vivo in mammals as ascites fluid.

[0497] Subclonal secreted monoclonal antibodies can be separated from the culture medium or ascites fluid through routine immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0498] Monoclonal antibodies can also be generated using recombinant DNA technology, such as that described in U.S. Patent 4,816,567. The DNA encoding the monoclonal antibodies of the present invention is easily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells of the present invention are a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into host cells that do not additionally produce immunoglobulin proteins, such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example by substitution, i.e., replacing the homologous murine sequence with the coding sequence of the human heavy and light chain constant domains (U.S. Patent 4,816,567; Morrison et al., see above), or by covalently conjugating the entire or partial coding sequence of immunoglobulin-coding sequences and non-immunoglobulin polypeptides. Such non-immunoglobulin polypeptides can be used to replace the constant domain of the antibodies of the present invention, or they can be used to replace the variable domain of an antigen-binding site of the antibodies of the present invention to produce chimeric bivalent antibodies.

[0499] Antibodies can be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves the recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, relevant cysteine ​​residues are substituted with or deleted from another amino acid residue to prevent cross-linking.

[0500] In vitro methods are also suitable for preparing monovalent antibodies. Digesting antibodies to generate their fragments, particularly Fab fragments, can be accomplished using conventional techniques known in the art.

[0501] Human antibodies and humanized antibodies

[0502] The anti-TIGIT antibodies of the present invention may also include humanized antibodies or human antibodies. Humanized forms of non-human (e.g., mouse) antibodies refer to chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that contain at least a sequence derived from a non-human immunoglobulin. Humanized antibodies comprise antibodies in which the complementarity-determining region (CDR) residues of a human immunoglobulin (receptor antibody) are replaced with CDR residues of a non-human species (donor antibody) such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, the Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Humanized antibodies may also contain residues not found in either the receptor antibody or the input CDR or framework sequence. Typically, humanized antibodies will contain at least one, usually two, substantially whole variable domains, wherein the entire or substantially whole CDR corresponds to the CDR of the non-human immunoglobulin, and the entire or substantially whole FR is the FR of the human immunoglobulin common sequence. Humanized antibodies will ideally also contain at least a portion of the immunoglobulin constant region (Fc), which is typically the constant region of human immunoglobulins (Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0503] Methods for humanizing nonhuman antibodies are well known in the art. Typically, one or more amino acid residues from a nonhuman source are introduced into a humanized antibody. These nonhuman amino acid residues are often referred to as “input” residues, and they are usually derived from the “input” variable domain. Humanization can be performed essentially following the approach of Winter et al. (Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyenet et al., Science 239: 1534-1536 (1988)), i.e., replacing the corresponding sequence of the human antibody with a rodent CDR or CDR sequence. Therefore, such “humanized” antibodies are chimeric antibodies (US Patent 4,816,567), in which essentially less than the entire human variable domain is replaced with a corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some of the CDR residues and possibly some of the FR residues are replaced with residues from similar sites in rodent antibodies.

[0504] Human antibodies can also be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991); Marks et al., J. Mol. Biol. 222: 581 (1991)). Techniques by Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol. 147(1): 86-95 (1991)). Similarly, human antibodies can be generated by introducing human immunoglobulin loci into transgenic animals, such as mice, whose endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody generation was observed, closely resembling what is seen in humans in all aspects, including gene rearrangement, assembly, and antibody ensemble. This method 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).

[0505] Antibodies can also be affinity-matured using known selection and / or mutagenesis methods as described above. Preferred affinity-matured antibodies have an affinity 5 times, more preferably 10 times, or even more preferably 20 or 30 times higher than the starting antibody (typically mouse, humanized, or human) used to prepare the mature antibody.

[0506] Bispecific antibodies

[0507] Bispecific antibodies are monoclonal antibodies that have binding specificity to at least two different antigens, preferably human or humanized antibodies. In this case, one binding specificity is against TIGIT, and the other is against any other antigen, preferably against a cell surface protein or a receptor or receptor subunit.

[0508] Methods for generating bispecific antibodies are known in the art. Traditionally, the recombinant generation of bispecific antibodies is based on the co-expression of two pairs of immunoglobulin heavy-light chains, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305: 537-539 (1983)). Due to the random allocation of the immunoglobulin heavy and light chains, these hybridomas (quadromas) generate a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is typically accomplished via an affinity chromatography step. Similar procedures are disclosed in WO 93 / 08829, published May 13, 1993, and in Traunecker et al., EMBO J.10: 3655-3659 (1991).

[0509] An antibody variable domain having the desired binding specificity (antibody-antigen binding site) can be fused to an immunoglobulin constant domain sequence. The fusion preferably uses an immunoglobulin heavy chain constant domain containing at least a portion of the hinge, CH2, and CH3 regions. Preferably, a first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into a suitable host organism. For more details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

[0510] According to another method described in WO 96 / 27011, the interface between a pair of antibody molecules can be modified to maximize the percentage of heterodimers recovered from recombinant cell cultures. The preferred interface comprises at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory “cavity” of the same or similar size for the large side chains is created at the interface of the second antibody molecule. This provides a mechanism for increasing heterodimer yield compared to other unwanted end products such as homodimers.

[0511] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, chemical linking can be used to prepare bispecific antibodies. Brennan et al., Science 229:81 (1985) described a procedure for generating F(ab')2 fragments by proteolytic cleavage of intact antibodies. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragment is then converted into a thionitrobenzoate (TNB) derivative. One of the Fab'-TNB derivatives is then reduced back to 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 antibody can be used as a selective immobilization reagent for enzymes.

[0512] The Fab' fragment can be directly recovered from *E. coli* and chemically conjugated to form bispecific antibodies. Shalaby et al., *J. Exp. Med.* 175: 217-225 (1992) described the generation of a fully humanized bispecific antibody molecule F(ab')2. Each Fab' fragment was separately secreted by *E. coli* and directionally chemically conjugated in vitro to form a bispecific antibody. The bispecific antibody thus formed can bind to cells overexpressing the ErbB2 receptor and normal human T cells, and trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0513] Several techniques for the direct preparation and isolation of bispecific antibody fragments from recombinant cell cultures are also described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portion of two different antibodies via gene fusion. The antibody homodimer is reduced in the hinge region to form a monomer, which is then re-oxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The “biantibody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides an alternative mechanism for the preparation of bispecific antibody fragments. The fragment contains a heavy chain variable domain (V) linked by a linker. H ) and light chain variable domain (V L The joint is too short, preventing pairing between two structural domains on the same chain. Therefore, it forces a V-shaped segment... H and V L The complementary V on the structural domain and another segmentL and V H Domain pairing forms two antigen-binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv(sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

[0514] This includes antibodies with more than two titers. As a non-limiting example, trispecific antibodies can be prepared. See, for example, Tutt et al., J. Immunol. 147:60 (1991).

[0515] Exemplary bispecific antibodies can bind to two different epitopes on a given TIGIT peptide described herein. Alternatively, the anti-TIGIT peptide arm can be conjugated with arms that bind to triggering molecules on leukocytes, such as T-cell receptor molecules (e.g., CD2, CD3, CD28, or B7) or Fc receptors (FcγRs) of IgG, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), thereby focusing cellular defense mechanisms on cells expressing a specific TIGIT peptide. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing a specific TIGIT peptide. These antibodies possess a TIGIT-binding arm and an arm that binds to cytotoxic agents or radionuclide chelators such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest that binds to a TIGIT peptide also binds to tissue factor (TF).

[0516] Heteroconjugated antibodies

[0517] Heteroconjugated antibodies are also within the scope of this invention. Heteroconjugated antibodies consist of two covalently linked antibodies. Such antibodies are proposed, for example, for targeting unwanted cells with immune system cells (US Patent 4,676,980) and for treating HIV infection (WO 91 / 00360; WO 92 / 200373; EP 03089). It is contemplated that antibodies can be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, disulfide exchange reactions or by forming thioether bonds can be used to construct immunotoxins. Examples of reagents suitable for this purpose include iminothiolates and methyl-4-mercaptobutyrimidates, such as those disclosed in US Patent 4,676,980.

[0518] Effector Functional Engineering Modification

[0519] It may be desirable to modify the antibodies of the present invention in terms of effector function, thereby enhancing, for example, the efficacy of the antibodies in treating cancer. For example, cysteine ​​residues may be introduced into the Fc region, thereby forming an interchain disulfide bond in that region. The homodimeric antibody thus generated may have improved internalization ability and / or enhanced complement-mediated cell killing and antibody-dependent cell-mediated cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, the antibody may be modified to have a dual Fc region, thereby possessing enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

[0520] In some implementations, anti-TIGIT antibodies were generated; these are hamster anti-mouse antibodies. Two antibodies, 10A7 and 1F4, also specifically bind to human TIGIT. The amino acid sequences of the light and heavy chains of the 10A7 antibody were determined using standard techniques. The light chain sequence of this antibody is as follows:

[0521] (SEQ ID NO:13), and the heavy chain sequence of this antibody is:

[0522] (SEQ ID NO:15), where the complementarity determination region (CDR) of each chain is presented in bold text. Thus, the CDR1 of the 10A7 light chain has the sequence KSSQSLYYSGVKENLLA (SEQ ID NO:1), the CDR2 of the 10A7 light chain has the sequence ASIRFT (SEQ ID NO:2), and the CDR3 of the 10A7 light chain has the sequence QQGINNPLT (SEQ ID NO:3). The CDR1 of the 10A7 heavy chain has the sequence GFTFSSFTMH (SEQ ID NO:4), the CDR2 of the 10A7 heavy chain has the sequence FIRSGGIVFYADAVRG (SEQ ID NO:5), and the CDR3 of the 10A7 heavy chain has the sequence RPLGHNTFDS (SEQ ID NO:6).

[0523] The amino acid sequences of the light and heavy chains of the 1F4 antibody were determined. The light chain sequence of this antibody is as follows:

[0524] (SEQ ID NO:14), and the heavy chain sequence of this antibody is:

[0525] (SEQ ID NO:16), where the complementarity determination region (CDR) of each chain is presented in bold text. Thus, the CDR1 of the 1F4 light chain has the sequence RSSQSLVNSYGNTFLS (SEQ ID NO:7), the CDR2 of the 1F4 light chain has the sequence GISNRFS (SEQ ID NO:8), and the CDR3 of the 1F4 light chain has the sequence LQGTHQPPT (SEQ ID NO:9). The CDR1 of the 1F4 heavy chain has the sequence GYSFTGHLMN (SEQ ID NO:10), the CDR2 of the 1F4 heavy chain has the sequence LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and the CDR3 of the 1F4 heavy chain has the sequence GLRGFYAMDY (SEQ ID NO:12).

[0526] The nucleotide sequence encoding the 1F4 light chain was determined as follows:

[0527] (SEQ ID NO:38), and the nucleotide sequence encoding the 1F4 heavy chain was determined to be...

[0528] (SEQ ID NO:39).

[0529] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises at least one HVR, which comprises an amino acid sequence selected from the following amino acid sequences: (1) KSSQSLYYSGVKENLLA (SEQ ID NO:1), ASIRFT (SEQ ID NO:2), QQGINNPLT (SEQ ID NO:3), GFTFSSFTMH (SEQ ID NO:4), FIRSGSGIVFYADAVRG (SEQ ID NO:5), and RPLGHNTFDS (SEQ ID NO:6), or (2) RSSQSLVNSYGNTFLS (SEQ ID NO:7), GISNRFS (SEQ ID NO:8), LQGTHQPPT (SEQ ID NO:9), GYSFTGHLMN (SEQ ID NO:10), LIIPYNGGTSYNQKFKG (SEQ ID NO:11), and GLRGFYAMDY (SEQ ID NO:12).

[0530] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody light chain, the antibody light chain comprising...

[0531] (SEQ ID NO:13) or

[0532] The amino acid sequence shown in (SEQ ID NO:14) is as follows.

[0533] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody heavy chain, which contains

[0534] (SEQ ID NO:15) or

[0535] The amino acid sequence shown in (SEQ ID NO:16) is as follows.

[0536] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises an antibody light chain and an antibody heavy chain, the antibody light chain comprising...

[0537] (SEQ ID NO:13) or

[0538] The antibody heavy chain contains the amino acid sequence shown in SEQ ID NO:14.

[0539] (SEQ ID NO:15) or

[0540] The amino acid sequence shown in (SEQ ID NO:16) is as follows.

[0541] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment is selected from humanized antibodies, chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, and immunotoxins.

[0542] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment comprises at least one HVR that is at least 90% identical to any of the following HVRs: (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).

[0543] In some embodiments, the anti-TIGIT antibody or a fragment thereof comprises a light chain and / or a heavy chain, the light chain and the heavy chain respectively comprising, with

[0544] (SEQ ID NO:13) or

[0545] The amino acid sequence shown in (SEQ ID NO:14) or related to

[0546] (SEQ ID NO:15) or

[0547] The amino acid sequence shown in (SEQ ID NO:16) is at least 90% identical to the amino acid sequence shown in the SEQ ID NO:16.

[0548] Drugs that regulate CD226 expression and / or activity

[0549] This article provides a method for treating cancer or delaying cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist and an agent that regulates CD226 expression and / or activity. This article also provides a method for reducing or inhibiting cancer recurrence or progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist and an agent that regulates CD226 expression and / or activity. This article also provides a method for treating immune-related diseases or delaying the progression of immune-related diseases in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist and an agent that regulates CD226 expression and / or activity. This article also provides a method for reducing or inhibiting the progression of immune-related diseases in an individual, comprising administering to the individual an effective amount of a PD-1 axis-binding antagonist and an agent that regulates CD226 expression and / or activity. This article further provides a method for enhancing, strengthening, or stimulating an immune response or function in an individual by administering to the individual an effective amount of a PD-1 axis-binding antagonist and an agent that regulates CD226 expression and / or activity.

[0550] For example, agents regulating CD226 expression and / or activity are those capable of increasing and / or stimulating CD226 expression and / or activity, increasing and / or stimulating the interaction of CD226 with PVR, PVRL2, and / or PVRL3, and increasing and / or stimulating intracellular signaling mediated by CD226 binding to PVR, PVRL2, and / or PVRL3. In some embodiments, agents capable of increasing and / or stimulating CD226 expression and / or activity are those that increase and / or stimulate CD226 expression and / or activity. In some embodiments, agents capable of increasing and / or stimulating the interaction of CD226 with PVR, PVRL2, and / or PVRL3 are those that increase and / or stimulate the interaction of CD226 with PVR, PVRL2, and / or PVRL3. In some implementations, 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 stimulate intracellular signaling mediated by CD226 binding to PVR, PVRL2, and / or PVRL3.

[0551] In some embodiments, the agent regulating CD226 expression and / or activity is selected from agents that inhibit and / or block the interaction between CD226 and TIGIT, antagonists of TIGIT expression and / or activity, antagonists of PVR expression and / or activity, agents that inhibit and / or block the interaction between TIGIT and PVR, agents that inhibit and / or block the interaction between TIGIT and PVR L2, agents that inhibit and / or block the interaction between TIGIT and PVR L3, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2, agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3, and combinations thereof. In some embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is selected from small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides. In some embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments, the agent inhibiting and / or blocking the interaction between CD226 and TIGIT is a repressive nucleic acid selected from antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras.

[0552] In some embodiments, the antagonist of TIGIT expression and / or activity is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment. In some embodiments, the antagonist of TIGIT expression and / or activity is an inhibitory nucleic acid selected from antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras. In some embodiments, the antagonist of PVR expression and / or activity is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the agent inhibiting and / or blocking the interaction between TIGIT and PVR is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the agent inhibiting and / or blocking the interaction between TIGIT and PVR L2 is a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the agents that inhibit and / or block the interaction between TIGIT and PVR L3 are small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides. In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR are small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides. In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L2 are small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides. In some embodiments, the agents that inhibit and / or block intracellular signaling mediated by TIGIT bound to PVR L3 are small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments, aptamers, inhibitory nucleic acids, and inhibitory peptides.

[0553] In some embodiments, the antagonist of TIGIT expression and / or activity comprises a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the antagonist of PVR expression and / or activity comprises a small molecule inhibitor, an inhibitory antibody or its antigen-binding fragment thereof, an aptamer, an inhibitory nucleic acid, and an inhibitory peptide. In some embodiments, the agent inhibiting intracellular signaling mediated by TIGIT bound to PVR is selected from the group consisting of: small molecule inhibitors, inhibitory antibodies or their antigen-binding fragments thereof, aptamers, inhibitory nucleic acids, and inhibitory peptides. In some embodiments, the antagonist of TIGIT expression and / or activity is an anti-TIGIT antibody or its antigen-binding fragment thereof. In some embodiments, the antagonist of TIGIT expression and / or activity is an inhibitory nucleic acid selected from antisense polynucleotides, interfering RNA, catalytic RNA, and RNA-DNA chimeras.

[0554] Combinations of T-cell targets for immunomodulatory antibody therapy

[0555] In addition to specific antigen recognition via the TCR, T cell activation is regulated by a balance of positive and negative signals provided by co-stimulatory receptors. These surface proteins are typically members of the TNF receptor or any of the B7 superfamily. Activating co-stimulatory receptors include CD226, CD28, OX40, GITR, CD137, CD27, HVEM, MICA, ICOS, NKG2D, and 2B4. Inhibitory co-stimulatory receptors include CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7H4, and CD96. Agonistant antibodies against activating co-stimulatory molecules and blocking antibodies against negative co-stimulatory molecules can enhance T cell stimulation to promote tumor destruction.

[0556] This document provides a method for enhancing, strengthening, or stimulating an immune response or 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 other immunosuppressive receptors. In some embodiments, the one or more other immunosuppressive receptors are selected from PD-1, CTLA-4, LAG3, TIM3, BTLA, VISTA, B7H4, and CD96.

[0557] This document also provides a method for enhancing, strengthening, or stimulating an immune response or 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 enhances or activates one or more other immune co-stimulatory receptors. In some embodiments, the one or more other immune co-stimulatory receptors are selected from CD226, OX-40, CD28, CD27, CD137, HVEM, GITR, MICA, ICOS, NKG2D, and 2B4. In some embodiments, the one or more other immune co-stimulatory receptors are selected from CD226, OX-40, CD27, CD137, HVEM, and GITR. In some embodiments, the one or more other immune co-stimulatory receptors are selected from OX-40 and CD27.

[0558] IV. Reagent Kit

[0559] In another aspect, a kit is provided comprising a PD-1 axis binding antagonist and a packaging insert containing instructions for the use of the PD-1 axis binding antagonist in combination with agents that reduce or inhibit TIGIT expression and / or activity to treat cancer in an individual or delay cancer progression or enhance the immune function of an individual with cancer. Any PD-1 axis binding antagonist and / or agents that reduce or inhibit TIGIT expression and / or activity described herein may be included in this kit.

[0560] 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 packaging insert containing instructions on using the PD-1 axis binding antagonist and the agent that reduces or inhibits TIGIT expression and / or activity in an individual to treat cancer or delay cancer progression or enhance the immune function of an individual with cancer. Any PD-1 axis binding antagonist and / or agent that reduces or inhibits TIGIT expression and / or activity described herein may be included in this kit.

[0561] In another aspect, a kit is provided comprising an agent that reduces or inhibits TIGIT expression and / or activity, and a packaging insert containing instructions regarding the use of the agent, in combination with a PD-1 axis binding antagonist, to treat cancer in an individual or delay cancer progres...

Claims

1. A method for treating cancer or delaying cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity.

2. A method for reducing or inhibiting cancer recurrence or cancer progression in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity.

3. A method for treating an immune-related disease or delaying the progression of an immune-related disease in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity.

4. A method for reducing or inhibiting the progression of immune-related diseases in an individual, comprising administering to the individual an effective amount of a PD-1 axis binding antagonist and an agent that reduces or inhibits TIGIT expression and / or activity.

5. The method of claim 3 or 4, wherein the immune-related disease is related to a T-cell dysfunction disorder.

6. The method of claim 5, wherein the T-cell dysfunction is characterized by reduced responsiveness to antigenic stimulation.

7. The method of claim 5, wherein the T cell dysfunction is characterized by T cell unresponsiveness or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities.

8. The method of claim 5, wherein the T cell dysfunction is characterized by T cell depletion.

9. The method of any one of claims 3-8, wherein the T cells are CD4+ and CD8+ T cells.

10. The method of any one of claims 3-9, wherein the immune-related disease is selected from the group consisting of: unexplained acute infections, chronic infections, and tumor immunity.

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