EA044486B1 - THREE-COMPONENT COMBINED ANTIBODY PREPARATIONS - Google Patents

Description

Cross reference to related applications

This patent application claims priority under 35 USC §119 to US Patent Application No. 62/513960 filed June 1, 2017, 62/515452 filed June 5, 2017, 62/538563 filed July 28, 2017 ., 62/547051 filed August 17, 2017, 62/582756 filed November 7, 2017, and 62/618005 filed January 16, 2018, all of which are expressly incorporated herein by reference in their entirety completeness.

I. State of the art

TIGIT is a coinhibitory receptor that is highly expressed on effector and regulatory (Treg) CD4+ T cells, effector CD8+ T cells, and NK cells. TIGIT has been demonstrated to attenuate the immune response through (1) direct signaling, (2) induction of ligand signaling, and (3) competition with disrupted signaling by the costimulatory receptor CD226 (also known as DNAM-1). TIGIT signaling has been most well studied in NK cells, where interaction with its cognate ligand, poliovirus receptor (PVR, also known as CD155), has been demonstrated to directly suppress NK cell cytotoxicity through its cytoplasmic ITIM domain. Knockout of the TIGIT gene or blockade of the TIGIT/PVR interaction with antibodies has been demonstrated to enhance NK cell killing in vitro and also exacerbate autoimmune diseases in vivo. In addition to direct effects on T and NK cells, TIGIT can induce PVR-mediated signaling in dendritic or tumor cells, leading to increased production of anti-inflammatory cytokines such as IL10. In T cells, TIGIT can also inhibit lymphocyte responses by disrupting homodimerization of the costimulatory receptor CD226 and competing with it for binding to PVR.

TIGIT is highly expressed on lymphocytes, including tumor-infiltrating lymphocytes (TILs) and Tregs, which infiltrate various tumor types. PVR is also widely expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be the dominant immune evasion mechanism in cancer. Notably, TIGIT expression is closely correlated with the expression of another important coinhibitory receptor, PD1. TIGIT and PD1 are coexpressed on TILs of numerous human and mouse tumors. Unlike TIGIT and CTLA4, PD1 inhibition by T cell responses does not involve competition for ligand binding to the costimulatory receptor.

Immune checkpoint domain-containing poliovirus receptor-associated immunoglobulin (PVRIG, also known as CD112R) is a novel inhibitory receptor in the TIGIT receptor family. PVRIG binds with high affinity to its cognate ligand, poliovirus receptor-related protein 2 (PVRL2, also known as CD112 or nectin-2) to deliver an inhibitory signal through its ITIM motif in T and NK cells. The affinity of TIGIT for PVR and PVRIG for PVRL2 is higher than that of CD226 for PVR or PVRL2, suggesting that TIGIT and PVRIG may outcompete PVR and PVRL2 from CD226, providing an indirect mechanism by which TIGIT and PVRIG may reduce lymphocyte function. Thus, two receptors from the same family, TIGIT and PVRIG, deliver inhibitory signals to dampen T and NK cell responses.

Accordingly, TIGIT and PVRIG are of interest for triplet therapy combinations with checkpoint inhibitors, including anti-PD-1 antibodies.

II. Summary of the invention

The present invention provides methods and compositions containing combinations of TIGIT antibodies, as described herein and as set forth in the claims, with PVRIG antibodies and checkpoint inhibitors, including anti-PD-1 antibodies. The present invention also provides nucleic acids encoding these antibodies and compositions thereof.

The present invention provides a method of treating cancer in said patient, comprising: a) obtaining a biopsy from said patient containing tumor cells; b) measuring the number of PD-L1-positive tumor cells or immune cells in the specified biopsy; c) if the indicated number of PD-L1-positive tumor cells or immune cells exceeds 1% compared to staining of the same tumor cells with the corresponding isotype control antibody for the antibodies used, administration of a three-component combination drug containing an anti-TIGIT antibody, an anti-PVRIG antibody and αntu-PD-1 antibody; and d) if the indicated number of PD-L1-positive tumor cells or immune cells is less than 1% compared to staining of the same tumor cells with the appropriate isotype control antibody for the antibodies used, administration of a two-component combination preparation containing the anti-TIGIT antibody and the antibody anti-PVRIG.

In some embodiments of the method, the anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 ( S241P) and CHA.9.547.13.H4 (S241P).

In some embodiments of the method, the anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA7.518.1.H4 (S241P) and

- 1 044486

SNA.7.538.1.2.N4 (S241P).

In some embodiments of the method, the anti-PD-1 antibody is an antibody selected from at least one of pembrolizumab, cemiplimab, and nivolumab.

In some embodiments of this method, the two-component combination therapy is selected from administering CPA.9.083.H4 (S241P) and CHA.7.518.1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SNA.9.547.13.N4 (S241P) and SNA.7.538.1.2.N4 (S241P).

In some embodiments of the method, the triple combination therapy is selected from administering CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P, nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.7.538 .1.2.H4 (S241P); CPA.9.083.H4 (S241P), emiplimab and CNA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CPA. 9.083.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); CHA9.547.7.H4 (S241P) , cemiplimab and CHA.7.538.1.2.H4 (S241P), as well as CHA.9.547.13.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P).

In some embodiments of this method, the antibodies are administered simultaneously.

In some embodiments of this method, the antibodies are administered sequentially.

In some embodiments of the method, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma , kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, cancer from Merkel cell, MSI-high cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS). In some embodiments of the method, the cancer is selected from the group consisting of triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merkel cell cancer, high MSI cancer, KRAS mutant tumors , adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS).In some embodiments of the method, the cancer is selected from the group consisting of triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer ), Merkel cell cancer, and high MSI cancer.

The present invention also provides a method of treating cancer in a specified patient, comprising administering a three-component combination therapy comprising an anti-TIGIT antibody, an anti-PVRIG antibody and an anti-PD-1 antibody.

In some embodiments of the method, the anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 ( S241P) and SNA.9.547.13.N4 (S241P).

In some embodiments of the method, the anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P).

In some embodiments of the method, the anti-PD-1 antibody is an antibody selected from at least one of pembrolizumab, cemiplimab, and nivolumab.

In some embodiments of the method, the three-component combination therapy includes administration of an anti-PD-1 antibody in combination with a two-component combination therapy.

- 2 044486 therapy selected from the administration of CPA.9.083.H4 (S241P) and CHA.7.518.1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SNA.9.547.13.N4 (S241P) and SNA.7.538.1.2.N4 (S241P).

In some embodiments of the method, the triple combination therapy is selected from administering CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P, nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.7.538 .1.2.H4 (S241P); CPA.9.083.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CHA.9.547.13.H4 (S241P), cemiplimab and CNA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CNA.7.538.1.2.H4 (S241P); CHA9.547.7.H4 ( S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P), as well as CHA.9.547.13.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P).

In some embodiments of this method, the antibodies are administered simultaneously.

In some embodiments of this method, the antibodies are administered sequentially.

In some embodiments of the method, the cancer for triple combination therapy is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach cancer (gastric cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloblastic leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel cell cancers, high-MSI cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS). In some embodiments of the method, the cancer is selected from the group consisting of triple negative breast cancer, stomach cancer (gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer)), Merkel cell cancer, high MSI cancer, KRAS -mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

The present invention also provides a pharmaceutical dosage kit comprising: a) a container containing a unit dose of an anti-TIGIT antibody; and b) a container containing a unit dose of anti-PVRIG antibody.

The present invention also provides a pharmaceutical dosage kit comprising: a) a container containing a unit dose of an anti-TIGIT antibody; and b) a container containing a unit dose of anti-PVRIG antibody; and c) a container containing αntu-PD-1 antibody.

In a further aspect, the present invention provides methods comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) TIGIT protein; ii) PVRIG protein; iii) PVR protein; iv) PD-1 protein; v) PD-L1 protein; vi) PVRL2; and vi) the appropriate isotype control for the antibodies in i)-vi); c) performing fluorescence activated cell sorting (FACS); d) for each of TIGIT, PVRIG, PVR, PD-1, PVRL2 and PD-L1, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; wherein, if the percentage of positive cells is > 1% for either TIGIT or PVR, and either PVRIG or PVRL2, and either PD-1 or PD-L1, proceed to step e); and e) administering antibodies to TIGIT, PVRIG and PD-1 to said patient.

In a further aspect, the present invention provides methods comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) TIGIT protein; ii) PVR protein; iii) PD-1 protein; iv) PD-L1 protein; and v) appropriate isotype control for antibodies in i)-iv); c) performing fluorescence activated cell sorting (FACS); d) for each of TIGIT, PVR, PD-1 and PD-L1, determining the percentage of cells in the indicated population that express the protein relative to the indicated isoantibody

- 3 044486 peak control; however, if the percentage of positive cells is > 1% for all 4 receptors, e) administration of antibodies to TIGIT and PD-1 to the specified patient.

In a further aspect, the present invention provides methods comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) PVRIG protein; ii) PVRL2 protein; iii) PD-1 protein; iv) PD-L1 protein; and v) appropriate isotype control for antibodies in i)-iv); c) performing fluorescence activated cell sorting (FACS); d) for each of PVRIG, PVRL2, PD-1 and PD-L1, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for all 4 receptors, e) administration of antibodies to PVRIG and PD-1 to the specified patient.

In a further aspect, the present invention provides methods comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) PVRIG protein; ii) PVRL2 protein; iii) TIGIT protein; iv) PVR protein; and v) appropriate isotype control for antibodies in i)-iv); c) performing fluorescence activated cell sorting (FACS); d) for each of PVRIG, PVRL2, TIGIT and PVR, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for all 4 receptors, e) administration of antibodies to PVRIG and TIGIT to the specified patient.

In a further aspect, the present invention provides methods comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) PVRIG protein; ii) TIGIT protein; iii) PVRL2 protein; iv) PD-1 protein; v) PD-L1 protein; and vi) appropriate isotype control for antibodies in i)-v); c) performing fluorescence activated cell sorting (FACS); d) for each of PVRIG, TIGIT, PVRL2, PD-1 and PDL1, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for all 5 receptors, e) administration of antibodies to PVRIG, TIGIT and PD-1 to the specified patient.

In a further aspect, the present invention provides methods comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) PVRIG protein; ii) PVRL2 protein; iii) TIGIT protein; iv) PVR protein; v) PD-1; and vi) appropriate isotype control for antibodies in i)-v); c) performing fluorescence activated cell sorting (FACS); d) for each of PVRIG, PVRL2, TIGIT and PVR, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for all 4 receptors, e) administration of antibodies to PVRIG, TIGIT and PD-1 to the specified patient.

In some embodiments, the present invention provides a method of treating cancer in a patient, comprising: a) obtaining a biopsy from said patient containing tumor cells; b) measuring the number of PD-L1-positive tumor cells or immune cells in the specified biopsy; c) if the indicated number of PD-L1-positive tumor cells or immune cells exceeds 1% compared to staining of the same tumor cells with the corresponding isotype control antibody for the antibodies used, administration of a three-component combination drug containing an anti-TIGIT antibody, an anti-PVRIG antibody and anti-PD-1 antibody; and d) if the indicated number of PD-L1-positive tumor cells or immune cells is less than 1% compared to staining of the same tumor cells with the appropriate isotype control antibody for the antibodies used, administration of a two-component combination preparation containing the anti-TIGIT antibody and the antibody anti-PVRIG.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

In some embodiments of the present invention, the anti-PD-1 antibody is an antibody selected from any of the anti-PD-1 antibodies described herein, including any of the antibodies described in FIG. 7.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 ( S241P) and CHA.9.547.13.H4 (S241P).

In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P).

In some embodiments of the present invention, the anti-PD-1 antibody is

- 4 044486 combat antibody selected from at least one of the following antibodies: pembrolizumab, cemiplimab and nivolumab.

In some embodiments of the present invention, the two-component combination therapy is selected from administering CPA.9.083.H4 (S241P) and CHA.7.518.1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SNA.9.547.13.N4 (S241P) and SNA.7.538.1.2.N4 (S241P).

In some embodiments of the present invention, the triple combination therapy is selected from administering CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P, nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.7.538 .1.2.H4 (S241P); CPA.9.083.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CNA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CPA. 9.083.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); CHA9.547.7.H4 (S241P) , cemiplimab and CHA.7.538.1.2.H4 (S241P), as well as CHA.9.547.13.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P).

In some embodiments of the present invention, the antibodies are administered simultaneously.

In some embodiments of the present invention, the antibodies are administered sequentially.

In some embodiments of the present invention, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma , kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, cancer of the Merkel cell, MSI-high cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS).

In some embodiments of the present invention, the cancer is selected from the group consisting of triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), as well as Merkel cell cancer, high MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

In some embodiments, the present invention provides a method of treating cancer in a patient, comprising administering a triple combination therapy comprising an anti-TIGIT antibody, an anti-PVRIG antibody, and an αntu-PD-1 antibody.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 ( S241P) and CHA.9.547.13.H4 (S241P).

In some embodiments of the present invention, said anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P).

In some embodiments of the present invention, the anti-PD-1 antibody is an antibody selected from the group consisting of pembrolizumab, cemiplimab, and nivolumab.

In some embodiments of the present invention, the triple combination therapy comprises administering an anti-PD-1 antibody in combination with a dual combination therapy selected from administering CPA.9.083.H4 (S241P) and CHA.7.518.1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SNA.9.547.13.N4 (S241P) and SNA.7.538.1.2.N4 (S241P).

In some embodiments of this method, a three-component combined

- 5 044486 therapy is selected from the administration of CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S24lP, nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.7.538 .1.2.H4 (S241P); CPA.9.083.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CNA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CHA9.547.13.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CPA. 9.083.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); CHA9.547.7.H4 (S241P) , cemiplimab and CHA.7.538.1.2.H4 (S241P), as well as CHA.9.547.13.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P).

In some embodiments of the present invention, the antibodies are administered simultaneously.

In some embodiments of the present invention, the antibodies are administered sequentially.

In some embodiments of the present invention, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma , kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, cancer from Merkel cell, MSI-high cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS).

In some embodiments of the present invention, the cancer is selected from the group consisting of triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), as well as Merkel cell cancer, high MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

In some embodiments, the present invention provides a pharmaceutical dosage kit comprising: a) a container containing a unit dose of an anti-TIGIT antibody; and b) a container containing a unit dose of anti-PVRIG antibody.

In some embodiments, the present invention provides a pharmaceutical dosage kit comprising: a) a container containing a unit dose of an anti-TIGIT antibody; b) a container containing a unit dose of anti-PVRIG antibody; and c) a container containing an anti-PD-l antibody.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

In some embodiments of the present invention, the anti-PD-l antibody is an antibody selected from any of the anti-PD-l antibodies described herein, including any of the antibodies described in FIG. 7.

In some embodiments, the present invention provides a method comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) TIGIT protein; ii) PVRIG protein; iii) PVR protein; iv) PD-1 protein; v) PD-L1 protein; and vi) appropriate isotype control for antibodies in i)-v); c) performing fluorescence activated cell sorting (FACS); d) for each of TIGIT, PVRIG, PVR, PD-1 and PD-L1, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for all 5 receptors, e) administration of antibodies to TIGIT, PVRIG and PD-1 to the specified patient.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is

- 6 044486 is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

In some embodiments of the present invention, the anti-PD-1 antibody is an antibody selected from any of the anti-PD-1 antibodies described herein, including any of the antibodies described in FIG. 7.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.

In some embodiments of the present invention, the anti-PD-1 antibody is selected from pembrolizumab and nivolumab.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P).

In some embodiments, the present invention provides a method comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) PVRIG protein; ii) TIGIT protein; iii) PVRL2 protein; iv) PD1 protein; v) PD-L1 protein; and vi) appropriate isotype control for antibodies in i)-v); c) performing flow cytometry/fluorescence activated cell sorting (FACS); d) for each of PVRIG, TIGIT, PVRL2, PD-1 and PD-L1, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for all 5 receptors, e) administration of antibodies to PVRIG and PD-1 to the specified patient.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

In some embodiments of the present invention, the anti-PD-1 antibody is an antibody selected from any of the anti-PD-1 antibodies described herein, including any of the antibodies described in FIG. 7.

In some embodiments of the present invention, the anti-PD-Ll antibody is an antibody selected from any of the anti-PD-Ll antibodies described herein, including any of the antibodies described in FIG. 62.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P).

In some embodiments of the present invention, the anti-PD-1 antibody is selected from pembrolizumab and nivolumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.

In some embodiments, the present invention provides a method comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) PVRIG protein; ii) PD-1 protein; iii) PVRL2 protein; iv) TIGIT protein; v) PVR protein; and vi) appropriate isotype control for antibodies in i)-v); c) performing fluorescence activated cell sorting (FACS); d) for each of PVRIG, PVRL2, TIGIT and PVR, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for all 5 receptors, e) administration of antibodies to PVRIG and TIGIT to the specified patient.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

In some embodiments of the present invention, the anti-PD-1 antibody is an antibody selected from any of the anti-PD-1 antibodies described herein, including any of the antibodies described in FIG. 7.

In some embodiments of the present invention, the anti-PD-Ll antibody is an antibody selected from any of the anti-PD-Ll antibodies described herein, including any of the antibodies described in FIG. 62.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P).

In some embodiments of the present invention, the anti-TIGIT antibody is CPA9.086.

- 7 044486

In some embodiments of the present invention, the anti-PD-1 antibody is selected from pembrolizumab and nivolumab.

In some embodiments, the present invention provides a method of treating cancer in a patient, comprising: a) obtaining a biopsy from said patient containing tumor cells; b) measuring the number of PD-L1 positive tumor cells or immune cells in said biopsy; c) if the indicated number of PD-L1-positive tumor cells or immune cells exceeds 1% compared to staining of the same tumor cells with the corresponding isotype control antibody for the antibodies used, administration of a three-component combination drug containing an anti-TIGIT antibody, an anti-PVRIG antibody and αntu-PD-L1 antibody; and d) if the indicated number of PD-L1-positive tumor cells or immune cells is less than 1% compared to staining of the same tumor cells with the appropriate isotype control antibody for the antibodies used, administration of a two-component combination preparation containing the anti-TIGIT antibody and the antibody anti-PVRIG.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

In some embodiments of the present invention, the anti-PD-1 antibody is an antibody selected from any of the anti-PD-L1 antibodies described herein, including any of the antibodies described in FIG. 62.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 ( S241P) and CHA.9.547.13.H4 (S241P).

In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P).

58. The method according to any one of claims 52-57, characterized in that said anti-PD-L1 antibody is an antibody selected from at least one of the following antibodies: atezolizumab, avelumab and durvalumab.

In some embodiments of the present invention, the two-component combination therapy is selected from administering CPA.9.083.H4 (S241P) and CHA.7.518.1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SNA.9.547.13.N4 (S241P) and SNA.7.538.1.2.N4 (S241P).

In some embodiments of the present invention, the triple combination therapy is selected from administering CPA.9.083.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CHA.9.547.7.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), atezolizumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); CHA9.547.7.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), atezolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), avelumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), avelumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), avelumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), avelumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P, avelumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), avelumab and SHA.7.538.1.2.H4 (S241P); SHA7.538.1 .2.H4 (S241P); CPA.9.083.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CHA.9.547.13.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), durvalumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), durvalumab and CHA.7.538.1.2.H4 (S241P); CHA.9.547.7. H4 (S241P), durvalumab and SHA.7.538.1.2.H4 (S241P), and SHA.9.547.13.H4 (S241P), durvalumab and SHA.7.538.1.2.H4 (S241P).

In some embodiments of the present invention, the antibodies are administered simultaneously.

In some embodiments of the present invention, the antibodies are administered sequentially.

In some embodiments of the present invention, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer),

- 8 044486 melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloblastic leukemia (AML), acute lymph regional leukemia T-cells (T-ALL ), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel cell cancer, MSI-high cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS).

In some embodiments of the present invention, the cancer is selected from the group consisting of triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), as well as Merkel cell cancer, high MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

In some embodiments, the present invention provides a method of treating cancer in a patient, comprising administering a triple combination therapy comprising an anti-TIGIT antibody, an anti-PVRIG antibody, and an αntu-PD-L1 antibody.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 ( S241P) and CHA.9.547.13.H4 (S241P).

In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P).

In some embodiments of the present invention, the anti-PD-L1 antibody is an antibody selected from the group consisting of atezolizumab, avelumab, and durvalumab.

In some embodiments of the present invention, the triple combination therapy comprises administering an anti-PD-L1 antibody in combination with a dual combination therapy selected from administering CPA.9.083.H4 (S241P) and CHA.7.518.1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SNA.9.547.13.N4 (S241P) and SNA.7.538.1.2.N4 (S241P).

In some embodiments of the present invention, the triple combination therapy is selected from administering CPA.9.083.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CHA.9.547.7.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CHA.9.547.13.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), atezolizumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), atezolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); CHA9.547.7.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), avelumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), avelumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), avelumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P, avelumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), avelumab and SHA.7.538.1.2.H4 (S241P); SHA7.538.1 .2.H4 (S241P); CPA.9.083.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CHA.9.547.13.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), durvalumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), durvalumab and CHA.7.538.1.2.H4 (S241P); CHA.9.547.7. H4 (S241P), durvalumab and SHA.7.538.1.2.H4 (S241P), and SHA.9.547.13.H4 (S241P), durvalumab and SHA.7.538.1.2.H4 (S241P).

In some embodiments of the present invention, the antibodies are administered simultaneously.

In some embodiments of the present invention, the antibodies are administered sequentially.

In some embodiments of the present invention, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma , kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, cancer of the Merkel cell, MSI-high cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS).

In some embodiments of the present invention, the cancer is selected from the group consisting of triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), as well as Merkel cell cancer, high MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplasia

- 9 044486 logical syndromes (MDS).

III. Brief description of graphic materials

In fig. 1A-1B show the amino acid sequences of the human IgG1 constant domains (with some suitable amino acid substitutions), human IgG2, IgG3, IgG4, IgG4 with a hinge variant that finds particular use in this invention, and the kappa and lambda light chain constant domains.

In fig. Figure 2 shows the sequence of the TIGIT, PVRIG and PD-1 proteins of humans and cynomolgus monkeys (referred to as Supo)

In fig. 3A-3SSSS show the sequences of four anti-TIGIT antibodies that block the interaction of TIGIT and PVR, CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 (S241P) and CHA.9.547 .13.H4 (S241P), as well as the reference antibodies, BM26 and BM29, and numerous other anti-TIGIT antibodies.

In fig. 4A-4C show FACS FACS KD results of anti-TIGIT antibodies (CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA.9.547.7.H4 (S241P) and CHA.9.547.13.H4 ( S241P), as well as reference antibodies, BM26 and BM29, binding to TIGIT-overexpressing Expi293 HEK cells from human (A), cynomolgus (B), and mouse (C).

In fig. 5A-5F show the sequences of two anti-PVRIG antibodies, CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 (S241P). Other anti-PVRIG antibodies are shown in FIG. 63.

In fig. 6 demonstrates the binding of CHA.7.518.1.H4 (S241P) to PVRIG using flow cytometry. (A) Binding to PVRIG-overexpressing HEK293 cells. CHA.7.518.1.H4 (S241P) binds human and cynomolgus PVRIG-overexpressing HEK293 cells, but not mouse PVRIG-overexpressing HEK293 cells or parental HEK293 cells. (B) Binding of CHA.7.518.1.H4 (S241P) to Jurkat cells. Specific binding was observed for CHA.7.518.1.H4 (S241P) but not for the irrelevant isotype control antibody. The dissociation constant (KD) relative to the binding of CHA.7.518.1.H4 (S241P) to targets expressed on cells is given in the table.

In fig. 7A-7F show the sequences of two anti-PD-1 antibodies.

In fig. 8 demonstrates expression of PVRIG, TIGIT, PD1 on CMVpp65-reactive T cells as described in the experiments of Example 1. (A) Gating strategy for detection of tetramer-stained CMV-CTL. Demonstrated gating hierarchy and tetramer-positive cells in three donors. Lymphocytes were gated in the FS/SS quadrant (top left), followed by singlet selection and subsequent removal of CD14-CD19-CD56- cells, and subsequent removal of CD3+ CD8+ positive cells. In the CD3+ CD8+ positive population, the percentage of cells binding each tetramer was determined from individual donors. Staining results using the HLA-A*02:01 CMV tetramer are demonstrated. (B) Expression of PVRIG, TIGIT, and PD-1 on CMVpp65-reactive T cells obtained from 3 donors is demonstrated.

In fig. Figure 9 demonstrates the kinetics of PVRIG, TIGIT, and PD-1 expression on CCD8 + CMV + T cells as described in the experiments in Example 1. (A) Shows the percentage of pp65 tetramer positive CD8 T cells after 0, 72, 144, 216, and 288 hours of stimulation. IL-2, IL-7 and CMV pp65 peptidam. (B) TIGIT, (C) CHA.7.518.1.H4 (S241P), (D) PD-1 expression on CMVpp65-reactive CD8 T cells at various time points after stimulation (n=3).

In fig. 10 shows the expression of PVRL2, PVR, PDL1 and HLA-A2 on Colo205 and Panc.04.05 cells assessed by flow cytometry as described in the experiments of Example 1. The number in the upper right corner indicates the percentage of ligand (PVRL2, PVR, PDL1) or HLA-A2 expressed on tumor cell lines compared with an isotype control antibody.

In fig. 11A-1Y demonstrate the effect of inhibitory receptor blockade on CMVpp65-reactive CD8 T cells in co-culture with cancer cell lines, as described in the experiments in Example 1. CMVpp65-reactive T cells for 2 donors (donor 4 and donor 156) were co-cultured with 0 .03 μg/ml Panc.04.05 or Colo205 cells loaded with CMVpp65 peptide for 24 h in the presence of 10 μg/ml CHA.7.518.1.H4 (S241P), anti-TIGIT, anti-PD-1, or isotype controls, either alone or in combination. (A) CHA.7.518.1.H4 (S241P), anti-TIGIT or anti-PD1 antibodies tested alone, in a two-component combination, or in a three-component combination. (B) CHA.7.518.1.H4 (S241P) and anti-TIGIT antibodies tested alone or in combination. (C) CHA.7.518.1.H4 (S241P) and anti-PD1 antibodies tested alone or in combination. (D) Anti-TIGIT and anti-PD1 antibodies tested alone or in combination. Conditioned media were analyzed for cytokine secretion. Histograms show mean + standard deviation for IFN-γ, where each point represents a technical replicate. Data are representative of n>2 experiments.

In fig. 12 demonstrates PVRIG expression on cells from dissected tumors as described in the experiments of Example 2. (A) Samples were grouped based on tumor type as determined by the histopathological report. Each sample demonstrated PVRIG expression at

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CD4+ T cells, CD8+ T cells, CD4-CD8- T cells and NK cells. Each point in the column represents a different sample. An MFIr value in a sample greater than 1 indicates detection of PVRIG expression. The median is depicted by the median line, and the upper and lower quartiles are represented by the gray space above and below the median line. (B) All tumor samples examined demonstrated PVRIG expression on CD4+ T cells, CD8+ T cells, CD4-CD8- T cells, and NK cells. The median is depicted by the median line, and the upper and lower quartiles are depicted by the light gray and dark gray space above and below the median line. Whiskers depict 1.5 times the interquartile range. (C) Representative FACS histograms for PVRIG (blue) versus isotype control (red) are shown for 4 subsets of cells isolated from lung and kidney tumors.

In fig. 13 demonstrates correlation analysis of PD1, TIGIT and PVRIG expression on CD4+ and CD8+ T cells from dissected endometrial tumors as described in the experiments of Example 2. For each endometrial sample, MFIr for PVRIG, PD1 and TIGIT on CD4 and CD8 T cells was calculated. Spearman correlation analysis was performed and r2 and p values were obtained.

In fig. 14 demonstrates coexpression analysis of PD1, TIGIT, and PVRIG expression on CD8+ T cells from a dissected lung and kidney cancer specimen as described in the experiments of Example 2.

In fig. 15 shows a comparison of PVRIG expression on T cells from dissected tumors with corresponding NCTs (normal adjacent tissues) as described in the experiments of Example 2. (A) Corresponding tumor and normal adjacent tissues from colon/stomach/rectal/endometrial tumors. uterus, kidney, lung, and ovary were assessed for PVRIG expression on CD4 and CD8 T cells. Each line represents the corresponding donor. A paired Student's t test was used for all samples, comparing PVRIG expression on CD4 and CD8 T cells in NCT and tumor. (B) PVRIG fold change (in NCT versus tumor) is plotted against PD1 fold change for CD4 and CD8 T cells. Spearman correlation analysis was performed and the r value and p value were obtained.

In fig. 16 demonstrates PVRL2 expression on immune and nonimmune cell subsets from all dissected tumor samples as described in the experiments of Example 2. PVRL2 expression on various tumor-derived cell subsets is demonstrated. MFIr values greater than 1 indicate detection of PVRL2 expression. The median is depicted by the median line, and the upper and lower quartiles are depicted by the light gray and dark gray space above and below the median line. Whiskers depict 1.5 times the interquartile range.

In fig. 17 demonstrates PVRL expression on non-immune cell subsets from dissected tumors as described in the experiments of Example 2. Samples were grouped based on tumor type, as determined by the histopathological report. Each sample demonstrated PVRL2 expression on CD45-nonimmune cells. Each point represents a different sample. The median is depicted by the median line, and the upper and lower quartiles are represented by the gray space above and below the median line.

In fig. 18 demonstrates PVRL2 expression on subsets of myeloid cells from dissected tumors as described in the experiments of Example 2. Samples were grouped based on tumor type, as determined by histopathological report. Each sample demonstrated PVRL2 expression on myeloid cells, which included monocyte, mDC, and pDC populations. Each point represents a different sample. The median is depicted by the median line, and the upper and lower quartiles are represented by the gray space above and below the median line.

In fig. 19 shows a comparison of PVRL2 expression on monocytes and ^CD45 tumor cells from dissected tumors and corresponding NCTs as described in the experiments of Example 2. (A) Corresponding tumor and normal adjacent tissues from colon/stomach/rectum, endometrial/uterine tumors, kidneys, lungs, and ovaries were assessed for PVRL2 expression on CD45 cells and on monocytes. Each line represents the corresponding donor. A paired Student's t test was used for all samples, comparing PVRL2 expression on CD45 cells and monocyte cells in NCT and tumor. (B) PVRL2 fold change (in NCT versus tumor) is plotted against PD-L1 fold change for CD45 cells and for monocytes. Spearman correlation analysis was performed and the r value and p value were obtained.

In fig. 20 demonstrates the coexpression of PVRIG on T cells with PVRL2 on monocytes and ^CD45 cells in tumor tissues, as described in the experiments in Example 2. The graph shows the expression of PVRIG on CD8 T cells and PVRL2 on monocytes or CD45 cells from the same sample. Tumors were grouped by type, with each dot representing an individual tumor. The reference lines are drawn at an MFIr value of 2.

In fig. 21A-21B demonstrate the expression of PVRL2 and PD-L1 in colon, skin and breast cancer, as described in the experiments of Example 3.

In fig. 22 demonstrates PVRL2 expression in PD-L1-negative and PD-L1-positive tumors as described in the experiments of Example 3. Based on PD-L1 staining, tumors were classified as PD-L1 negative (no PD-L1 staining was observed in both duplicate cores for each tumor), or PD-L1 positive (positive staining

- 11 044486 was observed in both duplicate nuclei for each tumor. A) PVRL2 expression analyzed and demonstrated for each cancer type. B) For PD-L1-negative tumors, the number of samples expressing PVRL2 (partially PVRL2-positive or more/total samples) for each cancer type is shown.

In fig. 23 demonstrates the expression of PVRL2 and PD-L1 on the invasive tumor surface as described in the experiments of Example 3. A. In this tumor sample, PVRL2 was expressed on both immune cells and tumor cells of the invasive tumor surface, as shown by blue and red lines . B) In this tumor sample, PD-L1 was expressed in the immune compartment.

In fig. 24 demonstrates antitumor responses to mono-, dual-, and triple-antibody combinations in the CT26 tumor model as described in the experiments in Example 4. Groups of 10-15 Balb/c mice were injected subcutaneously with 5x105 CT26 cells. Mice received the assigned antibody combination 2 times per week for 3 weeks, starting on day 7 post-inoculation. A) Tumor volumes of all analyzed groups were measured 2 times a week, including the positive control group (anti-PDL-1 + anti-CTLA-4 antibodies). The TGI and p values for the triple combination therapy group compared with the indicated groups are shown in the table. B) Relative survival rate in certain groups. C) Spider plots show individual response in intervention groups, while PR indicates partial responders with tumor size less than or equal to 1000 mm ³ . Fig. 25 depicts expression profiles for PVRIG and PVRL2, as well as PD-L1 in various human tumors as described in the experiments of Examples 2 and 3.

In fig. 26 demonstrates in vivo data using an anti-PVRIG antibody in TIGIT −/− mice or a combination of anti-PVRIG and anti-PD-1 antibodies in wild-type Balb/c mice to reduce syngeneic tumor growth as described in the experiments of Example 4.

Fig. 27 PVRIG is highly expressed in subsets of cytotoxic lymphocytes from a human cancer sample. A) Demonstrated expression of PVRIG on subsets of leukocytes from PBMCs from 5-8 healthy donors. PVRIG expression is defined as the ratio of the MFI of PVRIG relative to the MFI of the isotype control. B) Demonstrated expression of PVRIG, TIGIT, CD96 and PD-1 on peripheral blood Tregs compared to CD8 T cell subsets from PBMCs from 5 healthy donors. C) CMV pp65-specific T cells from 3 healthy donors were expanded in vitro using pp65 peptide (495-503), IL-2 and IL-7 for up to 7 days. Expression of TIGIT (blue) and PVRIG (black) on HLA-A2/pp65 tetramer-positive cells (495-503) is demonstrated. D) Human T cells cultured with allogeneic DCs and expression of TIGIT and PVRIG on CD4+ T cells at days 0, 1, 2 and 7 after activation are demonstrated. E) Representative FACS plots showing PVRIG expression (blue) versus isotype control (red) on TILs (CD4 T cells, CD8 T cells, and NK cells) from representative lung and kidney cancers. F) Demonstrated co-expression of PVRIG, TIGIT and PD-1 on CD4 and CD8 TILs from a lung cancer sample. G) Demonstrated expression of PVRIG on CD8+ and CD4+ TILs from dissected human cancer tumors of various types. Each point represents an individual tumor from an individual patient. H) Relative expression of CD8 TILs was assessed in comparison with Treg TILs for PVRIG, TIGIT and PD-1 from endometrial, kidney and lung tumors. For each tumor, fold expression on CD8 TILs was normalized to fold expression on Treg TILs and plotted. For A, B, C, G and H, the mean ± SEM is shown using error bars.

Fig. 28A - Fig. 28F. PVRL2 expression is increased in the tumor microenvironment. A) PVRL2 expression was assessed by IHC in lung, ovarian/endometrial, breast, colon, and kidney tumor cells. For each tumor, 2 independent observers scored 2 cores. Representative staining for each descriptor is demonstrated in Figure B) Representative melanoma tumor showing PVRL2 expression on tumor cells as well as immune cells in the stroma. C) PVRL2 expression from dissected tumors was examined by FACS on subsets of CD45 ^- , CD14 + and CD14'CD33 ^b “ ^c mDC cells. For each cancer type, the mean ± SEM is given. D) Representative FACS plots of PVRL2 (blue) versus IgG (red) expression for lung cancer are shown. E) For tumor samples in which we were able to assess both PVRIG and PVRL2 expression, PVRIG expression on CD8 T cells is plotted against PVRL2 expression on CD14+ TAMs for each tumor. Each dot represents a different tumor sample. The red line represents 2-fold expression of PVRIG or PVRL2 compared to IgG.

Fig. 29. Distinct regulation of PVRL2 and PD-L1 on tumor cells. A) Expression of PD-L1 and PVRL2 was assessed by IHC on serial sections. Shown is PVRL2 expression on PDL1-negative (left) and PD-L1-positive (tumors). Tumors were defined as PD-L1 negative if there was no staining on duplicate nuclei for a given tumor. PD-L1 positive staining was defined as at least partial positive staining on both duplicate cores for a given tumor. The number of PVRL2-positive tumors from PDL1-positive and PD-L1-negative tumors is shown in the table (positive/total). B, C)

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Representative expression of PVRL2+PD-L1' endometrial tumor (B) and PVRL2+PD-L1' lung tumor (C). D) Immature BM-DCs were cultured with the indicated stimuli and expression of PVR, PVRL2, PD-L1 was assessed by FACS on day 2 of culture. For each treatment, expression was normalized to the control treatment alone. E) Demonstrated expression of PVR, PVRL2 and PD-L1 on HT-29 cells treated with IFN-γ or medium alone. PD-L1 or PVRL2 is shown in blue, and IgG isotype control staining is shown in red.

Fig. 30. CHA.7.518.1.H4 (S241P) is a high affinity antibody that enhances T cell activation. A) Demonstrated binding of CHA.7.518.1.H4 (S241P) or isotype control IgG to parental HEK293 PVRIG or HEK293 cells by FACS. FACS KD values are given for binding of CHA.7.518.1.H4 (S241P) to HEK293 hPVRIG, HEK293 cPVRIG and Jurkat cells. B) CHA.7.518.1.H4 (S241P) disrupts PVRL2 Fc binding to HEK293 cells ectopically expressing PVRIG. Given is the mean value ± St Off for the values of three repetitions. C) CHA.7.518.1.H4 (S241P) blocks PVRIG Fc binding to HEK293 cells that endogenously express PVRL2. D) Human CD4 T cells were cocultured with aAPC CHO cells expressing cell surface bound anti-CD3 and hPVRL2 antibody in the presence of 10 μg/ml anti-PVRIG antibody and human IgG isotype control antibodies.

The effect of anti-PVRIG Ab on the proliferation of CD4 T cells isolated from 11 different donors was demonstrated. Error bars show mean ± SEM. E) gp100-specific T cell lines (TIL-209, TIL-463) were co-cultured with CHO cells engineered to express HLA-A2 and PVRL2 along with 10 μg/ml anti-PVRIG or isotype control IgG antibody. The production of IFN-γ and TNF-α was analyzed at a time point of 24 h from the start of co-culture. The mean value + St Off is given for the values of three repetitions. The percentage change in IFN-γ and TNF-α for each treatment modality relative to the isotype control is indicated by the number above each bar. F) Demonstrated expression of PVR, PVRL2 and PD-L1 (red) relative to IgG (blue) on MEL624, Colo205 and Panc.05.04 cells. For T cells, expression of PVRIG, TIGIT and PD-1 (red) relative to IgG (blue) was demonstrated on TIL-209 and TIL-463 gp100-specific T cells and on CMVpp65-specific T cells. To expand CMVpp65 reactive T cells, PBMCs were cultured with pp65 peptide (495-503), IL-2 and IL-7 for 10 days. Expression of PVRIG, TIGIT, PD-1 was demonstrated on HLA-A2/pp65-tetramer-positive cells. G) gp100-specific T cells (TIL-209, TIL-463) expanded from TILs derived from melanoma tumors were co-cultured with MEL624 cells in the presence of 10 μg/ml of the indicated antibodies. The concentration of IFN-γ in the conditioned media was determined after 24 hours. H, I) Expanded CMVpp65-specific T cells were co-cultured with Colo205 and Panc.05.04 cells, CMVpp65 peptide and the indicated antibodies at a concentration of 10 μg/ml. The concentration of IFN-γ in conditioned media was determined after 24 hours. For E, G, H, I, the mean value ± St Off for three repetitions is given. The percentage change in IFN-γ for each treatment relative to the isotype control is indicated by the number above each bar.

Fig. 31. Enhanced T cell function in PVRIG-deficient mice. A) Assessment of RNA expression of PVRIG measured by qRT-PCR from purified mouse immune cell subsets. Relative expression relative to the constitutive gene was determined using the ACt method. B) pmel CD8 ⁺ TCR transgenic T cells were activated by the levels of gp100 (25-33), PVRIG and TIGIT RNA transcripts assessed by qRT-PCR at the indicated time points. The graph shows the mean ± SEM of the results of 5 different experiments. C) Spleens were collected from littermates of PVRIG'/' and WT mice and analyzed by flow cytometry for PVRIG expression on NK, CD4+ and CD8+ T cells (resting cells). In addition, CD3+ T cells were isolated from splenocytes and activated for 11 days using anti-CD3/anti-CD28 beads. After activation, PVRIG expression on CD4+ and CD8+ T cells (activated cells) was analyzed by flow cytometry. Each dot represents cells obtained from an individual mouse. D) Splenocytes from WT and PVRIG'/' mice were labeled with Cell Proliferation Dye eFluor450 and cultured in the presence of control Fc (mouse IgG2a) or mouse PVRL2 Fc. After 4 days of culture, cell division was analyzed by flow cytometry. Shown are representative FACS plots from the experiment (left) and summary data of percent inhibition using PVRL2 Fc (defined as % proliferation of control Fc subtracted from % proliferation of PVRL2 Fc) in 3 independent experiments (right). * indicates p value < 0.05, paired Student's t test to analyze the change in proliferation in the presence of PVRL2-FC relative to proliferation in the presence of protein control in WT T cells compared to PVRIG'/' T cells. E) pmel CD8 ⁺ T cells obtained from pmel PVRIG'/' or pmel PVRIG WT mice were activated for 11 days with their cognate peptide and IL2. Activated pmel CD8 ⁺ cells were then cocultured with B16-Db/gp100 cells for 18 h, and CD107 expression and cytokine production were assessed after coculture. Introducing

- 13 044486 Lena four independent experiments, as indicated by each paired dot. * indicates p value < 0.05, Student's t test comparing PVRIG ^- / ^- with WT.

Fig. 32. PVRIG deficiency results in decreased tumor growth and increased CD8 effector T cell machinery. (A) C57BL/6 WT or PVRIG ⁻ / ⁻ mice were injected subcutaneously with 5x105 MC38 cells. Tumor volumes were measured 2 times a week. * indicates p value <0.05 for WT mice compared to PVRIG ⁻ / ⁻ mice (ANOVA). (B) Growth curves of individual tumors are shown. One representative experiment out of 2 performed is demonstrated. (C) C57BL/6 WT or PVRIG ⁻ / ⁻ mice were injected subcutaneously with 5x105 MC38 cells. On day 14 post-inoculation, mice were treated with anti-PDL1, twice a week for 2 weeks. Tumor volumes were measured twice a week. P value = 0.052 for WT mice compared to PVRIG ⁻ / ⁻ mice treated with anti-PD-L1. (D) Growth curves of individual tumors are shown. One representative experiment out of 2 performed is demonstrated. (E) Demonstrated number of CD8 ⁺ IFN-γ ⁺ TNF-a ⁺ effector cells in tumor-draining lymph nodes of mice from the 4 treatment groups on day 18. (F) Demonstrated total number of CD8 + IFN-γ + TNF-a + effector cells per mg tumor tissue on day 18. (GH) Total TIL score and cytotoxic T cell score relative to TIL obtained from nSolver 3.0 advanced analysis of the mouse pan-cancer immune codeset panel (Nanostring Technologies, Seattle, Washington state) conducted on CD45 ⁺ -enriched cells from MC38 day 18 TILs isolated from mice of 2 treatment groups, wild-type mice and PVRIG-deficient mice.

Fig. 33. Anti-PVRIG antagonist antibodies synergistically inhibit tumors grown in combination with PD-1 inhibitors or genetic TIGIT deficiency. A) The mPVRL2 Fc fusion protein was demonstrated to bind to engineered mPVRIG HEK293 cells that were preincubated with serial dilutions of anti-mPVRIG mAb or isotype control IgG Ab. B) BALB/c mice were injected subcutaneously with 5x105 CT26 cells. At day 14 postinoculation, mice were sacrificed and spleens as well as draining lymph nodes and tumors were collected. Cells were analyzed by flow cytometry for PVRIG expression on CD3 ⁺ CD4 + T cells, CD3 + CD8 + T cells, CD3 ⁻ CD49b ⁺ NK cells, CD11b ⁺ Gr-1 ⁺ myeloid-derived suppressor cells (MDSCs), and CD11b ⁺ F4 /80 ⁺ macrophages. C, D) BALB/c mice were injected subcutaneously with 5x105 CT26 cells. On day 7 postinoculation, mice were treated with anti-PD-L1 and/or anti-PVRIG Ab, 2 times per week for 3 weeks (arrows indicate Ab administration). C) Tumor volumes are demonstrated. *** indicates p value < 0.001 (ANOVA) for the anti-PD-L1 + Rat IgG2b group compared with the anti-PD-Ll + aPVRIG group. Arrows indicate the period when antibodies were administered. D. Survival analysis of mice with complete response. * indicates p value < 0.05 (log rank test) for the anti-PD-L1 + Rat IgG2b group compared with the anti-PD-L1 + anti-PVRIG group. One representative study of 3 studies was shown. E. C57BL/6 or TIGIT' ^/ 'mice were injected subcutaneously with 1x105 B16/Db-hmgp100 cells. Mice received the prescribed mAb twice a week for 3 weeks, starting on the day of inoculation (day 0). E. Tumor volumes were measured 2 times a week; the mean value ± SEM is given. Tumor growth inhibition measured on the indicated days was compared with WT + mIgG1 isotype control. *** indicates p value < 0.001 for TIGIT ^−/− + aPVRIG compared to WT + mIgG1 isotype control. Arrows indicate the period when antibodies were administered. F. Growth curves of individual tumors for each mouse are shown. One representative experiment out of 2 performed is demonstrated.

Fig. 34. PVRIG is expressed on T and NK cell TILs in human cancer. A) Demonstrated expression of PVRIG, TIGIT, CD96 and PD-1 on subsets of CD4 T cells from PBMCs from healthy donors. The mean value ± SEM is shown. B) Human T cells were co-cultured with allogeneic PBMCs; demonstrated PVRIG protein expression on CD4 and CD8 T cells (top). C) Tumors were dissected and individual cells were activated using anti-CD3 and anti-CD28. Expression of PVRIG (blue) relative to isotype control IgG (red) was assessed at day O (directly ex vivo) and day 5 postactivation. D) Demonstrated expression of PVRIG on NK cells from dissected human tumors. Each point represents an individual tumor from an individual patient. The mean ± 95% confidence interval is shown. D) Cells from dissected tumors were activated with anti-CD3 and anti-CD28 beads for 5 days. Expression of PVRIG (blue) relative to control IgG (red) on CD4 and CD8 T cells at day 0 directly ex vivo and at day 5 after activation is demonstrated for 2 dissected tumor samples. E) PVRIG expression was assessed on CD4 and CD8 T cells from dissected tumors and from dissected normal adjacent tissue from a matched donor. Each line represents corresponding tissue obtained from an individual patient. Paired Student's t test was used. F) Correlation analysis of fold expression of PVRIG, TIGIT, and PD-1 relative to isotype control IgG on CD4 and CD8 T cells from tumors was demonstrated. Each dot represents an individual tumor sample. The Spearman correlation coefficient and p value are given.

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Fig. 35. PVRL2 expression is upregulated in colon, skin, and breast cancers. A) Photomicrographs illustrating the binding of anti-human PVRL2 antibody Sigma to FFPE sections of positive cells, human PVRL2 CHO-S (right) compared to negative cells, CHO-S (left), after antigen retrieval at pH 9. B) Antibody anti- PVRL2 was analyzed in a panel of PVRL2+ (HT29, MCF7, PC3, PANC1, RT4, NCI-H1573) and PVRL2 (Jurkat, 0PM2, Daudi, CA46) cell lines. C-F) Patterned expression of PVRL2 in normal and cancerous lung tissues. C) Normal tissue showing no staining. D) Lung adenocarcinoma with partial positive staining. E) Lung adenocarcinoma showing positive staining. F) Lung adenocarcinoma with strong positive staining.

Fig. 36. Increased expression of PVRL2 on TAM and ^CD45 tumor cells compared to normal adjacent tissue. Expression of PVRL2 on ^CD45 cells and TAMs from the donor matched tumor and normal adjacent tissue was demonstrated. The p value of paired Student's t test is given.

Fig. 37. PVRIG and PVRL2 are coexpressed in the same tumor sample. PVRIG expression on CD4 T cells (A) and NK cells (B) was plotted against PVRL2 expression on TAMs for an individual tumor.

Fig. 38. Activity of CHA.7.518.1.H4 (S241P) on human T cells. A) Expression of PVRIG on CD4 T cells activated by CHO cells expressing anti-CD3 and PVRL2 associated with the cell surface. B) Demonstrated expression of HLA-A2, B-2m and PVRL2 in parental and engineered CHO-S cell lines. The expression fold relative to the isotype is indicated using a number. C) CHO cells ectopically expressing cell surface associated anti-CD3 and PVRL2 were co-cultured with purified CD8 T cells in the presence of varying concentrations of anti-PVRIG Ab or appropriate IgG control. % proliferation is given. Each point represents the average of three replicates. D) CHO cells with ectopic expression of HLA-A2/B2m and PVRL2 were co-cultured with 2 gp100-specific T cell lines (TIL F4, TIL 209) in the presence of 1 μg/ml gp100 and various concentrations of anti-PVRIG antibody or corresponding IgG control . TNF-α concentrations decrease on day 3 of coculture. Each value represents the average of three replicates.

Fig. 39. Characterization of binding interactions between mPVRIG and anti-mPVRIG surrogate antibody. A, B) Binding of mPVRIG to mPVRL2 was assessed using surface plasmon resonance. C) Soluble Fc receptor or control proteins were incubated in a dose-dependent manner with immobilized mPVRL2 HIS in ELISA format. Demonstrated Fc receptor binding. D) Soluble HRL protein PVRL2 was incubated in a dose-dependent manner on PVRIG Fc or DNAM Fc coated plates. E) Demonstrated binding of mPVRIG Fc or control Fc fusion protein to B16-F10 cell line transfected with mPVRL2 siRNA, mPVRsRNA or scrambled siRNA transfection. F) Affinity characterization of the rat anti-mouse PVRIG mAb was performed by examining the binding of anti-mPVRIG to HEK293 cells overexpressing mPVRIG. G) Affinity characterization of the rat anti-mouse PVRIG mAb was performed by examining the binding of anti-mPVRIG to the D10.G4.1 cell line endogenously expressing mPVRIG compared to a rat IgG isotype control. H) Binding of anti-mPVRIG to D10.G4.1 cells transfected with mouse PVRIG-siRNA (green histogram) compared to scr siRNA (orange histogram). I) Binding of mPVRIG Fc preincubated with anti-mPVRIG Ab to B16-F10 cells that endogenously express PVRL2.

Fig. 40. Generation of transgenic mice with knockout of PVRIG and TIGIT. PVRIG conditional knockout and Tigit knockout mouse lines were generated by Ozgene Pty Ltd (Bentley WA, Australia). A) A targeting construct in which PVRIG exons 1-4 were flanked by loxP sites was electroporated into the ES cell line C57BL/6, Bruce4 (Koentgen et al., Int Immunol 5: 957964, 1993). B) A targeting construct in which the coding region of Tigit exon 1 (including ATG) and exons 2 and 3 were replaced with an FR-flanked neo-cassette was electroporated into the ES cell line C57BL/6, Bruce4. Homologous recombinant ES cell clones were identified by Southern hybridization and introduced into goGermline blastocysts (Koentgen et al., Genesis 54: 326-333, 2016). Chimeric male mice were generated and crossed with C57BL/6J females to establish C57BL/6-based heterozygous germline progeny. Germline mice were crossed with the ubiquitous FLP C57BL/6 mouse line to remove the FRT-flanked selective marker cassette and generate conditional or knockout alleles (for PVRIG and Tigit, respectively). For PVRIG knockout, mice were further crossed with the ubiquitous Cre C57BL/6 mouse line to remove loxP -flanked exons and generate a knockout allele.

Fig. 41. PVRIG knockout mice are immunophenotypically similar to wild-type mice. Mice (n=5 per wild-type and PVRIG knockout cohort) were euthanized prior to venous blood collection into anticoagulant-coated tubes and organ collection. Individual cells were extracted from freshly collected bone marrow, thymus, spleen, skin and mesenteric lymph nodes. Cells were stained with fluorochrome-conjugated surface marker antibodies

- 15 044486 lami and analyzed on a BD LSR Fortessa flow cytometer. Panels illustrate comparable numbers of myeloid cells (A), dendritic cells (B), B cells (C), T cells (D), CD4 T cells (E), CD8 T cells (F), and NK cells ( G) by type of lymphoid tissue. (H-I) Venous whole blood was analyzed in the Hemavet 950 veterinary hematology system to compare differential rates and numbers of blood cell subsets between wild-type and PVRIG-deficient mice.

Fig. 42. Increased T cell effector function in PVRIG'/' mice treated with αntu-PDL1 compared to WT mice treated with anti-PD-Ll.

MC38 tumors were inoculated into WT mice or PVRIG'/' mice and then treated with anti-PD-Ll or rat isotype control IgG2b. At day 18, tumor-infiltrating CD45+ lymphocytes were purified from tumors, RNA was extracted, and transcript profiling was performed. Several T cell-associated genes are demonstrated, with each dot representing an individual mouse. Student's t-test p values are shown.

Fig. 43. Anti-TIGIT and anti-PVRIG antibodies induce the destruction of tumor cells. An in vitro coculture assay with expanded CMV-specific human CD8+ T cells was used to evaluate the effect of reference antibodies anti-TIGIT and CHA.7.518.1.H4 (S241P) on killing antigen-specific tumor cells. The HLA-A2+ target cell lines used in the assay were Me1624 (A) and Panc.05.04 (B). Synagis hIgG4 is an isotype control antibody. Luciferase activity in target cells was measured using Bio-Glo luciferase substrate. Representative data (n > 2) show the percentage of specific killing (mean +/- standard deviation) of Me1624 or Panc.05.04 cells after 16 hours coculture with CMV-specific human CD8+ T cells from three different donors.

Fig. 44. Dose-dependent destruction of tumor cells by anti-TIGIT antibodies with CHA.7.518.1.H4 (S241P). An in vitro coculture assay with expanded CMV-specific human CD8+ T cells was used to evaluate the effect of two different anti-TIGIT antibodies, BM26 and CPA.9.086, in combination with CHA.7.518.1.H4 (S241P), on antigen-specific killing of Mel624 cells. Luciferase activity in target cells was measured using Bio-Glo luciferase substrate. Representative data (n>2) demonstrate the percentage of specific killing (mean +/- standard deviation) of Mel624 cells after 16 hours of coculture with CMV-specific human CD8+ T cells from a single donor.

Fig. 45. CDR sequences CPA.9.086, IMGT and Kabat numbering.

Fig. 46. The combination of anti-TIGIT hIgG4 + CHA.7.518.1.H4 (S241P) induces the destruction of tumor cells. Co-culture of CMV-reactive CD8+ T cells with Me1624 PVR, PVRL2 and OE luciferase. Single dose of 10 μg/mL anti-TIGIT Ab and 10 μg/mL CHA.7.518.1.H4 (S241P) with CMV-reactive donor 4 with dose titration starting at 0.5 μg/mL aTIGIT Ab and 10 μg/mL SNA. 7.518.1.H4 (S241P) with CMV-reactive donor 156.

Fig. 47. Anti-TIGIT antibodies enhance IFN-γ in combination with anti-PD-1 antibody. An in vitro coculture assay with expanded CMV-specific human CD8+ T cells was used to evaluate the effect of CPA.9.086 compared with the reference antibodies BM26 and BM29 on the secretion of antigen-specific cytokines in combination with the anti-PD-1 antibody, pembrolizumab. The target cell line used in the assay was HLA-A2+ pancreatic adenocarcinoma cells, Panc.05.04, which endogenously express human PVR and PD-L1. Panc.05.04 cells were injected with CMV pp65 peptide at a concentration of 0.01 μg/ml at 37°C for 1 hour. Cells were then washed and seeded at a concentration of 50,000 cells/well in 96-well round-bottom plates treated with tissue culture. Human anti-TIGIT antibody or hIgG4 isotype control antibody (anti-Synagis) was added at a concentration of 0.1 μg/ml in combination with anti-PD-1 antibody (shaded bars) or hIgG4 isotype control antibody at a concentration of 10 μg/ml (solid bars). columns). CMV-specific human CD8+ T cells from a single donor were expanded according to the protocol above. 50,000 human CD8+ T cells were added to each well. Co-cultures were incubated at 37°C with 5% CO2 for 24 hours. The amount of human IFN-γ in the co-culture supernatant was measured by flow cytometry using a bead cytometric assay (BD Biosciences).

In fig. 48 depicts expression profiling of the PVRIG/TIGIT axis in tumors; Lung and endometrial cancers have high rates of both the PVRIG-PVRL2 and TIGITPVR pathways. (A,B) Expression of PVRIG and TIGIT was analyzed on CD4+ and CD8+ T cells from dissected human tumors by FACS. Fold expression was calculated by dividing the MFI value for PVRIG or TIGIT by the MFI value for control IgG. Gray line = no expression detected. Each orange dot represents an individual tumor sample, and the median of the samples is shown in the blue bar. C, D) PVRIG expression on CD8+ T cells versus PVRL2 expression on CD45' cells, or TIGIT expression on CD8+ T cells versus PVR expression on CD45' cells

- 16 044486 cells from dissected tumors are plotted. Each dot represents an individual tumor sample.

In fig. 49 depicts expression data for PD-1, PVRIG and TIGIT on CD8 T cells, which demonstrates that PVRIG+TIGIT+PD-1+CD8+TIL are widespread and have a depleted profile. A) TILs from human cancer tissues were stained for expression of PD1, PVRIG and TIGIT on CD8 T cells. The percentage of cells that express combinations of PD-1, PVRIG, or TIGIT on CD8+ T cells was determined using Boolean gating. B) Representative expression of PD-1, PVRIG and TIGIT on CD4+ and CD8+ T cells from a lung tumor is demonstrated. C) TILs from human cancers were stained for cell surface PD1, PVRIG and TIGIT on CD8+ T cells, permeabilized, and stained for Eomes and T-bet. The percentage of Eomes+ T-bet cells in each cell subset is shown. Paired Student's t test was used; p values are given. D) Representative FACS plots illustrating Eomes and T-bet expression on CD8+ T cells expressing PD-1, PVRIG, or TIGIT from an ovarian and bladder tumor are shown. E) The percentage of cells expressing Eomes+ T-bet- was determined based on the expression of PD-1, PVRIG, and TIGIT. Thus, PVRIG expression correlates with expression of the transcription factor Eomes+ T-bet-, a phenotype known to be associated with T cell exhaustion. Triple positive PVRIG+ TIGIT+ PD-1+ cells also had a high percentage of Eomes+ T-bet cells.

In fig. 50 demonstrated that PVRL2 is induced in cancer and expressed in PD‐L1 tumors. A) PVRL2 expression was assessed by IHC in lung, ovarian/endometrial, breast, colon, kidney, and skin tumors. Error bars show mean ± SEM. For each tumor, 2 nuclei were assessed by a pathologist and a score was calculated based on the extent and intensity of membranous staining on the tumor cells. The average score of 2 cores is given for each tumor. B) Expression of PD-L1 and PVRL2 was assessed by IHC on serial sections. Tumors were grouped by tissue type; demonstrated PVRL2 expression on PD-L1-negative and PD-L1-positive tumors. Tumors were defined as PD-L1 negative if there was no membranous staining on tumor or immune cells from any duplicate nucleus for a given tumor. PD-L1 positive staining was defined as membranous staining in at least 1 tumor core. Error bars show the mean ± SEM for each group. C) Representative expression of PVRL2 + PD-L1- endometrial carcinoma and PVRL2 + PD-L1- lung tumor.

In fig. 51. Anti-PVRIG/TIGIT/PD-1 has been demonstrated to synergistically enhance T cell function. (A) CMVpp65 CD8 T cells were stained for TIGIT/PD-1/PVRIG expression, and tumor cell lines were stained for PD-L1, HLA-A2, PVR, and PVRL2. Representative histograms of FACs are shown. B) CMVpp65-specific T cells were cocultured with Panc0504 and Colo205 cells, CMVpp65 peptide and the indicated antibodies at a concentration of 10 μg/ml. IFN-γ concentrations in conditioned media were determined at the 18 h time point. The percentage above the bar represents the % increase in IFN-γ secretion compared to the IgG isotype.

In fig. 52 demonstrated that blockade of the PVRIG‐PVRL2 interaction induces the expression of PD‐1 and TIGIT. CMVpp65-specific T cells from 1-2 donors were co-cultured with Panc0504, CMVpp65 peptide and the indicated antibodies at a concentration of 10 μg/ml for 18 hours. The cells were then stained for FAC; the percentage of PD-1, TIGIT and LAG3 cells for each treatment modality was demonstrated. Histograms for each receptor are presented. Red = Isotype, Blue = Target expression. A) TIGIT expression was induced by CHA7.518.1.H4 (S241P) or anti-PD1. (B) PD-1 expression was induced by CHA7.518.1.H4 (S241P) and/or CPA.9.083.H4 (S241P). (C) LAG-3 expression was not induced by CHA7.518.1.H4 (S241P), anti-TIGIT, or anti-PD-1.

Fig. 53. Tumors obtained within 24 hours of surgical resection were dissected and purified. CD3+ TILs were cocultured with MEL624 cells expressing surface bound anti-CD3 and the indicated antibodies at a concentration of 10 μg/ml. IFN-γ concentrations in conditioned media were determined at the 72-hour time point. The % change in IFN-γ for each method of exposure relative to hIgG4 was demonstrated.

Fig. 54. Blockade or deficiency of antibodies to PVRIG causes a decrease in tumor growth. Blockade or deficiency of PVRIG antibodies inhibit tumor growth. A) BALB/c mice were injected subcutaneously with 5x105 CT26 cells. On day 7 postinoculation, mice were treated with anti-PD-L1 and/or anti-PVRIG twice a week for 3 weeks. Tumor volumes are demonstrated. n=10 mice per group. The average value +/- SEM is shown. *** indicates p value < 0.001 (repeated measures ANOVA) for anti-PD-L1 ⁺ Rat IgG2b compared with anti-PD-L1 ⁺ anti-PVRIG groups. B) C57BL/6 WT or PVRIG ^- / ^- mice were injected subcutaneously with 5x105 MC38 cells. n=10 mice per group. The average value +/- SEM is shown. * indicates p value < 0.05 for WT versus PVRIG −/− mice (repeated measures ANOVA). The growth curves of individual

- 17 044486 tumors. Representative data from n=2 experiments.

Fig. 55. Expression profiling of the PVRIG/TIGIT axis in human tumors. Lung and endometrial cancers have high rates of both the PVRIG-PVRL2 and TIGITPVR pathways. (A,B) Expression of PVRIG and TIGIT was analyzed on CD4+ and CD8+ T cells from dissected human tumors by FACS. Fold expression was calculated by dividing the MFI value for PVRIG or TIGIT by the MFI value for control IgG. Gray line = no expression detected. Each orange dot represents an individual tumor sample, and the median of the samples is shown in the blue bar. C, D) PVRIG expression on CD8+ T cells versus PVRL2 expression on CD45 ⁻ cells, or TIGIT expression on CD8+ T cells versus PVR expression on CD45 ⁻ cells from dissected human tumors are plotted. Each dot represents an individual tumor sample.

Fig. 56. PVRIG+ TIGIT+ PD1+ cells have the highest % and highest level of depletion of CD8+ TILs. PVRIG+ TIGIT+ PD1+ CD8+ TILs are widespread and have an attrition phenotype. A) CD8 ⁺ TILs from human cancer tissues were stained for expression of PD-1, PVRIG, and TIGIT. The percentage of CD8 ⁺ TILs that expressed PD-1, PVRIG, or TIGIT combinations on CD8 + T cells was determined using Boolean gating. Each dot represents an individual tumor sample. B) CD8 ⁺ TILs from human cancers were stained for cell surface PD1, PVRIG and TIGIT, permeabilized, and stained for intracellular Eomes and T-bet. The percentage of Eomes+ T-bet- CD8 ⁺ T cells is shown. Paired Student's t test was used; p values are given. C) The percentage of Eomes ⁺ T-bet-CD8 ⁺ T cells expressing PD-1, PVRIG, and TIGIT was determined in a variety of human cancers.

Fig. 57. Relative expression differences of PVRL2 versus PVR depending on cancer type. Relative RNA and protein expression of PVRL2 and PVR in different human tumors. RNA expression of PVRL2 and PVR from TCGA was plotted as the ratio of PVRL2 to PVR in a variety of human cancers (left panel). Tumors with higher RNA expression of PVRL2 compared to PVR include breast, ovarian, prostate, endometrial, bladder, pancreatic, and lung tumors. The protein expression ratio (gMFI) of PVRL2 relative to PVR on CD45 tumor cells from dissected human tumors is plotted (right panel). Each dot represents an individual tumor sample. Tumors with higher expression of PVRL2 protein compared to PVR include ovarian, breast, endometrial, lung, prostate, oral, and gastric tumors. Higher RNA expression correlates with higher protein levels for PVRL2 in several tumors, including breast, ovarian, endometrial, prostate and lung cancers.

Fig. 58. PVRL2+ PVR- tumor cells and APCs are present in human tumors. PVRL2+ PVR- tumor cells and APCs are present in human tumors. The expression of PVRL2 and PVR from dissected tumors determined by FACS on A) CD45 tumor cells, and B) cDC2 (CD1c ⁺ CD14-HLA-DR ^high LinCD141) and CD14 + TAMs is plotted. PVRL2+ PVR- tumor cells and APC are represented on the graph as red dots in areas with a positive percentage. Representative FACS plots of PVRL2 and PVR (blue) expression versus IgG isotype control (red) for ovarian and endometrial tumors are shown.

Fig. 59 The combination CHA7.518.1.H4 (S241P) + CPA.9.083.H4 (S241P) is more active than pembrolizumab on primary CD3+ TILs. CHA7.518.1.H4 (S241P) and/or CPA.9.083.H4 (S241P) have similar or greater activity than pembrolizumab on freshly isolated human TILs. Human tumors obtained within 24 hours of surgical resection were dissected and CD3+ TILs were purified. Isolated CD3+ TILs were co-cultured with a modified Mel-624 tumor cell line expressing surface-bound anti-CD3 and the indicated antibodies at a concentration of 10 μg/ml. Secretion of IFN-γ in the conditioned medium was measured at the 72 hour time point. The percentage change in IFN-γ for each treatment compared to the hIgG isotype control was demonstrated.

Fig. 60. PVRIG/PVRL2 blockade induces PD-1 and TIGIT expression. PVRIG/PVRL2 blockade induces PD-1 and TIGIT expression. CMVpp65 ^- specific CD8 ⁺ T cells from 2 donors were co-cultured with Panc.05.04, CMVpp65 peptide, and the indicated antibodies at a concentration of 10 μg/ml for 18 hours. Cells were stained; The percentages of A) TIGIT+, B) PD ^{- 1+} and C) LAG3 ⁺ CD8+ T cells after each treatment are shown.

Fig. 61A-61C. The three-component combination demonstrates improved antitumor efficacy. A) Growth kinetics of CT26 tumors in the minimal disease model. Groups of 10 Balb/c females were inoculated with CT26 cells in the right flank. IV administration of antibodies began when the tumors reached the desired average volume (30-60 mm ³ ). Mice were administered anti-TIGIT mIgG1 or anti-PVRIG mIgG1 at a dose of 10 mg/kg, anti-PD-L1 mIgG1 at a dose of 3 mg/kg and isotype control at a dose of 10 mg/kg in the form of a two-component or three-component combination, 3 times every two weeks. a total of 6 doses over two weeks. TGI with anti-TIGIT mIgG1 in combination was calculated by % TGI = [1- (average volume

- 18 044486 tumor of the test product, divided by the average tumor volume of the control product) * 100]. An asterisk (***, ****) indicates p < 0.001 or p < 0.0001, respectively, for differences between the two-component or tripartite combination and isotype control compared with the two-component or tripartite combination, using two-way ANOVA. B) Spider plots of individual tumor volumes of each mouse in the three treatment groups were measured until tumor volumes >1500 mm ³ or 45 days were achieved (the main parameters assessed in the study). C) Kaplan-Meier survival curves for mice treated with drugs in three different treatment groups. The log-rank test (Cox-Mantel test) revealed a p value of <0.0001, 90% survival in mice treated with the triple antibody combination compared with 40% survival in mice treated with the dual antibody combination.

In fig. 62A-62I show sequences of typical anti-PD-L1 antibodies.

In fig. 63A-63AAA show the sequences of numerous typical antibodies to PVRIG.

IV. Detailed description of the invention

A. Introduction.

Therapeutic antibodies directed against immune checkpoint inhibitors such as PD-1 are showing great promise in cancer treatment in clinical practice. Cancer can be thought of as the patient's inability to recognize and eliminate cancer cells. In many cases, these transformed (eg, cancer) cells antagonize immune surveillance. There are natural control mechanisms that limit the activation of T cells in the body to prevent runaway T cell activity, which can be used by cancer cells to evade or suppress the immune response. The goal of immunotherapy is to restore the ability of immune effector cells, especially T cells, to recognize and eliminate cancer. The field of immuno-oncology, sometimes called immunotherapy, is rapidly evolving, and several T-cell inhibitory antibodies have recently been approved, such as Yervoy®, Keytruda®, and Opdivo®. These antibodies are commonly called checkpoint inhibitors because they block normally negative regulators of T-cell immunity. It is generally believed that various immunomodulatory signals, both co-stimulatory and co-inhibitory, can be used to elicit an optimal antigen-specific immune response.

Typically, these monoclonal antibodies bind to checkpoint inhibitor proteins such as CTLA-4 and PD-1, which normally prevent or suppress cytotoxic T cell (CTL) activation. By inhibiting checkpoint proteins, for example by using antibodies that bind these proteins, an enhanced antitumor T cell response can be achieved. That is, these cancer checkpoint proteins suppress the immune response; When the proteins are blocked, for example by anti-checkpoint protein antibodies, the immune system is activated, resulting in immunostimulation and the ability to treat pathological conditions such as cancer and infectious diseases.

This invention is directed to compositions and methods of using multiple anti-checkpoint inhibitors in combination to obtain better patient outcomes. In particular, combinations of anti-TIGIT, anti-PVRIG and αntu-PD-1 antibodies are being considered. In addition, these methods are particularly useful in combination with assessing PD-L1 expression levels in a patient's tumor. If the percentage of PD-L1-positive tumor cells or immune cells exceeds 1% (> 1%) compared with the same tumor cells stained with an antibody corresponding to the isotype control antibody for the antibodies used, then a three-component combination of anti-TIGIT antibodies should be administered, anti-PVRIG and anti-PD-1. Whereas, patients with PD-L1-positive tumor cells or immune cells in abundance below 1% (<1%) of isotype controls should be treated with a two-component combination of anti-TIGIT and anti-PVRIG antibodies.

As discussed herein, TIGIT is a coinhibitory receptor that is highly expressed on effector and regulatory (Treg) CD4+ T cells, effector CD8+ T cells, and NK cells. TIGIT has been demonstrated to attenuate the immune response through (1) direct signaling, (2) induction of ligand signaling, and (3) competition with disrupted signaling by the costimulatory receptor CD226 (also known as DNAM-1).

Human poliovirus receptor-associated immunoglobulin domain-containing protein, or PVRIG, is expressed on the cell surface of NK and T cells and shares several similarities with other known immune checkpoints. PVRIG has been recognized as a checkpoint inhibitor, see USSNs 62/118,208, 62/141,120, 62/235,823, 62/376,343, 15/048,967, 62/376,335, 62/417,217, and 62/477,974, all of which are explicitly included herein by reference in their entirety and, in particular, for antibody sequences, figures, and figure legends. As demonstrated in these papers, PVRL2 has been identified/confirmed as an analogue of PVRIG. Antibodies that bind to PVRIG were generated and a subset of

- 19 044486 set of antibodies that bind to PVRIG and block the interaction of PVRIG and PVLR2. When PVRIG is bound by its ligand (PVRL2), an inhibitory signal is generated that acts to dampen the NK and T cell immune response against the target cell (i.e., similar to PD-1/PDL1). Blocking PVRL2 binding to PVRIG turns off this inhibitory PVRIG signal and, as a result, modulates the immune response of NK and T cells.

PD-1, or programmed cell death protein 1, is a known checkpoint inhibitor. There are two approved αntu-PD-1 antibodies, pembrolizumab (Keytruda®), cemiplimab (REGN2810), and nivolumab (Opdivo®), with many others in clinical development (including, but not limited to, pidilizumab, clones of BAP049, as set forth in WO 2015/112900 (the sequences of which are expressly incorporated herein by reference), antibody 317-4B6 as set forth in WO 2015/035606 (the sequence of which is expressly incorporated herein by reference), antibody ARE2058 as set forth in US 2016/0075783 (the sequence of which is expressly incorporated herein by reference).

Three approved anti-PD-L1 antibodies are in clinical development: atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®) and durvalumab, as well as other anti-PD-L1 antibodies.

The functional effects of combinations of these antibodies on NK and T cells can be assessed in vitro (and in some cases in vivo, as described in more detail below) by measuring changes in the following parameters: proliferation, cytokine release, and cell surface markers. Accordingly, the functional effects of anti-TIGIT antibodies on NK, effector T cells and Treg cells can be assessed in vitro (and in some cases in vivo, as described in more detail below) by measuring changes in the following parameters: proliferation, cytokine release and markers cell surface. For NK cells, increased cell proliferation, cytotoxicity (the ability to kill target cells, measured by increased expression of CD107a, granzyme, and perforin or by directly measuring target cell killing), cytokine production (eg, IFN-γ and TNF), and receptor expression cell surface (eg CD25) indicates immune modulation, such as enhanced killing of cancer cells. For effector T and Treg cells, increased proliferation, increased expression of cell surface activation receptors (e.g. CD25, CD69, CD137 and PD-1), cytotoxicity (ability to kill target cells as mentioned above) and cytokine production (e.g. IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-10, IL-17A) indicates immune modulation, such as enhanced killing of cancer cells. Accordingly, treatment evaluation can be performed using assays that assess one or more of the following mechanisms: (i) increased immune response, (ii) increased αβ and/or γδ T cell activation, (iii) increased cytotoxic T cell activity, ( iv) increased activity of NK and/or NKT cells, (v) decreased suppression of αβ and/or γδ T cells, (vi) increased secretion of anti-inflammatory cytokines, (vii) increased secretion of IL-2; (viii) increased production of interferon-γ, (ix) increased Th1 response, (x) decreased Th2 response, (xi) decreased number or elimination of cells and/or activity of at least one of the regulatory T cells.

In particular, any of the assays described in Example 1 can be used to measure T cell activation and/or T cell inhibition.

Thus, some embodiments of the present invention provide the use of a combination therapy of anti-TIGIT, anti-PVRIG and anti-PD-1 antibodies (or only anti-TIGIT and anti-PVRIG antibodies in some cases, as described herein) for implementation in the body subject to one or more of the following mechanisms: (a) activation of anti-inflammatory cytokines; (b) increasing proliferation and/or expansion of T cells; (c) increased production of interferon or TNF-α by T cells; (d) increased secretion of IL-2; (f) stimulating antibody responses; (f) inhibiting the growth of cancer cells; (g) stimulation of antigen-specific T-cell immunity; (h) promoting activation of CD4+ and/or CD8+ T cells; (i) attenuation of Treg-mediated suppression; (j) promoting NK cell activity; (k) promoting apoptosis or lysis of cancer cells; and/or (l) cytotoxic or cytostatic effect on cancer cells.

Accordingly, the present invention provides anti-TIGIT, anti-PVRIG and anti-PD1 antibodies for use in combination therapy and in combination with diagnostic assays that measure the expression levels of one or more of the following proteins: TIGIT, PVRIG and PD-1, and /or measuring levels of TIGIT ligands (eg, PVR), PVRIG (PVRL2) and PD-1 (PD-L1).

B. Definitions.

For the purpose of a more complete understanding of this application, several definitions are provided below. Such definitions are intended to cover grammatical equivalents.

In this context, ablation refers to the reduction or elimination of activity. In some embodiments of the present invention, it is useful to remove activity from the constant domains of antibodies. For example, elimination of Fr/R binding means that the amino acid variant of the Fc region is characterized by less than 50% of the initial binding compared to an Fc region that does not contain

- 20 044486 specific variant, with a loss of less than 70-80-90-95-98% of activity, which is preferred, and, as a rule, activity below the level of detectable binding in the Biacore assay. As shown in FIG. 1, one of the ablation options in the IgG1 constant region is the N297A variant, which removes the native glycosylation site and significantly reduces the binding of FcyRIIIa and, thus, reduces antibody-dependent cellular cytotoxicity (ADCC).

By antigen binding domain or ABD, as used herein, is meant a set of six Complementary Determining Regions (CDRs) that, when present as part of a polypeptide sequence, specifically bind a target antigen as discussed herein. Thus, the TIGIT antigen binding domain binds the TIGIT antigen (the sequence of which is shown in FIG. 2) as described herein. As is known in the art, these CDRs are typically present as a first set of heavy chain variable region CDRs (vhCDR or VhCDR) and a second set of light chain variable region CDRs (vlCDR or VlCDR), each containing three CDRs: vhCDR1 , vhCDR2, vhCDR3 for the heavy chain and vlCDR1, vlCDR2 and vlCDR3 for the light chain. CDRs are present in the heavy chain variable domains and light chain variable domains, respectively, and together form the Fv region. Thus, in some cases, the six CDRs of the antigen binding domain are formed by a variable heavy chain and a variable light chain. In Fab format, a set of 6 CDRs includes two different polypeptide sequences: a heavy chain variable domain (vh or VH; containing vhCDR1, vhCDR2 and vhCDR3) and a light chain variable domain (vl or _VL ; containing vlCDR1, vlCDR2 and vlCDR3), with the C-terminus of the vh domain attached to the N-terminus of the heavy chain CH1 domain, and the C-terminus of the vl domain attached to the N-terminus of the light chain constant domain (thus forming the light chain).

As used herein, the term modification refers to an amino acid substitution, insertion and/or deletion in a polypeptide sequence, or a change in a moiety chemically linked to a protein. For example, the modification may be an altered carbohydrate or PEG structure attached to the protein. As used herein, the term amino acid modification refers to an amino acid substitution, insertion and/or deletion in a polypeptide sequence. For clarity, unless otherwise noted, an amino acid modification always refers to the amino acid encoded by DNA, for example, the 20 amino acids that have codons in DNA and RNA.

As used herein, the term amino acid substitution or substitution refers to the replacement of an amino acid at a specific position in the original polypeptide sequence with another amino acid. In particular, in some embodiments of the present invention, the substitution is an amino acid that does not occur naturally at the particular position, or does not occur naturally in the body, or in any organism. For example, the N297A substitution refers to a polypeptide variant, in this case an Fc variant, in which the asparagine at position 297 is replaced by alanine. To be clear, a protein that has been engineered to change the nucleic acid coding sequence but not to replace the original amino acid (for example, exchanging CGG (encoding arginine) for CGA (still encoding arginine) to increase expression level in the host) is not amino acid replacement; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the specific position at which it began, it is not an amino acid substitution.

As used herein, the term amino acid insertion or insertion refers to the addition of an amino acid sequence at a specific position to the original polypeptide sequence. For example, -233E or 233E indicates a glutamic acid insertion after position 233 and before position 234. Additionally, -233ADE or A233ADE indicates an AlaAspGlu insertion after position 233 and before position 234.

As used herein, the term amino acid deletion or deletion refers to the removal of an amino acid sequence at a specific position in the original polypeptide sequence. For example, E233- or E233#, E233() or E233del denotes a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233# denotes a deletion of the GluAspAla sequence that begins at position 233.

As used herein, the term variant protein or protein variant or variant refers to a protein that differs from the parent protein by at least one amino acid modification. A protein variant may refer to the protein itself, a composition containing the protein, or the amino acid sequence that encodes it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, for example, from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent protein. As described below, in some embodiments of the present invention, the parent polypeptide, such as the parent Fc polypeptide, is a human wild-type sequence, such as an IgG1, IgG2, IgG3, or IgG4 Fc region, although human sequences with variants may also serve as parent polypeptides . Variant protein sequence in this

- 21 044486 document will preferably have at least about 80% sequence identity to the parent protein, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity. A protein variant may refer to the protein variant itself, compositions containing the protein variant, or the DNA sequence that encodes it. Accordingly, the term antibody variant or variant antibody as used herein means an antibody that differs from the parent antibody by at least one amino acid modification, and the term IgG variant or IgG variant as used herein means an antibody that differs from the parent IgG ( again, in many cases from a human IgG sequence) due to at least one amino acid modification, and the term immunoglobulin variant or immunoglobulin variant as used herein means an immunoglobulin sequence that differs from the parent immunoglobulin sequence due to at least one amino acid modification . As used herein, the term Fc variant or Fc variant refers to a protein containing an amino acid modification in the Fc domain. The Fc variants of this invention are defined according to the modifications of the amino acids that make them up. Thus, for example, S241P or S228P is a hinge variant with a substituted proline at position 228 relative to the original IgG4 hinge polypeptide, with S228P numbering corresponding to the EU index and S241P corresponding to Kabat numbering. The EU index or EU-index, as in Kabat or the EU numbering scheme, refers to the numbering of EU antibodies (Edelman et al., 1969, Proc Natl Acad Sci USA 63: 78-85, which is incorporated herein by reference in its entirety) . The modification may be an addition, deletion or substitution. Substitutions may include naturally occurring amino acids and, in some cases, synthetic amino acids. Examples include US Pat. No. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of America 99:11020-11024; and L. Wang & P. G. Schultz (2002), Chem. 1-10, which are incorporated herein by reference in their entirety.

As used herein, the term protein refers to at least two covalently linked amino acids, and the term includes proteins, polypeptides, oligopeptides and peptides. The peptidyl group may contain naturally occurring amino acids and peptide bonds or synthetic peptidomimetic structures, that is, analogues such as peptoids (see Simon et al., PNAS USA 89 (20): 9367 (1992), which is incorporated herein by reference in full). Amino acids can be either naturally occurring or synthetic (eg, not an amino acid encoded by DNA); as will be appreciated by those skilled in the art. For example, for purposes of the invention, homophenylalanine, citrulline, ornithine and noreucine are considered synthetic amino acids, and both D- and L- (R or S) configured amino acids can be used. Variants of the present invention may contain modifications that include the use of synthetic amino acids incorporated using, for example, technologies developed by Schultz and colleagues, including, but not limited to, methods described in Cropp & Shultz, 2004, Trends Genet. 20 (12): 625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101 (2): 7566-71, Zhang et al., 2003, 303 (5656): 371-3 and Chin et al. , 2003, Science 301 (5635): 964-7, all of which are incorporated herein by reference in their entirety. In addition, polypeptides may include synthetic derivatization of one or more side chains or ends, glycosylation, PEGylation, cyclic rearrangement, cyclization, linkages to other molecules, fusion with proteins or protein domains, and the addition of peptide tags or tags.

As used herein, the term residue refers to a position in a protein and its associated amino acid identity. For example, asparagine 297 (also called Asn297 or N297) is the residue at position 297 of the human IgG1 antibody.

As used herein, the term Fab or Fab region refers to a polypeptide that contains immunoglobulin domains VH, CH1, VL and CL. Fab may refer to this region in the isolation, or may refer to this region in the context of a full-length antibody or antibody fragment.

As used herein, the term Fv, Fv fragment, or Fv region refers to a polypeptide that contains the VL and VH domains of a single antibody. As will be appreciated by those skilled in the art, they typically consist of two circuits.

As used herein, the term single chain Fv or scFv refers to a heavy chain variable domain covalently attached to a light chain variable domain, typically via an scFv linker as described herein, to form an scFv or scFv domain. The scFv domain can have any orientation from N- to C-terminus (vh-linker-vl or vl-linker-vh). Typically, the linker is a scFv linker, as is well known in the art, the linker peptide preferably comprising the following amino acid residues: Gly, Ser, Ala or Thr. The linker peptide must have a length that is sufficient to link two

- 22 044486 molecules so that they take the correct conformation relative to each other in order to maintain the required activity. In one embodiment of the present invention, the linker is about 1-50 amino acids in length, preferably about 1-30 amino acids in length. In one embodiment of the present invention, linkers from 1 to 20 amino acids in length can be used, while in some embodiments of the present invention linkers from about 5 to about 10 amino acids in length are used. Suitable linkers include glycineserine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n and (GGGS)n, where n is an integer of at least one (and typically , 3 to 4), glycine-alanine polymers, alanine-serine polymers and other flexible linkers. Alternatively, various non-protein polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers, that is, may find use as linkers.

The term IgG subclass modification or isotype modification as used herein refers to an amino acid modification that converts one amino acid of one IgG isotype into the corresponding amino acid in another aligned IgG isotype. For example, since IgG1 contains tyrosine and IgG2 phenylalanine at EU position 296, the F296Y substitution in IgG2 is considered a modification of the IgG subclass. Similarly, since IgG1 has a proline at position 241 and IgG4 has a serine at this position, the IgG4 molecule with S241P is considered a modification of the IgG subclass. It should be noted that subclass modifications are considered herein as amino acid substitutions.

As used herein, the term non-naturally occurring modification refers to an amino acid modification that is not isotypic. For example, since none of the IgGs contain an asparagine at position 297, the N297A substitution in IgG1, IgG2, IgG3, or IgG4 (or their hybrids) is considered a non-naturally occurring modification.

As used herein, the term amino acid and amino acid identity refers to one of the 20 naturally occurring amino acids that are encoded by DNA and RNA.

As used herein, the term effector function refers to a biochemical phenomenon that results from the interaction of the Fc region of an antibody with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and KZC. In many cases, it is desirable to eliminate most or all effector functions by either different IgG isotypes (eg, IgG4) or amino acid substitutions in the Fc domain; however, it is desirable to maintain binding to the FcRn receptor, as this helps to prolong the half-life of antibodies in human serum.

As used herein, the term IgG Fc ligand refers to a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include, but are not limited to, FcyRI, FcyRIIs, FcyRIIIs, FcRn, C1q, C3, MBL, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcyR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to FcyR (Davis et al., 2002, Immunological Reviews 190: 123-136, which is incorporated herein by reference in its entirety volume). Fc ligands may include uncharacterized molecules that bind Fc. Specific IgG Fc ligands are FcRn and Fc gamma receptors. As used herein, the term Fc ligand refers to a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc/Fc ligand complex.

As used herein, the term parent polypeptide refers to the original polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a naturally occurring polypeptide or a variant, or an engineered variant of a naturally occurring polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that contain the parent polypeptide, or the amino acid sequence that encodes it. Accordingly, the term parental immunoglobulin as used herein refers to an unmodified immunoglobulin polypeptide that is modified to produce a variant antibody, and the term parent antibody as used herein refers to an unmodified antibody that is modified to produce a variant antibody. It should be noted that the term parent antibody includes known commercial, recombinantly produced antibodies, as defined below.

As used herein, the term Fc or Fc region or Fc domain refers to a polypeptide containing the constant region of an antibody, excluding the first constant domain of an immunoglobulin domain and, in some cases, a hinge portion. Thus, Fc refers to the last two constant regions of the immunoglobulin domains IgA, IgD and IgG, the last three constant regions of the immunoglobulin domains IgE and IgM, and the flexible hinge N-terminus of these domains. In IgA and IgM, the Fc may include the J chain. In IgG, the Fc domain contains immunoglobulin domains

- 23 044486

Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cyl (Cy1) and Cy2 (Cy2). Although the boundaries of the Fc region may vary, the Fc region of the human IgG heavy chain is generally defined to include residues C226 or P230 to its carboxyl terminus, numbering according to the EU index, as in Kabat. In some embodiments of the present invention, as more fully described below, amino acid modifications are made in the Fc region, for example, to alter binding to one or more FcyR receptors or an FcRn receptor.

As used herein, the term heavy chain constant region refers to the CH1-hinge-CH2-CH3 portion of an antibody.

As used herein, the term position refers to a location in a protein sequence. The provisions may be numbered sequentially or according to a specified format, such as the EU suffix for antibody numbering.

As used herein, the term target antigen means a molecule that is specifically associated with the variable region of a given antibody. The target antigen of interest in the context of this document is TIGIT, typically human TIGIT and, optionally, cynomolgus TIGIT, the sequences of which are shown in the figures.

As used herein, the term target cell refers to a cell that expresses the target antigen.

As used herein, the term variable region means a region of an immunoglobulin that contains one or more Ig domains substantially encoded by any of the Vk (V. kappa), Vλ, (V. lamda) and/or VH genes that constitute the genetic loci kappa, lambda and immunoglobulin heavy chain, respectively.

As used herein, the term wild type or WT refers to an amino acid sequence or nucleotide sequence that occurs in nature, including allelic variations. The WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

Antibodies of this invention are typically isolated or recombinant. The term isolated, when used to describe the various polypeptides characterized herein, means a polypeptide that has been identified and separated and/or isolated from the cell or cell culture from which it was expressed. Typically, the isolated polypeptide will be obtained through at least one purification step. The term isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities. The term recombinant means that antibodies are generated using recombinant nucleic acid techniques in exogenous host cells.

The term specific binding or specifically binds or is specific for a particular antigen or epitope means binding that is measurably different from a nonspecific interaction. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure but lacking binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

Specific binding for a particular antigen or epitope may occur, for example, in the presence of an antibody having a KD for the antigen or epitope of at least about 10 ^-9 M, at least about 10 ^-10 M, at least about 10 ^-11 M, at least about 10 ^-12 M, at least about 10 ^-13 M, at least about 10 ^-14 M, at least about 10 ^-15 M, where KD means the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20, 50, 100, 500, 1000, 5000, 10,000 or more times higher for the control molecule relative to the antigen or epitope.

In addition, specific binding for a particular antigen or epitope can occur, for example, in the presence of an antibody having a KA or Ka value for the antigen or epitope that is at least 20, 50, 100, 500, 1000, 5000 - 10,000 times or more for the epitope compared to the control, where KA or Ka means the rate of association of a particular antibody-antigen interaction. Binding affinity is typically measured using surface plasmon resonance (eg, Biacore assay) and flow cytometry with antigen-expressing cells.

V. Antibodies

As discussed below, the term antibody is used generally. Typically, traditional antibody structural units contain a tetramer. Typically, each tetramer consists of two identical pairs of polypeptide chains, each pair having one light chain (typically having a molecular weight of about 25 kDa) and one heavy chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. This invention is directed to antibodies that are generally based on the IgG class, which has several subclasses, including

- 24 044486 tea, but not limited to, IgG1, IgG2, IgG3 and IgG4. In general, IgG1, IgG2 and IgG4 are used more often than IgG3. It should be noted that IgG1 has different allotypes with polymorphisms at positions 356 (D or E) and 358 (L or M). The sequences reported herein use the 356D/358M allotype, but another allotype is also included herein. That is, any sequence comprising the IgG1 Fc domain described herein may have 356E/358L replacing the 356D/358M allotype.

The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, commonly referred to in the art and herein as the Fv domain or Fv region. In the variable region, three loops assemble for each of the heavy chain and light chain V domains to form an antigen binding site. Each of the loops is called a complementarity determining region (hereinafter referred to as CDR) in which the change in amino acid sequence is most significant. The term variable refers to the fact that certain segments of the variable region vary widely in sequence among antibodies. Variability within a variable region is not evenly distributed. In contrast, V regions are composed of relatively invariant regions called framework regions (FRs) of 15-30 amino acids separated by shorter regions of excessive variability called hypervariable regions, each 9-15 amino acids or more in length.

Each VH and VL consists of three hypervariable regions (complementarity determining regions, CDRs) and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region typically covers amino acid residues from about amino acid residues 24-34 (LCDR1; L is light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and from about 31-35B (HCDR1; H stands for heavy chain), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and/or residues forming a hypervariable loop (for example, residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53 -55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region Chothia and Lesk (1987) J Mol Biol 196: 901-917 Specific CDRs of the present invention are described below.

As those skilled in the art will appreciate, the exact numbering and placement of CDRs may vary between numbering systems. However, it should be understood that the description of the sequence of the heavy chain variable region and/or the light chain variable region includes a description of the associated CDRs. Accordingly, the description of each heavy chain variable region is a vhCDR description (eg, vhCDR1, vhCDR2 and vhCDR3), and the description of each light chain variable region is a vlCDR description (eg, vlCDR1, vlCDR2 and vlCDR3). A useful comparison of CDR numbering is provided below, see Lafranc et al., Dev. Sotr. Immunol. 27 (1): 55-77 (2003):___________________________________________________________

Kabat + Chotia IMGT Kabat AbM Chotia Contact vhCDRl 26-35 27-38 31-35 26-35 26-32 30-35 vhCDR2 50-65 56-65 50-65 50-58 53-55 47-58 vhCDR3 95-102 105-117 95-102 95-102 96-101 93-101 vlCDRl 24-34 27-38 24-34 24-34 26-32 30-36 V1CDR2 50-56 56-65 50-56 50-56 50-52 46-55 V1CDR3 89-97 105-117 89-97 89-97 91-96 89-96

In this specification, the Kabat numbering system is generally used to designate the residue in the variable domain (approximately light chain variable region residues 1-107 and heavy chain variable region residues 1-113) and hinge, and the EU numbering system is used for Fc regions (eg , Kabat et al., supra (1991)).

The present invention provides a large number of different sets of CDRs. In this case, the complete set of CDRs includes three light chain variable CDRs and three heavy chain variable CDRs, for example, vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. Accordingly, they may be part of a larger light or heavy chain variable domain. Additionally, as more fully stated herein, the heavy chain variable domains and the light chain variable domains may be on separate polypeptide chains in the case of a heavy and light chain, or on a single polypeptide chain in the case of scFv sequences.

CDRs contribute to the formation of the antigen-binding or, more specifically, epitope-binding site of antibodies. The term epitope refers to a determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as a paratope. Epitopes are groups of molecules, such as amino acids or sugar side chains, and usually have specific structural characteristics as well as specific charge characteristics. One antigen can have more than one epitope.

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An epitope may contain amino acid residues directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen binding peptide; in other words, the amino acid residue is within the range of action of the specific antigen-binding peptide.

Epitopes can be conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes may differ in that in the presence of denaturing solvents, binding to the former, as opposed to the latter, is lost.

Typically, an epitope includes at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified by a simple immunoassay demonstrating the ability of one antibody to block the binding of another antibody to a target antigen, such as binning. As stated below, this invention includes not only the antigen binding domains and antibodies listed herein, but also those that compete for binding to epitopes associated with the listed antigen binding domains.

The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected numerous primary sequences of heavy chain and light chain variable regions. Based on the degree of sequence conservation, they classified individual primary sequences into CDR sequences and scaffold sequences, and also compiled a list of them (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat et al., which incorporated herein by reference in its entirety).

The IgG subclass of immunoglobulins has several immunoglobulin domains in the heavy chain. As used herein, the term immunoglobulin (Ig) domain refers to a region of an immunoglobulin having a distinct tertiary structure. Heavy chain domains of interest in this invention include heavy chain constant (CH) domains and hinge domains. In the context of IgG antibodies, each IgG isotype has three CH regions. Accordingly, the CH domains in the context of IgG are as follows: CH1 refers to positions 118-220 according to the EU index, as per Kabat. CH2 refers to positions 237-340 according to the EU index as per Kabat, and CH3 refers to positions 341-447 according to the EU index as per Kabat. As demonstrated herein and described below, pI variants can be found in one or more regions of the SN, as well as in the hinge region, discussed below.

Another type of heavy chain Ig domain is the hinge region. As used herein, the term hinge or hinge region or antibody hinge region or immunoglobulin hinge region refers to a flexible polypeptide containing amino acids between the first and second constant domains of an antibody. Structurally, the CH1 domain of IgG ends at EU-position 220, and the CH2 domain of IgG begins at EU-position 237 residue. Thus, for IgG, the antibody hinge is herein defined to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), with the numbering corresponding to the EU index as per Kabat's K. In some embodiments of the present invention, for example in the context of an Fc region, a bottom hinge is included, with the bottom hinge typically being at positions 226 or 230.

A light chain typically contains two domains: a light chain variable domain (containing the light chain CDR and, together with the heavy chain variable domains, forming the Fv region) and a light chain constant region (often called CL or Cλ).

Another region of interest for additional substitutions described below is the Fc region.

A. Chimeric and humanized antibodies.

In some embodiments of the present invention, the antibodies in the present invention can be obtained from a mixture of different species, for example, a chimeric antibody and/or a humanized antibody. In general, both chimeric antibodies and humanized antibodies refer to antibodies that combine regions of more than one species. For example, chimeric antibodies traditionally include variable region(s) from mouse (or rat, in some cases) and constant region(s) from human. Humanized antibodies generally refer to non-human antibodies in which the variable domain framework regions have been replaced with sequences found in human antibodies. Typically, in a humanized antibody, all of the antibody except the CDR is encoded by a polynucleotide of human origin or is identical to such an antibody except for its CDR. CDRs, some or all of which are encoded by nucleic acids originating from a non-human organism, are grafted into the beta sheet framework of a human antibody variable region to create an antibody whose specificity is determined by the grafted CDRs. Pops

- 26 044486 the production of such antibodies is described, for example, in WO 92/11018, Jones, 1986, Nature 321: 522-525, Verhoeyen et al., 1988, Science 239: 1534-1536, all of which are incorporated herein by reference in full. Back mutation of selected acceptor scaffold residues into corresponding donor residues is often required to restore the affinity that is lost in the original graft construct (US 5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205; US 5821337; US 60542 97; US 6407213, all of which are incorporated herein by reference in their entirety). An optimally humanized antibody will also contain at least a portion of an immunoglobulin constant region, typically a human immunoglobulin region, and thus will typically comprise a human Fc region. Humanized antibodies can also be generated using mice with genetically engineered immune systems. Roque et al., 2004, Biotechnol. Prog. 20: 639-654, which is incorporated herein by reference in its entirety. A variety of methods and techniques for humanizing and reshaping non-human antibodies are well known in the art (see Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited in that document are incorporated herein by reference in their entirety). Humanization methods include, but are not limited to, those described in Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, all of which are incorporated herein by reference in their entirety. Humanization or other methods of reducing the immunogenicity of non-human antibody variable regions may include surface modification methods, as described, for example, in Roguska et al., 1994, Proc. Natl. Academician Sci. USA 91: 969-973, which is incorporated herein by reference in its entirety.

In certain embodiments of the present invention, the antibodies of the present invention comprise a heavy chain variable region from a particular germline immunoglobulin heavy chain gene and/or a light chain variable region from a particular germline immunoglobulin light chain gene (with optional mutations, as generally described herein). document). For example, such antibodies may contain or consist of a human antibody containing heavy or light chain variable regions that are the product of or derived from a particular germline sequence. A human antibody that is the product of or derived from a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is most similar in sequence (i.e., has the greatest % identity) to the human antibody sequence. A human antibody that is the product of or derived from a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to naturally occurring somatic mutations or the deliberate introduction of a site-directed mutation. However, a humanized antibody typically is at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene, and contains amino acid residues that identify the antibody as being derived from human sequences as compared to the germline immunoglobulin amino acid sequences other species (eg, mouse germline sequences) In certain cases, a humanized antibody may be at least 95, 96, 97, 98, or 99%, or even at least 96, 97, 98, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a humanized antibody derived from a particular human germline sequence will exhibit no more than 10-20 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In some cases, a humanized antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences from the amino acid sequence encoded by the germline immunoglobulin gene (again, prior to the introduction of any variant described herein, i.e., the number of variants , is generally small prior to the introduction of the variants of this invention).

In one embodiment of the present invention, the parent antibody is affinity mature, as is known in the art. Structure-based methods, for example, as described in USSN 11/004590, can be used for humanization and affinity maturation. For humanization and/or affinity maturation of antibody variable regions, selection-based methods can be used, including, but not limited to, those described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16): 10678-10684; Rosok et al., 1996, J. Biol. Chem.

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271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all of which are incorporated herein by reference in their entirety. Other humanization methods may include grafting only parts of the CDR, including, but not limited to, the methods described in USSN 09/810,510; Tan et al., 2002, J. Immunol. 169: 11191125; De Pascalis et al., 2002, J. Immunol. 169: 3076-3084, all of which are incorporated herein by reference in their entirety.

A. Specific anti-TIGIT antibodies.

This invention provides antigen binding domains, including full length antibodies, that contain a number of specific, listed sets of 6 CDRs and specific heavy chain variable regions (vh, VH or VH) and light chain variable regions (vl, VL or VL) that bind to TIGIT.

In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a set of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) from CPA.9.083.H4 (S241P), as demonstrated in FIG. 3. In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) from CPA.9.083.H4 (S241P), as demonstrated in FIG. 3 related to human IgG constant domain IgG1, IgG2, IgG3, IgG4 and IgG4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P).

In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a set of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) from CPA.9.086.H4 (S241P), as demonstrated in FIG. 3. In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) from CPA.9.086.H4 (S241P), (VL) in FIG. H, associated with the constant domain of human IgG IgG1, IgG2, IgG3, IgG4 and IgG4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P).

In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a set of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) from CHA.9.547.7.H4 (S241P), as demonstrated in FIG. 3. In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) from CHA.9.547.7.H4 (S241P), as demonstrated in FIG. 3 related to human IgG constant domain IgG1, IgG2, IgG3, IgG4 and IgG4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P).

In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a set of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) from CHA.9.547.13.H4 (S241P), as demonstrated in FIG. 3. In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) from CHA.9.547.13.H4 (S241P), as demonstrated in FIG. 3 related to human IgG constant domain IgG1, IgG2, IgG3, IgG4 and IgG4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P).

Other anti-TIGIT antibodies that find use in combinations with anti-PVRIG antibodies as described herein are presented in FIG. 4 in USSN 62/513916 entitled Anti-TIGIT Antibodies and Methods for Their Use, filed June 1, 2017 by assignee Compugen, as well as those included in FIG. 3.

B. Additional anti-TIGIT antibodies for use in combination therapy.

Also included are additional anti-TIGIT antibodies that can be used in combination with anti-PVRIG antibodies and optionally with anti-PD-1 antibodies as specified herein. As discussed in more detail below, anti-TIGIT antibodies are particularly effective when combined with anti-PVRIG antibodies. Thus, in some embodiments of the present invention, alternative anti-TIGIT antibodies are used in combination with anti-PVRIG antibodies specified herein, and in particular, either CHA.7.538.1.2.H4 (S241P) or CHA. 7.518.1.Н4 (S241P).

Accordingly, in one embodiment of the present invention, the anti-TIGIT antibodies described in US Pat. No. 9,499,596 (incorporated herein by reference in its entirety and specifically for SEQ ID NO: listed below) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having the light chain sequence SEQ ID NO: 21 and the heavy chain sequence SEQ ID NO: 22 (from USP 9499596) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518. 1.H4 (S241P). In addition, an anti-TIGIT antibody having a light chain sequence of SEQ ID NO: 29 and a heavy chain sequence of SEQ ID NO: 30 (from USP 9499596) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1. H4

- 28 044486 (S241P).

Likewise, in one embodiment of the present invention, the anti-TIGIT antibodies described in WO 2016/191643 (incorporated herein by reference in their entirety and specifically for SEQ ID NO: listed below, and in particular for the OMP- antibody sequences) 313M32) can be combined with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody having a light chain sequence of SEQ ID NO: 72 and a heavy chain sequence of SEQ ID NO: 70 (from WO 2016/191643) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA. 7.518.1.Н4 (S241P).

Accordingly, in one embodiment of the present invention, the anti-TIGIT antibodies described in WO 2017/053748 (incorporated herein by reference in its entirety and specifically for SEQ ID NO: listed below) can be combined with CHA.7.538.1.2. H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 36 and a heavy chain variable sequence of SEQ ID NO: 34 (from WO 2017/053748) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In addition, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 36 and a heavy chain variable sequence of SEQ ID NO: 35 (from WO 2017/053748) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In addition, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 38 and a heavy chain variable sequence of SEQ ID NO: 37 (from WO 2017/053748) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA. 7.518.1.Н4 (S241P). In addition, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 40 and a heavy chain variable sequence of SEQ ID NO: 39 (from WO 2017/053748) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibodies include a Genentech antibody, MTIG7192A, currently in clinical development (available on the World Wide Web at clintrials.gov/ct2/show/NCT02794571?term=MTIG7192A&rank=1). In one embodiment of the present invention, the anti-TIGIT antibody MTIG7192A can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P).

Likewise, in one embodiment, the anti-TIGIT antibodies described in WO 2016/191643 (incorporated herein by reference in its entirety and specifically for SEQ ID NO: listed below, and in particular for the OMP-313M32 antibody sequences) , can be combined with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody having a light chain sequence of SEQ ID NO: 72 and a heavy chain sequence of SEQ ID NO: 70 (from WO 2016/191643) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518. 1.H4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibodies include the Oncomed antibody, OMP-313M32, currently in clinical development (available on the World Wide Web at clintrials.gov/ct2/show/NCT03119428?term=OMP313M32&rank=1 ). In one embodiment of the present invention, the anti-TIGIT antibody OMP313M32 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P).

Additionally, in one embodiment of the present invention, the anti-TIGIT antibodies described in WO 2016/028656 (incorporated herein by reference in their entirety and specifically for SEQ ID NO: listed below, and in particular for the MEB125 antibody sequences .31C6.A1.205 VH4/VL1 (VH SEQ ID NO: 127, VL sequence SEQ ID NO: 130 with human IgG1 constant domain), MEB 125.31C6.A1.205 VH5/VL4 (VH SEQ ID NO: 128, VL sequence SEQ ID NO: 133 and human IgG1 constant region) and MEB125.31.C6, A1.205 VH5/VL3 (VH sequence SEQ ID NO: 128, VL sequence SEQ ID NO: 132 and human IgG1 constant region)), can combine with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, the anti-TIGIT antibody MEB125.31C6.A1.205 VH4/VL1 (VH sequence SEQ ID NO: 127, VL sequence SEQ ID NO: 130 with human IgG1 constant domain) (from WO 2016/028656), can be combined with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, the anti-TIGIT antibody MEB 125.31C6.A1.205 VH5/VL4 (VH sequence SEQ ID NO: 128, VL sequence SEQ ID NO: 133 and human IgG1 constant region (from WO 2016/028656), can be combined with SNA .7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P) Specifically, anti-TIGIT antibody MEB 125.31.C6.A1.2O5 VH5/VL3 (VH sequence SEQ ID NO: 128, VL sequence SEQ ID NO: 132 and human IgG1 constant region (from WO 2016/028656), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, anti-TIGIT antibody , containing the VH sequences SEQ ID NO: 7, the VL sequences SEQ ID NO: 8 and the human IgG1 constant region (from WO 2016/028656), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1. H4 (S241P) In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 63, VL sequences SEQ ID NO: 64 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2 .H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody containing the VH sequences of SEQ ID NO: 94, VL sequences

- 29 044486

SEQ ID NO: 95 and human IgG1 constant region (from WO 2016/028656), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 126, VL sequences SEQ ID NO: 131 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 128, VL sequences SEQ ID NO: 131 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 125, VL sequences SEQ ID NO: 133 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 126, VL sequences SEQ ID NO: 130 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 125, VL sequences SEQ ID NO: 132 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody containing the VH sequences of SEQ ID NO: 143, the VL sequences of SEQ ID NO: 145 and the human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 144, VL sequences SEQ ID NO: 146 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 149, VL sequences SEQ ID NO: 151 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody containing VH sequences SEQ ID NO: 150, VL sequences SEQ ID NO: 152 and a human IgG1 constant region (from WO 2016/028656) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P).

Additionally, in one embodiment of the present invention, the anti-TIGIT antibodies described in WO 2017/030823 (incorporated herein by reference in its entirety and specifically for SEQ ID NO: listed below) can be combined with CHA.7.538.1.2 .H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 8 and a heavy chain variable sequence of SEQ ID NO: 7 (from WO 2017/030823) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 13 and a heavy chain variable sequence of SEQ ID NO: 9 (from WO 2017/030823) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 24 and a heavy chain variable sequence of SEQ ID NO: 23 (from WO 2017/030823) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 29 and a heavy chain variable sequence of SEQ ID NO: 25 (from WO 2017/030823) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In some embodiments of the present invention, an anti-TIGIT antibody having a light chain variable sequence selected from SEQ ID NO: 14, 15, 16, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80, and a heavy chain variable sequence selected from SEQ ID NO: 10, 11, 12, 48, 49, 50, 51, 52, 53, 54, 55 or 56 (from WO 2017/030823), can be combined with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). In some embodiments of the present invention, an anti-TIGIT antibody having a light chain variable sequence selected from SEQ ID NO: 14, 15, 16, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80, and a heavy chain variable sequence selected from SEQ ID NO: 10, 11, 12, 48, 49, 50, 51, 52, 53, 54, 55 or 56 (from WO2017/030823), can be combined with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). In some embodiments of the present invention, an anti-TIGIT antibody having a light chain variable sequence selected from SEQ ID NO: 30, 31, or 32 and a heavy chain variable sequence selected from SEQ ID NO: 26, 27, 28, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 or 112 (from WO2017/ 030823), can be combined with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibodies include a Merck antibody, MK7684, currently in clinical development (available on the World Wide Web at clintrials.gov/ct2/show/NCT02964013?term=MK-7684&rank=1 ). In one embodiment of the present invention, the anti-TIGIT antibody MK-7684 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P).

Accordingly, in one embodiment of the present invention, anti-TIGIT antibodies, as described in US Patent Application No. 2016/0176963 (incorporated herein by reference

- 30 044486 in full and specifically for SEQ ID NO: listed below), can be combined with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). Specifically, an anti-TIGIT antibody having a light chain variable sequence of SEQ ID NO: 6, 9, 11 or 13 and a heavy chain variable sequence of SEQ ID NO: 2, 3, 4, 5, 7, 8, 10 or 12 (from US 2016/0176963), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having a light chain variable sequence from SEQ ID NO: 6 and a heavy chain variable sequence from SEQ ID NO: 2, 3, 4 or 5 (from US 2016/0176963) can be combined with CHA.7.538 .1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having a light chain variable sequence from SEQ ID NO: 9 and a heavy chain variable sequence from SEQ ID NO: 7 or 8 (from US 2016/0176963) can be combined with CHA.7.538.1.2.H4 (S241P) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having the light chain variable sequence of SEQ ID NO: 11 and the heavy chain variable sequence of SEQ ID NO: 10 (from US 2016/0176963) can be combined with CHA.7.538.1.2.H4 (S241P ) or SNA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody having the light chain variable sequence of SEQ ID NO: 13 and the heavy chain variable sequence of SEQ ID NO: 12 (from US 2016/0176963) can be combined with CHA.7.538.1.2.H4 (S241P ) or SNA.7.518.1.H4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibodies include the BMS antibody, BMS98620, currently in clinical trials (available on the World Wide Web at clinicaltrials.gov/ct2/show/NCT02913313?term=BMS-986207&rank=1 ). In one embodiment of the present invention, the anti-TIGIT antibody BMS-98620 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P).

In one embodiment of the present invention, anti-TIGIT antibodies include Arcus Bio antibody, AB154. In one embodiment of the present invention, the anti-TIGIT antibody AB154 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P).

Additionally, in one embodiment of the present invention, the anti-TIGIT antibodies described in WO 2017/037707 (incorporated herein by reference in their entirety and specifically for SEQ ID NO: listed below, and in particular for the sequences of SEQ ID NOs: listed below, and in particular for antibody VSIG9#1 (SEQ ID NO: 7 VH and SEQ ID NO: 8 VL) and antibody 258-csl#4 (SEQ ID NO: 18 VH and SEQ ID NO: 19 VL) In particular, the anti-TIGIT antibody VSIG9#1 (from WO 2017/037707; SEQ ID NO: 7 VH and SEQ ID NO: 8 VL) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA .7.518.1.H4 (S241P) In particular, the anti-TIGIT antibody 258-csl#4 (from WO 2017/037707; SEQ ID NO: 18 VH and SEQ ID NO: 19 VL) can be combined with CHA.7.538. 1.2.Н4 (S241P) or SNA.7.518.1.Н4 (S241P).

Additionally, in one embodiment of the present invention, the anti-TIGIT antibodies described in WO 2017/059095 (incorporated herein by reference in its entirety and specifically for SEQ ID NO: listed below, and in particular for the sequences described in that document. In particular, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 13 and the V _L sequence of SEQ ID NO: 26 (from WO 2017/059095) can be combined with CHA.7.538.1.2.H4 ( S241P) or CHA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody containing the V _H sequence of SEQ ID NO: 12 and the V _L sequence of SEQ ID NO: 26 (from WO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P) Specifically, an anti-TIGIT antibody containing the _VH sequence of SEQ ID NO: 14 and the VL sequence of SEQ ID NO : 26 (from wO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P) In particular, an anti-TIGIT antibody containing the VH sequence from SEQ ID NO : 15 and the VL sequence from SEQ ID NO: 26 (from WO 2017/059095), can be combined with SHA.7.538.1.2.H4 (S241P) or SHA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 9 and the VL sequence of SEQ ID NO: 26 (from WO 2017/059095) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA. 7.518.1.Н4 (S241P). In particular, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 10 and the VL sequence of SEQ ID NO: 26 (from wO 2017/059095) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA. 7.518.1.Н4 (S241P). In particular, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO: 26 (from WO 2017/059095) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA. 7.518.1.Н4 (S241P). Specifically, an anti-TIGIT antibody comprising the heavy chain of SEQ ID NO: 99 and the light chain of SEQ ID NO: 92; or (it) the heavy chain from SEQ ID NO: 100 and the light chain from SEQ ID NO: 92 (from WO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). Specifically, an anti-TIGIT antibody comprising the heavy chain of SEQ ID NO: 97 and the light chain of SEQ ID NO: 92; or (ii) the heavy chain from SEQ ID NO: 98 and the light chain from SEQ ID NO: 92 (from WO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). Specifically, an anti-TIGIT antibody comprising the heavy chain of SEQ ID NO: 101 and the light chain of SEQ ID NO: 92; or (ii) the heavy chain from SEQ ID NO: 102 and the light chain from SEQ ID NO: 92 (from WO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). Specifically, an anti-TIGIT antibody comprising the heavy chain of SEQ ID NO: 103 and the light chain

- 31 044486 from SEQ ID NO: 92; or (ii) the heavy chain from SEQ ID NO: 104 and the light chain from SEQ ID NO: 92 (from WO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). Specifically, an anti-TIGIT antibody comprising the heavy chain of SEQ ID NO: 90 and the light chain of SEQ ID NO: 92; or (ii) the heavy chain from SEQ ID NO: 91 and the light chain from SEQ ID NO: 92 (from WO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). Specifically, an anti-TIGIT antibody comprising the heavy chain of SEQ ID NO: 93 and the light chain of SEQ ID NO: 92; or (ii) the heavy chain of SEQ ID NO: 94 and the light chain of SEQ ID NO: 92 (from WO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). Specifically, an anti-TIGIT antibody comprising the heavy chain of SEQ ID NO: 95 and the light chain of SEQ ID NO: 92; or (ii) the heavy chain of SEQ ID NO: 96 and the light chain of SEQ ID NO: 92 (from WO 2017/059095), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). Additionally, in one embodiment of the present invention, anti-TIGIT antibodies as described in WO 2016/106302 (incorporated herein by reference in their entirety and specifically for SEQ ID NO: listed below, and in particular for sequence specific sequences described in that publication. In particular, an anti-TIGIT antibody containing a heavy chain sequence selected from SEQ ID NO: 2, 3, 4, 5, 7, 8, 10 or 12 (from WO 2016/106302), and the light chain sequence from SEQ ID NO: 6, 9, 11 or 13 (from WO 2016/106302) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). , the anti-TIGIT antibody 22G2 (from WO 2016/106302) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody 11G11 (from WO 2016 /106302) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P) In particular, the anti-TIGIT antibody 15A6 (from WO 2016/106302) can be combined with CHA.7.538. 1.2.Н4 (S241P) or SNA.7.518.1.Н4 (S241P).

Additionally, in one embodiment of the present invention, the anti-TIGIT antibodies described in US Patent Publication No. 2017281764 (incorporated herein by reference in their entirety and specifically for SEQ ID NO: listed below, and in particular for the sequences described in that publication. In particular, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 10 and the V _L sequence of SEQ ID NO: 14 (from US 2017281764) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P) In particular, an anti-TIGIT antibody containing the V _H sequence of SEQ ID NO: 18 and the V _L sequence of SEQ ID NO: 22 (from US 2017281764) can be combined with CHA .7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P) Specifically, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 26 and the _VL sequence of SEQ ID NO: 30 (from US 2017281764), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, an anti-TIGIT antibody containing the VH sequence from SEQ ID NO: 35 and the VL sequence from SEQ ID NO: 37 (from US 2017281764), can be combined with SNA.7.538.1.2.N4 (S241P) or SNA.7.518.1.N4 (S241P). In particular, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 34 and the VL sequence of SEQ ID NO: 36 (from US 2017281764) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518. 1.H4 (S241P).

In another embodiment of the present invention, anti-TIGIT antibodies are described in International Patent Publication No. WO 2015/009856 (incorporated herein by reference in its entirety and specifically for SEQ ID NO: listed below, and in particular for the sequences described in that document (see also International Patent Publication No. WO 2016/011264) Specifically, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 15 and the V _L sequence of SEQ ID NO: 13 (from WO 2015/009856 ), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). Specifically, an anti-TIGIT antibody containing the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 14 (from WO 2015/009856), can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P).

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody described in any of the following: US Patent Application No. 20170037133, International Patent Publication No. WO 2017/048824; MBSA43 (commercially available from eBioscience), is an anti-TIGIT antibody pab2197 or pab2196 (US Patent Application No. 2017/0081409), EOS084448, CASC-674 (available from Adimab LLC). In particular, the anti-TIGIT antibody, as described in US patent application No. 2017/0037133, can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody, as described in international patent publication No. WO 2017/048824, can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MBSA43 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody pab2197 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody pab2196 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody EOS084448 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody CASC-674 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P).

In some embodiments of the present invention, the anti-TIGIT antibody is

- 32 044486 combat antibody described in US patent No. 9713364 (incorporated herein by reference in its entirety). In some embodiments of the present invention, the anti-TIGIT antibody is PTZ-201 (ASP8374). In particular, the anti-TIGIT antibody PTZ-201 (ASP8374) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from the group consisting of mAb1, MAB2, MAV3, MAB4, MAB5, MAB6, MAB7, MAB8, MAB9, MAB10, MAB11, MAB12, MAB13, MAB14, MAB15, MAB16, MAB17, MAB18, 40 MAB19, MAB20 or MAB21, as described in US Patent No. 9713364. In particular, the anti-TIGIT antibody MAB1 from US Patent No. 9713364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA. 7.518.1.Н4 (S241P). In particular, the anti-TIGIT antibody MAB2 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT MAB3 antibody from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT MAB4 antibody from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB5 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB6 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB7 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB8 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB9 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB10 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB11 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB12 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB13 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB14 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB15 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB16 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB17 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB18 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB19 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB20 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody MAB21 from US Pat. No. 9,713,364 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA7.518.1.H4 (S241P).

In some embodiments of the present invention, the anti-TIGIT antibody is 10A7, 1F4, 14A6, 28H5, 31C6, 15A6, 22G2, 11G11, and/or 10D7, each of which is incorporated herein by reference in its entirety. In particular, the anti-TIGIT 10A7 antibody can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT 1F4 antibody can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT 14A6 antibody can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody 28H5 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody 31C6 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT 15A6 antibody can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody 22G2 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT antibody 11G11 can be combined with CHA.7.538.1.2.H4 (S241P) or CHA.7.518.1.H4 (S241P). In particular, the anti-TIGIT 10D7 antibody can be combined with CHA.7.538.1.2.H4 (S241P) or CHA7.518.1.H4 (S241P).

In some embodiments of the present invention, the anti-TIGIT antibody is one of the antibodies described in international patent publication WO 2016/028656, incorporated herein in its entirety. In particular, the anti-TIGIT antibodies presented in International Patent Publication No. WO 2016/028656 (and reproduced herein in FIG. 3) can be combined with CHA.7.538.1.2.H4 (S241P) or CHA. 7.518.1.Н4 (S241P).

The anti-TIGIT antibodies described herein may find use in the triple combination therapy methods of this invention, which are labeled as follows. Such anti-TIGIT antibodies have reference numbers, for example, CPA.9.086. They represent a combination of variable heavy chains and variable light chains as a demonstration

- 33 044486 illustrated, for example, in Fig. 3, with the understanding that these antibodies comprise two heavy chains and two light chains. CPA.9.086.VH refers to the variable heavy chain portion of CPA.9.086, while CPA.9.O86.VL is the variable light chain. CPA.9.086.vhCDR1, CPA.9.086.vhCDR2, CPA.9.086.vhCDR3, CPA.9.086.vlCDR1, CPA. 9.086.vlCDR2 and

CPA.9.086.v1CDR3, refer to the specified CDRs. CPA.9.086.HC refers to the entire heavy chain (eg, variable and constant domain) of that molecule, and CPA.9.O86.LC refers to the entire light chain (eg, variable and constant domain) of the same molecule. In general, human kappa light chain is used in this invention for the constant domain of each phage antibody (or humanized hybridoma antibody), although in some embodiments a lambda light chain constant domain is used. CPA.9.086.III refers to a full-length antibody containing heavy and light chain variable domains, including the human IgG1 constant domain (hence H1; IgG1, IgG2, IgG3 and IgG4 sequences are shown in Fig. 1, for example). Accordingly, CPA.9.086.H2 will be an antibody containing the variable domains of CPA.9.086 associated with human IgG2. CPA.9.O86.H3 will be an antibody containing CPA.9.086 variable domains associated with human IgG3, and CPA.9.086.H4 will be an antibody containing CPA.9.086 variable domains associated with human IgG4. It should be noted that in some cases human IgG may have additional mutations, such as those described below, and this may be designated accordingly. For example, in many embodiments of the present invention, human IgG4 may have the S241P mutation, and this may be designated, for example, CPA.9.086.H4 (S241P). The sequence of human IgG4 with this S241P hinge variant is shown in FIG. 1. Other potential variants are IgG1 (N297A) (or other variants that eliminate glycosylation at this site and thus many effector functions associated with FcYRIIIa binding), and IgG1 (D265A), which reduces binding to FcyR receptors. Anti-TIGIT antibodies for use in the present invention may comprise any of the anti-TIGIT antibody domain sequences. Anti-TIGIT antibodies for use in this invention may contain any of the TIGIT antigen binding domains.

The present invention further provides heavy and light chain variable domains, as well as full-length heavy and light chains.

Some embodiments of the present invention provide scFvs that bind to TIGIT, comprising a heavy chain variable domain and a light chain variable domain linked through a scFv linker as described above. The VL and VH domains can be in any orientation, for example N- to C-terminal VH-linker-VL or VL-VH linker. They are named after their constituent parts; for example, scFv-CPA.9.086.VH-linker-VL or scFv-CPA.9.086.VL-linker-VH. Thus, scFv-CPA.9.086 can be in any orientation. Anti-TIGIT antibodies for use in this invention may contain any scFv that binds to TIGIT. Anti-TIGIT antibodies for use in this invention may contain any scFv that binds to TIGIT. Anti-TIGIT antibodies for use in this invention may include any of the following:

CPA.9.018, CPA.9.018.VH, CPA.9.018.VL, CPA.9.018.NS, CPA.9.018.LC,

SRA.9.018.N1, SRA.9.018.N2, SRA.9.018.NZ, SRA.9.018.N4; CPA.9.018.H4(S241P);

CPA.9.018.vhCDRl, CPA.9.018.vhCDR2, CPA.9.018.vhCDR3, CPA.9.018.vlCDRl,

CPA.9.018.V1CDR2, CPA.9.018.vlCDR3 and scFv-CPA.9.018;

CPA.9.027, CPA.9.027.VH, CPA.9.027.VL, CPA.9.027.HC, CPA.9.027.LC,

CPA.9.027.H1, CPA.9.027.H2, CPA.9.027.H3, CPA.9.027.H4; CPA.9.018.H4(S241P);

- 34 044486

CPA.9.027.vhCDRl, CPA.9.027.vhCDR2, CPA.9.027.vhCDR3, CPA.9.027.vlCDRl,

CPA.9.027.vlCDR2, CPA.9.027.vlCDR3 and scFv-CPA.9.027;

CPA.9.049, CPA.9.049.VH, CPA.9.049.VL, CPA.9.049.HC, CPA.9.049.LC,

CPA.9.049.H1, CPA.9.049.H2, CPA.9.049.H3; CPA.9.049.H4; CPA.9.049.H4(S241P);

CPA.9.049.vhCDRl, CPA.9.049.vhCDR2, CPA.9.049.vhCDR3, CPA.9.049.vlCDRl,

CPA.9.049.vlCDR2, CPA.9.049.vlCDR3 and scFv-CPA.9.049;

CPA.9.057, CPA.9.057.VH, CPA.9.057.VL, CPA.9.057.HC, CPA.9.057.LC,

CPA.9.057.H1, CPA.9.057.H2, CPA.9.057.H3; CPA.9.057.H4; CPA.9.057.H4(S241P);

CPA.9.057.vhCDRl, CPA.9.057.vhCDR2, CPA.9.057.vhCDR3, CPA.9.057.vlCDRl,

CPA.9.057.vlCDR2, CPA.9.057.vlCDR3 and scFv-CPA.9.057;

CPA.9.059, CPA.9.059.VH, CPA.9.059.VL, CPA.9.059.HC, CPA.9.059.LC,

CPA.9.059.H1, CPA.9.059.H2, CPA.9.059.H3; CPA.9.059.H4; CPA.9.059.H4(S241P);

CPA.9.059.vhCDRl, CPA.9.059.vhCDR2, CPA.9.059.vhCDR3, CPA.9.059.vlCDRl,

CPA.9.059.vlCDR2, CPA.9.059.vlCDR3 and scFv-CPA.9.059;

CPA.9.083, CPA.9.083.VH, CPA.9.083.VL, CPA.9.083.HC, CPA.9.083.LC,

CPA.9.083.H1, CPA.9.083.H2, CPA.9.083.H3; CPA.9.083.H4; CPA.9.083.H4(S241P);

CPA.9.083.vhCDRl, CPA.9.083 ,vhCDR2, CPA.9.083.vhCDR3, CPA.9.083 vlCDRl,

CPA.9.083. vlCDR2, CPA.9.083.vlCDR3 and scFv-CPA.9.083;

CPA.9.086, CPA.9.086.VH, CPA.9.086.VL, CPA.9.086.HC, CPA.9.086.LC,

CPA.9.086.H1, CPA.9.086.H2, CPA.9.086.H3; CPA.9.086.H4; CPA.9.086.H4(S241P);

CPA.9.086.vhCDRl, CPA.9.086.vhCDR2, CPA.9.086.vhCDR3, CPA.9.086.vlCDRl,

CPA.9.086.vlCDR2, CPA.9.086.vlCDR3 and scFv-CPA.9.086;

CPA.9.089, CPA.9.089.VH, CPA.9.089.VL, CPA.9.089.HC, CPA.9.089.LC,

CPA.9.089.H1, CPA.9.089.H2, CPA.9.089.H3; CPA.9.089.H4; CPA.9.089.H4(S241P);

CPA.9.089.vhCDRl, CPA.9.089.vhCDR2, CPA.9.089.vhCDR3, CPA.9.089.vlCDRl,

CPA.9.089.vlCDR2, CPA.9.089.vlCDR3 and scFv-CPA.9.089;

CPA.9.093, CPA.9.093. VH, CPA.9.093.VL, CPA.9.093.HC, CPA.9.093.LC,

CPA.9.093.H1, CPA.9.093.H2, CPA.9.093 H3; CPA.9.093.H4; CPA.9.093.H4(S241P);

CPA.9.093.vhCDRl, CPA.9.093 ,vhCDR2, CPA.9.093.vhCDR3, CPA.9.093 .vlCDRl,

CPA.9.093. vlCDR2, CPA.9.093, vlCDR3 and scFv-CPA.9.093;

CPA.9.101, CPA.9.101.VH, CPA.9.101.VL, CPA.9.101.HC, CPA.9.101.LC,

CPA.9.101.H1, CPA.9.101.H2, CPA.9.101.H3; CPA.9.101.H4; CPA.9.101.H4(S241P);

CPA.9.101.vhCDRl, CPA.9.101.vhCDR2, CPA.9.101.vhCDR3, CPA.9.101.vlCDRl,

CPA.9.101.V1CDR2, CPA.9.101.vlCDR3 and scFv-CPA.9.101; and

CPA.9.103, CPA.9.103.VH, CPA.9.103.VL, CPA.9.103.HC, CPA.9.103.LC,

CPA.9.103.H1, CPA.9.103.H2, CPA.9.103 H3; CPA.9.103 H4;

CPA.9.103.H4(S241P); CPA.9.103.vhCDRl, CPA.9.103.vhCDR2, CPA.9.103.vhCDR3,

CPA.9.103.vlCDRl, CPA.9.103.vlCDR2, CPA.9.103.vlCDR3 and scFv-CPA.9.103.

In addition, the present invention provides a series of anti-CHA antibodies, which are murine antibodies derived from hybridomas. As is well known in the art, the six CDRs are useful when placed in either a human heavy and light chain variable region framework or when the heavy and light chain variable regions are humanized. Accordingly, the present invention provides antibodies, typically full-length or scFv domains, that contain the following sets of CDRs, the sequences of which are shown in FIG. 3 and/or in the list of sequences:

-35 044486

CHA.9.536.1, CHA.9.536. l.VH, CHA.9.536.1. VL, CHA.9.536.1.HC, CHA.9.536.1.LC, CHA.9.536.1.Hl, CHA.9.536.1. H2, CHA.9.536.1. H3; CHA.9.536.1.H4,

CHA.9.536.1.H4(S241P), CHA.9.536.1.vhCDRl, CHA.9.536.1.vhCDR2,

CHA.9.536.1.vhCDR3, CHA.9.536.1.vlCDRl, CHA.9.536.1.vlCDR2 and CHA.9.536.1.vhCDR3;

CHA.9.536.3, CHA.9.536.3.VH, CHA.9.536.3.VL, CHA.9.536.3.HC, CHA.9.536.3.LC,

CHA.9.536.3.H1, CHA.9.536.3.H2, CHA.9.536.3.H3; CHA.9.536.3.H4,

CHA.9.536.3.H4(S241P); CHA.9.536.3.vhCDRl, CHA.9.536.3.vhCDR2,

CHA.9.536.3.vhCDR3, CHA.9.536.3.vlCDRl, CHA.9.536.3.vlCDR2 and CHA.9.536.3.vhCDR3;

CHA.9.536.4, CHA.9.536.4.VH, CHA.9.536.4.VL, CHA.9.536.4.HC, CHA.9.536.4.LC,

CHA.9.536.4.H1, CHA.9.536.4.H2, CHA.9.536.4.H3; CHA.9.536.4.H4,

CHA.9.536.4.H4(S241P), CHA.9.536.4.vhCDRl, CHA.9.536.4.vhCDR2,

CHA.9.536.4.vhCDR3, CHA.9.536.4.vlCDRl, CHA.9.536.4.vlCDR2 and CHA.9.536.4.vhCDR3;

CHA.9.536.5, CHA.9.536.5.VH, CHA.9.536.5.VL, CHA.9.536.5.HC, CHA.9.536.5.LC,

CHA.9.536.5.H1, CHA.9.536.5.H2, CHA.9.536.5.H3; CHA.9.536.5.H4,

CHA.9.536.5.H4(S241P), CHA.9.536.5.vhCDRl, CHA.9.536.5.vhCDR2,

CHA.9.536.5.vhCDR3, CHA.9.536.5.vlCDRl, CHA.9.536.5.vlCDR2 and CHA.9.536.5.vhCDR3;

CHA.9.536.6, CHA.9.536.6.VH, CHA.9.536.6.VL, CHA.9.536.6.HC, CHA.9.536.6.LC, CHA.9.536.6.H1, CHA.9.536. 6.H2, CHA.9.536.6.H3; CHA.9.536.6.H4, CHA.9.536.6.vhCDRl, CHA.9.536.6.vhCDR2, CHA.9.536.6.vhCDR3, CHA.9.536.6.vlCDRl, CHA.9.536.6.vlCDR2 and CHA. 9.536.6.vhCDR3;

CHA.9.536.7, CHA.9.536.7.VH, CHA.9.536.7.VL, CHA.9.536.7.HC, CHA.9.536.7.LC,

CHA.9.536.7.H1, CHA.9.536.7.H2, CHA.9.536.7.H3; CHA.9.536.7.H4,

CHA.9.536.5.H4(S241P); CHA.9.536.7.vhCDRl, CHA.9.536.7.vhCDR2,

CHA.9.536.7.vhCDR3, CHA.9.536.7.vlCDRl, CHA.9.536.7.vlCDR2 and CHA.9.536.7.vhCDR3;

- 36 044486

CHA.9.536.8, CHA.9.536.8.VH, CHA.9.536.8.VL, CHA.9.536.8.HC,

CHA.9.536.8.LC, CHA.9.536.8.H1, CHA.9.536.8.H2, CHA.9.536.8.H3; CHA.9.536.8.H4, CHA.9.536.8.H4(S241P), CHA.9.536.8.vhCDRl, CHA.9.536.8.vhCDR2,

HA.9.536.8.vhCDR3, CHA.9.536.8.vlCDRl, CHA.9.536.8.vlCDR2 and

CHA.9.536.8.vhCDR3; CHA.9.560.1, CHA. 9,560.1VH, CHA. 9.560. l.VL, CHA. 9.560. l.HC, CHA. 9.560.1.LC, SNA. 9.560.1.H1, SNA. 9.560.1.H2, SNA. 9.560.1.H3; CHA. 9.560.1.H4, CHA. 9.560.1.H4 (S241P), CHA. 9.560.l.vhCDRl, CHA. 9.560.l.vhCDR2, CHA. 9.560.l.vhCDR3, CHA. 9.560.l.vlCDRl, CHA. 9.560. LvlCDR2 and CHA. 9.560.l.vhCDR3;

CHA.9.560.3, CHA. 9.560. 3VH, CHA. 9.560. 3.VL, CHA. 9.560. 3.HC, CHA. 9.560. 3.LC, CHA. 9.560. 3.H1, CHA. 9.560. 3.H2, CHA. 9.560. Z.NZ; CHA.9.560.3.H4, CHA.9.560.3.H4 (S241P); CHA. 9.560. 3.vhCDRl, CHA. 9.560. 3.vhCDR2, CHA. 9.560. 3.vhCDR3, CHA. 9.560. 3.vlCDRl, CHA. 9.560. 3.vlCDR2 and CHA. 9.560. 3.vhCDR3;

CHA.9.560.4, CHA. 9.560. 4VH, CHA. 9.560. 4.VL, CHA. 9.560. 4.HC, CHA. 9.560. 4.LC, CHA. 9.560. 4.H1, CHA. 9.560. 4.H2, CHA. 9.560. 4.H3; CHA.9.560.4.H4, CHA.9.560.4.H4 (S241P), CHA. 9.560. 4.vhCDRl, CHA. 9.560. 4.vhCDR2, CHA. 9.560. 4.vhCDR3, CHA. 9.560. 4.vlCDRl, CHA. 9.560. 4.vlCDR2 and CHA. 9.560. 4.vhCDR3;

CHA.9.560.5, CHA. 9.560. 5VH, CHA. 9.560. 5.VL, CHA. 9.560. 5.HC, CHA. 9.560. 5.LC, CHA. 9.560. 5.H1, CHA. 9.560. 5.H2, CHA. 9.560. 5.H3; CHA. 9.560. 5.H4, CHA. 9.560. 5.vhCDRl, CHA. 9.560. 5.vhCDR2, CHA. 9.560. 5.vhCDR3, CHA. 9.560. 5.vlCDRl, CHA. 9.560. 5.V1CDR2 and CHA. 9.560. 5.vhCDR3;

CHA.9.560.6, CHA. 9.560. 6VH, CHA. 9.560. 6.VL, CHA. 9.560. 6.HC, CHA. 9.560. 6.LC, CHA. 9.560. 6.H1, CHA. 9.560. 6.H2, CHA. 9.560. 6.H3; CHA.9.560.6.H4, CHA.9.560.6.H4 (S241P), CHA. 9.560. 6.vhCDRl, CHA. 9.560. 6.vhCDR2, CHA. 9.560. 6.vhCDR3, CHA. 9.560. 6.vlCDRl, CHA. 9.560. 6.vlCDR2 and CHA. 9.560. 6.vhCDR3;

CHA.9.560.7, CHA. 9.560. 7VH, CHA. 9.560. 7.VL, CHA. 9.560. 7.HC, CHA. 9.560. 7.LC, CHA. 9.560. 7.H1, CHA. 9.560. 7.H2, CHA. 9.560. 7.H3; CHA.9.560.7.H4; CHA.9.560.7.H4 (S241P); CHA. 9.560. 7.vhCDRl, CHA. 9.560. 7.vhCDR2, CHA. 9.560. 7.vhCDR3, CHA. 9.560. 7.vlCDRl, CHA. 9.560. 7.vlCDR2 and CHA. 9.560. 7.vhCDR3;

CHA.9.560.8, CHA. 9.560. 8VH, CHA. 9.560. 8.VL, CHA. 9.560. 8.HC, CHA. 9.560. 8.LC, CHA. 9.560. 8.H1, CHA. 9.560. 8.H2, CHA. 9.560. 8.H3; CHA.9.560.8.H4, CHA.9.560.8.H4 (S241P); CHA. 9.560. 8.vhCDRl, CHA. 9.560. 8.vhCDR2, CHA. 9.560. 8.vhCDR3, CHA. 9.560. 8.vlCDRl, CHA. 9.560. 8.vlCDR2 and CHA. 9.560. 8.vhCDR3;

CHA.9.546.1, CHA. 9. 546.1VH, CHA. 9. 546.l.VL, CHA. 9. 546. l.HC, CHA. 9. 546.1.LC, CHA. 9. 546.1.Hl, CHA. 9. 546.1.H2, CHA. 9.546.1.H3; CHA.9.546.1.H4,

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CHA.9.546.1.H4 (S241P), SHA. 9. 546.1.vhCDRl, SNA. 9. 546.l.vhCDR2, SNA.

9. 546.1.vhCDR3, SNA. 9. 546.1.vlCDRl, SNA. 9. 546.1.vlCDR2 and SNA. 9.546.1.vhCDR3;

SNA.9.547.1, SNA. 9. 547.1VH, SNA. 9. 547.1.VL, SNA. 9. 547.1.NS, SNA. 9. 547.1.LC, SNA. 9. 547.1.N1, SNA. 9. 547.1.N2, SNA. 9. 547.1.NZ; SNA.9.547.1.N4, SNA.9.547.1.N4 (S241P), SNA. 9. 547.1.vhCDRl, SNA. 9. 547.1.vhCDR2, SNA. 9. 547.1.vhCDR3, SNA. 9. 547.1.vlCDRl, SNA. 9. 547.1.vlCDR2 and SNA. 9.547.1.vhCDR3;

SNA.9.547,2, SNA. 9. 547, 2VH, SNA. 9. 547, 2.VL, SNA. 9. 547, 2.NS, SNA. 9.547, 2.LC, SNA. 9. 547. 2.N1, SNA. 9. 547. 2.H2, SNA. 9. 547. 2.NZ; SNA.9.547.2.N4, SNA.9.547.2.N4 (S241P), SNA. 9. 547. 2, vhCDRl, SNA. 9. 547. 2.vhCDR2, SNA. 9. 547. 2.vhCDR3, SNA. 9. 547. 2.V1CDR1, SNA. 9. 547. 2.V1CDR2 and SNA. 9.547.2.vhCDR3;

SNA.9.547.3, SNA. 9. 547. 3VH, SNA. 9. 547. 3.VL, SNA. 9. 547. Z.NS, SNA. 9. 547. 3.LC, SNA. 9. 547. Z.N1, SNA. 9. 547. Z.N2, SNA. 9. 547. Z.NZ; SNA.9.547.3.N4, SNA.9.547.3.N4 (S241P), SNA. 9. 547. 3.vhCDRl, SNA. 9.547. 3.vhCDR2, SNA. 9. 547. 3.vhCDR3, SNA. 9. 547. 3.V1CDR1, SNA. 9. 547. 3.V1CDR2 and SNA. 9.547.3.vhCDR3;

SNA.9.547.4, SNA. 9. 547. 4VH, SNA. 9. 547. 4.VL, SNA. 9. 547. 4.NS, SNA. 9.547. 4.LC, SNA. 9. 547. 4.N1, SNA. 9. 547. 4.H2, SNA. 9. 547. 4.NZ; SNA.9.547.4.N4, SNA.9.547.4.N4 (S241P), SNA. 9. 547. 4.vhCDRl, SNA. 9. 547. 4.vhCDR2, SNA. 9. 547. 4.vhCDR3, SNA. 9. 547. 4.V1CDR1, SNA. 9. 547. 4.vlCDR2 and SNA. 9.547.4.vhCDR3;

SNA.9.547.6, SNA. 9. 547. 6 VH, SNA. 9. 547. 6.VL, SNA. 9. 547. 6.NS, SNA. 9. 547. 6.LC, SNA. 9. 547. 6.N1, SNA. 9. 547. 6.N2, SNA. 9. 547. 6.NZ; SNA.9.547.6.N4, SNA.9.547.6.N4 (S241P), SNA. 9. 547. 6, vhCDRl, SNA. 9. 547. 6.vhCDR2, SNA. 9. 547. 6.vhCDR3, SNA. 9. 547. 6.V1CDR1, SNA. 9. 547. 6.V1CDR2 and SNA. 9.547.6.vhCDR3;

SNA.9.547.7, SNA. 9.547.7VH, SNA. 9. 547. 7.VL, SNA. 9. 547. 7.NS, SNA. 9. 547. 7.LC, SNA. 9. 547. 7.N1, SNA. 9. 547. 7.N2, SNA. 9. 547. 7.NZ; SNA.9.547.7.N4, SNA.9.547.7.N4 (S241P), SNA. 9. 547. 7, vhCDRl, SNA. 9. 547. 7.vhCDR2, SNA. 9. 547. 7.vhCDR3, SNA. 9. 547. 7.V1CDR1, SNA. 9. 547. 7.V1CDR2 and SNA. 9.547.7.vhCDR3;

SNA.9.547.8, SNA. 9.547.8VH, SNA. 9. 547. 8.VL, SNA. 9. 547. 8.NS, CHA.9.547.8.LC, SNA. 9. 547. 8.N1, SNA. 9. 547. 8.H2, SNA. 9. 547. 8.NZ; SNA.9.547.8.N4, SNA.9.547.8.N4 (S241P), SNA. 9. 547. 8, vhCDRl, SNA. 9. 547. 8.vhCDR2, SNA. 9. 547. 8.vhCDR3, SNA. 9. 547. 8.V1CDR1, SNA. 9. 547. 8.V1CDR2 and SNA. 9.547.8.vhCDR3;

SNA.9.547.9, SNA.9.547.9, SNA.9.547.9VH, CHA.9.547.9.VL, SNA.9. 547.9.NS, CHA.9.547.9.LC, SNA.9.547.9.N1, SNA.9.547.9.N2, SNA.9.547.9.NZ; SNA.9.547.9.H4, SNA.9.547.9.H4, CHA.9.547.9.H4(S241P), CHA.9.547.9.H4(S241P), CHA.9.547.9.vhCDRl, SNA.9.547. 9.vhCDR2, CHA.9.547.9.vhCDR3, CHA.9.547.9.vlCDRl, CHA.9.547.9.vlCDR2 and CHA.9.547.9.vhCDR3;

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CHA.9.547.13, CHA.9.547.13, CHA.9.547. 13VH, CHA.9. 547.13.VL,

CHA.9. 547.13.HC, CHA. 9.547.13.LC, SNA. 9.547.13.H1, CHA.9.547.13.H2, CHA.9.

47.13.H3; CHA.9.547.13.H4, CHA.9.547.13.H4, CHA.9.547.13.H4 (S241P),

CHA.9.547.13.H4 (S241P), CHA. 9. 547.13.vhCDRl, CHA.9.547.13. vhCDR2, CHA.9.547.

.vhCDR3, CHA. 9. 547.13.vlCDRl, CHA. 9. 547.13.vlCDR2 and CHA. 9.547.13.vhCDR3;

CHA.9.541.1, CHA. 9. 541.1.VH, CHA. 9. 541.1.VL, CHA. 9. 541.1.HC, CHA. 9.

1.1.LC, CHA. 9. 541.1.Hl, CHA. 9.541.1.H2, CHA. 9.541.1.H3; CHA.9.541.1.H4, CHA.9.541.1.H4 (S241P), CHA. 9. 541.1.vhCDRl, CHA. 9. 541.1.vhCDR2, CHA. 9.

1.1.vhCDR3, CHA. 9. 541.1.vlCDRl, CHA. 9. 541.1.vlCDR2 and CHA. 9.541.l.vhCDR3;

CHA.9.541.3, CHA. 9.541.3.VH, CHA. 9. 541. 3.VL, CHA. 9.541.3.HC, CHA. 9.541.

.LC, CHA. 9.541.3.H1, CHA. 9.541.3.H2, CHA. 9.541.3.H3; CHA.9.541.3.H4, CHA.9.541.3.H4 (S241P), CHA. 9. 541. 3.vhCDRl, CHA. 9.541.3.vhCDR2, CHA. 9.541.

.vhCDR3, CHA. 9. 541. 3.vlCDRl, CHA. 9. 541. 3.vlCDR2 and CHA. 9.541. 3.vhCDR3;

CHA.9.541.4, CHA. 9. 541.4.VH, CHA. 9. 541. 4.VL, CHA. 9.541.4.HC, CHA. 9.541.

.LC, CHA. 9.541.4.H1, CHA. 9.541.4.H2, CHA. 9.541.4.H3; CHA.9.541.4.H4, CHA.9.541.4.H4 (S241P), CHA. 9. 541. 4.vhCDRl, CHA. 9.541.4.vhCDR2, CHA. 9.541.

.vhCDR3, CHA. 9. 541. 4.vlCDRl, CHA. 9. 541. 4.vlCDR2 and CHA. 9.541. 4.vhCDR3;

CHA.9.541.5, CHA. 9.541.5.VH, CHA. 9. 541. 5.VL, CHA. 9.541.5.HC, CHA. 9.541.

.LC, CHA. 9.541.5.H1, CHA. 9.541.5.H2, CHA. 9.541.5.H3; CHA.9.541.5.H4, CHA.9.541.5.H4 (S241P), CHA. 9. 541. 5.vhCDRl, CHA. 9.541.5.vhCDR2, CHA. 9.541.

.vhCDR3, CHA. 9. 541. 5.vlCDRl, CHA. 9. 541. 5.vlCDR2 and CHA. 9.541. 5.vhCDR3;

CHA.9.541.6, CHA. 9.541.6.VH, CHA. 9. 541. 6.VL, CHA. 9.541.6.HC, CHA. 9.541.

.LC, CHA. 9.541.6.H1, CHA. 9.541.6.H2, CHA. 9.541.6.H3; CHA.9.541.6.H4, CHA.9.541.6.H4 (S241P), CHA. 9. 541. 6.vhCDRl, CHA. 9. 541. 6.vhCDR2, CHA. 9.541.

.vhCDR3, CHA. 9. 541. 6.vlCDRl, CHA. 9. 541. 6.vlCDR2 and CHA. 9.541. 6.vhCDR3;

CHA.9.541.7, CHA. 9.541.7.VH, CHA. 9. 541. 7.VL, CHA. 9.541.7.HC, CHA. 9.541.

.LC, CHA. 9.541.7.H1, CHA. 9.541.7.H2, CHA. 9.541.7.H3; CHA.9.541.7.H4, CHA.9.541.7.H4 (S241P), CHA. 9. 541. 7.vhCDRl, CHA. 9.541.7.vhCDR2, CHA. 9.541.

.vhCDR3, CHA. 9. 541. 7.vlCDRl, CHA. 9. 541. 7.vlCDR2 and CHA. 9.541. 7.vhCDR3; And

CHA.9.541.8, CHA. 9.541.8.VH, CHA. 9. 541. 8.VL, CHA. 9.541.8.HC, CHA. 9.541.

.LC, CHA. 9.541.8.H1, CHA. 9.541.8.H2, CHA. 9.541.8.H3; CHA.9.541.8.H4, CHA.9.541.8.H4 (S241P); CHA. 9.541.8vhCDRl, CHA. 9.541.8.vhCDR2, CHA. 9.541.

.vhCDR3, CHA. 9. 541. 8.vlCDRl, CHA. 9. 541. 8.vlCDR2, as well as CHA. 9.541. 8.vhCDR3.

In the case of scFvs containing the CDRs of the above antibodies, they are labeled as scFvs that include a scFv containing a heavy chain variable domain with a vhCDR, a linker, and a light chain variable domain with a vlCDR, again as above in either orientation. Thus, the invention includes the use of scFv-CHA.9.536.3, scFv-CHA.9.536.4, scFv-CHA.9.536.5, scFv-CHA.9.536.7, scFvCHA.9.536.8, scFv-CHA.9.560. 1, scFv-CHA.9.560.3, scFv-CHA.9.560.4, scFv-CHA.9.560.5, scFvCHA.9.560.6, scFv-CHA.9.560.7, scFv-CHA.9.560.8, scFv- CHA.9.546.1, scFv-CHA.9.547.1, scFvCHA.9.547.2, scFv-CHA.9.547.3, scFv-CHA.9.547.4, scFv-CHA.9.547.6, scFv-CHA.9.547. 7,scFvCHA.9.547.8, scFv-CHA.9.547.9, scFv-CHA.9.547.13, scFv-CHA.9.541.1, scFv-CHA.9.541.3, scFvCHA.9.541.4, scFv-CHA. 9.541.5, scFv-CHA.9.541.6, scFv-CHA.9.541.7 and scFv-CHA.9.541.8.

In addition, CHA.9.543 binds to TIGIT but does not block TIGIT-PVR interaction.

As described herein, the invention further provides the use of variants of the above components (CPA and CHA), including variants in the CDR, as described above. Thus, the present invention provides antibodies containing a set of 6 CDRs as defined herein, which may contain one, two or three amino acid differences in the set of CDRs, provided that the antibody still binds to TIGIT. Suitable assays, such as Biacore assays, are known in the art for testing anti-TIGIT antibodies that contain mutations compared to the CDR sequences described herein.

In addition, the present invention further provides the use of variants of the above variable heavy and light chains. In this case, the variable heavy chains may be at

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80, 90, 95, 98 or 99% identical to the VH sequences of the present invention and/or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions or more when Fc variants are used. Variable light chains are provided which may be 80, 90, 95, 98 or 99% identical to the VL sequences of the present invention (and in particular CPA.9.086) and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions or more when Fc variants are used. In these embodiments of the present invention, the anti-TIGIT antibodies for use in the present invention still bind to TIGIT. Suitable assays, such as Biacore assays, are known in the art for testing anti-TIGIT antibodies that contain mutations compared to the CDR sequences described herein.

Likewise, heavy and light chains are provided that are 80, 90, 95, 98 or 99% identical to the full-length HC and LC sequences of the present invention (and in particular CPA.9.086) and/or contain from 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10 amino acid substitutions or more when Fc variants are used. These embodiments of the present invention include the above embodiments as long as the anti-TIGIT antibody still binds to TIGIT. Suitable assays, such as Biacore assays, are known in the art for testing anti-TIGIT antibodies that contain mutations compared to the CDR sequences described herein.

In addition, the framework regions of the variable heavy and variable light chains of either CPA or CHA antibodies in the present invention may be humanized (or, in the case of CHA antibodies, rehumanized) to the extent that alternative humanization methods can be performed), as is known in the art. of the art (random variants are generated in the CDR as needed) and thus humanized variants of the VH and VL circuits shown in FIG. 23 (and in particular CPA.9.086). In addition, the humanized heavy and light chain variable domains can then be fused to human constant regions, such as the constant regions from IgG1, IgG2, IgG3 and IgG4 (including IgG4 (S241P)).

In particular, mouse VH and VL chains can be humanized as known in the art, for example, using the IgBLAST program on the NCBI website, as described in Ye et al. Nucleic Acids Res. 41: W34-W40 (2013), incorporated herein by reference in its entirety for humanization methods. IgBLAST analyzes the mouse VH and/or mouse VL sequence and compares it to a library of known human germline sequences. As demonstrated herein, for the humanized sequences generated in this invention, human IMGT VH genes (F+ORF, 273 germline sequences) and human IMGT VL kappa genes (F+ORF, 74 germline sequences) were used as databases. . Five typical SHA sequences were selected: SHA.9.536, SHA9.560, SHA.9.546, SHA.9.547 and SHA.9.541 (see Fig. 3). For this humanization option, human germline IGHV1-46 (allele 1) was selected for all 5 as the acceptor sequence and heavy chain attachment region of human IGHJ4 (allele 1) (J gene). For three of the four (CHA.7.518, CHA.7.530, CHA.7.538_1 and CHA.7.538_2), the human germline IGKV1-39 (allele 1) was selected as the acceptor sequence and the human IGKJ2 (allele 1) light chain (gene J). The J gene was selected from human attachment region sequences collected in IMGT®, the international information system ImMunoGeneTics, at www.imgt.org. CDRs were defined according to the AbM definition (see www.bioinfo.org.uk/abs/). In some embodiments of the present invention, anti-TIGIT antibodies for use in the present invention include TIGIT binding regions or antigen binding domains, wherein the V _H and V _L sequences of different TIGIT binding regions or antigen binding domains can be mixed and matched to create other TIGIT binding regions or antigen binding domains. The binding of TIGIT to such mixed and matched anti-TIGIT antibodies can be tested using the binding assays described above, such as ELISA or Biacore assays). In some embodiments of the present invention, when the VH and VL chains are mixed and matched, the VH sequence from a particular VH/VL pairing is replaced with a structurally similar VH sequence. Likewise, in some embodiments of the present invention, the V _L sequence from a particular V _H /V _L pairing is replaced with a structurally similar V _L sequence. For example, the V _H and V _L sequences of homologous antibodies are particularly suitable for mixing and matching.

Accordingly, anti-TIGIT antibodies for use in this invention may contain CDR amino acid sequences selected from the group consisting of (a) sequences listed herein; (b) sequences that differ from the CDR amino acid sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; (c) amino acid sequences having 90% or more, 95% or more, 98% or more, or 99% or more identity with the sequences specified in (a) or (b); (d) a polypeptide having an amino acid sequence encoded by a polynucleotide having a nucleic acid sequence encoding amino acids as defined herein. In particular, the anti-TIGIT antibody may contain an antigen binding domain from the antibody

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CPA.9.086, which may have sequences selected from (a), (b), (c) or (d).

Also included within the definition of anti-TIGIT antibodies for use in this invention are antibodies containing TIGIT binding domains that are identical to the binding domains of the TIGIT antibodies listed herein. That is, in certain embodiments of the present invention, the anti-TIGIT antibody of the present invention comprises heavy and light chain variable regions containing amino acid sequences that are identical to all or portions of the binding domains of the anti-TIGIT amino acid sequences of the preferred anti-TIGIT antibodies, respectively, wherein these antibodies retain the desired functional properties of the parent anti-TIGIT antibodies. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology = number of identical positions/total number of positions X 100), taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below.

The percentage identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which is included in the ALIGN program (version 2.0), using a weight table residues of PAM120, a gap length of 12, and a sequence gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444453 (1970)), which is included in the program GAP in the GCG software package (commercially available), using either a Blossum 62 matrix or a PAM250 matrix, gap weight of 16, 14, 12, 10, 8, 6 or 4, and gap extension penalty of 1, 2, 3, 4, 5 or 6.

In general, the percentage identity for comparison between TIGIT binding domains or antigen binding domains is at least 75%, at least 80%, at least 90%, with a percentage identity of at least about 95, 96, 97 being preferred. 98 or 99%. The percentage identity may be along the entire amino acid sequence, for example, the entire heavy or light chain or along a portion of the chains. For example, the definitions of anti-TIGIT antibodies for use in this invention include antibodies whose TIGIT-binding portion or antigen-binding domains have identity along the entire variable region (for example, when the identity is 95 or 98% identity along the variable regions), or along the entire constant region region, or along only the Fc region. In particular, anti-TIGIT antibodies for use in the present invention include antibodies that have a TIGIT binding portion or antigen binding domains having a percentage identity with antibody CPA.9.086 of at least 75%, at least 80%, at least 90% , with a percent identity of at least about 95, 96, 97, 98, or 99% preferred.

In addition, also included are sequences that may have identical CDRs, but at the same time be changed in the framework portions of the variable domain (or the entire heavy or light chain). For example, anti-TIGIT antibodies for use in the present invention include antibodies with CDRs identical to those shown in FIG. 3, but whose identity along the variable region may be lower, for example with a percent identity of 95% or 98%. In particular, the present invention provides the use of anti-TIGIT antibodies that have TIGIT binding moieties or antigen binding domains whose CDRs are identical to CPA.9.086, but whose framework regions are 95 or 98% identical to CPA.9.086.

C. Anti-TIGIT antibodies in combination with αntu-PD-1 antibodies.

In another embodiment, the present invention provides combinations of anti-TIGIT antibodies of the present invention and anti-PD-1 antibodies. There are two approved anti-PD-1 antibodies, pembrolizumab (Keytruda®) and nivolumab (Opdivo®) and many others in clinical development that can be used in combination with the anti-TIGIT antibodies of this invention .

Accordingly, the present invention provides specific combinations: CPA.9.083.H4 (S241P), as shown in FIG. 3F, with pembrolizumab; CPA.9.083.H4 (S241P), as shown in FIG. 3F, with nivolumab; CPA.9.086.H4 (S241P), as shown in FIG. 3G, with pembrolizumab; CPA.9.086.H4 (S241P), as shown in FIG. 3G, with nivolumab; SNA.9.547.7N4 (S241P), as shown in FIG. 4НН, with pembrolizumab; SNA.9.547.7N4 (S241P), as shown in FIG. 3NN, with nivolumab; SNA.9.547.13.H4 (S241P), as shown in FIG. 3VV, with pembrolizumab, and CHA.9.547.13.H4 (S241P), as demonstrated in FIG. 3VV, with nivolumab; all from fig. 4 USSN 62/513916, entitled Anti-TIGIT Antibodies and Uses Thereof, filed June 1, 2017 by assignee Compugen. Other anti-TIGIT antibodies that can be combined with anti-PD-1 antibodies are also shown in FIG. 3.

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D. Specific anti-PVRIG antibodies.

This invention provides antigen binding domains, including full length antibodies, that contain a number of specific, listed sets of 6 CDRs and specific heavy chain variable regions (vh, VH or VH) and light chain variable regions (vl, VL or VL) that bind to PVRIG.

In one embodiment, the anti-PVRIG antibody is an antibody comprising a set of six CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) from CHA.7.518.1.H4 (S241P), as shown in FIG. 5. In one embodiment of the present invention, the anti-TIGIT antibody is an antibody comprising a heavy chain variable domain (vh) and a light chain variable domain (vl) from CHA.7.518.1.H4 (S241P), as demonstrated in FIG. 5 related to human IgG constant domain IgG1, IgG2, IgG3, IgG4 and IgG4 (S241P). In one embodiment of the present invention, the anti-TIGIT antibody is CHA.7.518.1.H4 (S241P). In some embodiments of the present invention, the anti-PVRIG antibody is an anti-PVRIG antibody, as demonstrated in FIG. 5 or FIG. 63.

In particular, the 2H6 anti-PVRIG antibody according to Zhu et al., WO 2017/041004, specifically incorporated herein by reference, which has vhCDR1 SEQ ID NO: 6, vhCDR2 SEQ ID NO: 7, vhCDR3 SEQ ID NO can be used : 8, vlCDR1 SEQ ID NO: 9, vlCDR2 SEQ ID NO: 10 and vhCDR3 SEQ ID NO: 11 from WO 2017/041004. 2H6 anti-PVRIG antibody according to Zhu et al. has a heavy chain variable domain containing SEQ ID NO: 6 and a light chain variable domain containing SEQ ID NO: 3, which can be linked to the IgG constant domain of IgG1, IgG2 , human IgG3, IgG4 and IgG4 (S241P). All SEQ IDs in this paragraph are taken from WO 2017/041004 and are also presented in FIG. 5.

In particular, the 334M5 anti-PVRIG antibody from WO 20180/017864, specifically incorporated herein by reference, which has vhCDR1 SEQ ID NO: 31, vhCDR2 SEQ ID NO: 32, vhCDR3 SEQ ID NO: 33: vlCDR1 SEQ can be used ID NO: 26, vlCDR2 SEQ ID NO: 27 and vhCDR3 SEQ ID NO: 28 from WO 2018/017864. The 334M5 anti-PVRIG antibody of WO 2018/017864 has a heavy chain variable domain containing SEQ ID NO: 30 and a light chain variable domain containing SEQ ID NO: 25, which can be linked to the IgG constant domain of IgG1, IgG2, IgG3 , IgG4 and human IgG4 (S241P). All SEQ IDs in this paragraph are taken from WO 2018/017864 and are also presented in FIG. 5.

E. Additional anti-PVRIG antibodies.

Antibodies to PVRIG that can find use in accordance with the three-component combinations of this invention are indicated as follows. These anti-PVRIG antibodies described herein are designated as follows. Antibodies to PVRIG have reference numbers, for example CPA.7.013. They are a combination of variable heavy chains and variable light chains, as shown in FIG. 63, for example.

CPA.7.013.VH refers to the variable heavy chain portion of CPA.7.013, while CPA.7.013.VL is the variable light chain. CPA.7.013.vhCDR1, CPA.7.013.vhCDR2, CPA.7.013.vhCDR3, CPA.7.013.vlCDR1, CPA.7.013.vlCDR2 and

CPA.7.013.vlCDR3, refers to the CDRs that are specified. CPA.7.013.HC refers to the entire heavy chain (eg, variable and constant domain) of that molecule, and CPA.7.013.LC refers to the entire light chain (eg, variable and constant domain) of the same molecule. CPA.7.013.H1 refers to a full-length antibody containing heavy and light chain variable domains, including the human IgG1 constant domain (hence H1; IgG1, IgG2, IgG3 and IgG4, as demonstrated in FIG. 1, for example). Accordingly, CPA.7.013.H2 will be an antibody containing the variable domains of CPA.7.013 associated with human IgG2. CPA.7.O13.H3 will be an antibody containing CPA.7.013 variable domains associated with human IgG3, and CPA.7.013.H4 will be an antibody containing CPA.7.013 variable domains associated with human IgG4.

Antibodies to PVRIG that can find use in accordance with the three-component combinations of this invention are indicated as follows. These antibodies have reference numbers, for example, CHA.7.518.1. They are a combination of variable heavy chains and variable light chains, as demonstrated, for example, in FIG. 5 and fig. 63, with the understanding that these antibodies comprise two heavy chains and two light chains. SRA. 7.518.1.VH refers to the variable heavy chain portion of CPA. 7.518.1, while CPA.7.518.1.VL is a variable light chain. SRA. 7.518.1.vhCDR1, CPA.7.518.1.vhCDR2, CPA. 7.518.1.vhCDR3, CPA. 7.518.1.vlCDR1, CPA. 7.518.1.vlCDR2 and CPA. 7.518.1.vlCDR3, refer to the specified CDRs. SRA. 7.518.1.HC refers to the entire heavy chain (eg, variable and constant domain) of this molecule, and CPA. 7.518.1.LC refers to the entire light chain (eg, variable and constant domain) of the same molecule. In general, the human kappa light chain is used in this invention for the constant domain of each phage antibody (or humanized hybridoma antibody), although in some embodiments of the present invention the lambda light chain constant domain is used. SRA. 7.518.1.H1 refers to a full-length antibody containing variable

- 42 044486 heavy and light chain domains, including the human IgG1 constant domain (hence H1; IgG1, IgG2, IgG3 and IgG4, as demonstrated in FIG. 1, for example). Accordingly, SRA. 7.518.1.H2 will be an antibody containing CPA variable domains. 7.518.1 related to human IgG2. SRA. 7.518.1.H3 will be an antibody containing CPA.7.518.1 variable domains associated with human IgG3, and CPA.7.518.1.H4 will be an antibody containing CPA variable domains. 7.518.1 related to human IgG4. It should be noted that in some cases human IgG may have additional mutations, such as those described below, and this may be designated accordingly. For example, in many embodiments of the present invention, human IgG4 may have the S241P mutation, and this may be referred to as CPA. 7.518.1.H4 (S241P), for example. The sequence of human IgG4 with this S241P hinge variant is shown in FIG. 1. Other potential variants are IgG1 (N297A) (or other variants that eliminate glycosylation at this site and thus many effector functions associated with FcYRIIIa binding), and IgG1 (D265A), which reduces binding to FcyR receptors. Anti-PVRIG antibodies for use in the present invention may comprise any of the anti-PVRIG antibody sequences. Anti-PVRIG antibodies for use in the present invention may contain any of the PVRIG antigen binding domain sequences.

The present invention further provides heavy and light chain variable domains, as well as full-length heavy and light chains, any of which can be used as part of anti-PVRIG antibodies for use in accordance with the present invention.

In some embodiments, the present invention provides scFvs that bind to PVRIG comprising a heavy chain variable domain and a light chain variable domain linked via a scFv linker as described above. The VL and VH domains can be in any orientation, for example N- to C-terminal 'VH-linker-VL' or VL-linker-VH. They are named after their constituent parts; for example, scFv-CHA.7.518.1VH-linker-VL or scFv-CPA. 7.518.1.VL-linker-VH. Thus, scFv-CPA. 7.518.1 can be in any orientation. Anti-PVRIG antibodies for use in this invention may contain a scFv that binds to PVRIG.

The present invention provides antigen binding domains, including full length antibodies, that contain a number of specific listed sets of 6 CDRs. Anti-PVRIG antibodies for use in the present invention may contain any of the sets of 6 CDRs of the anti-PVRIG antibody sequences provided herein.

The present invention further provides heavy and light chain variable domains, as well as full-length heavy and light chains.

In many embodiments of the present invention, anti-PVRIG antibodies for use in the present invention are human antibodies (derived from phage) and block the binding of PVRIG and PVLR2. The anti-PVRIG antibodies of the present invention may contain an anti-PVRIG antibody and/or antigen binding domain sequence capable of either binding or blocking receptor-ligand interaction. An anti-PVRIG antibody may comprise a CDR from an anti-PVRIG antibody sequence capable of both binding and blocking receptor-ligand interaction. The anti-CPA antibodies, as well as the CDR sequences that bind and block receptor ligand interaction, are as follows, with their components identified in sequence as shown in FIG. 63:

CPA.7.001, CPA.7.001.VH, CPA.7.001.VL, CPA.7.001.NS, CPA.7.001.LC and

SPA.7.001.N1, SPA.7.001. N2, SPA.7.001.NZ, SPA.7.001.N4; SPA.7.001. vhCDRl, CPA.7.001.vhCDR2, CPA.7.001.vhCDR3, CPA.7.001.vlCDRl, CPA.7.001.vlCDR2 and CPA.7.001.V1CDR3;

SPA.7.003, SPA.7.003. VH, CPA.7.003.VL, CPA.7.003.NS, CPA.7.003.LC,

SRA.7.003.N1, SRA.7.003.N2, SRA.7.003.NZ, SRA.7.003.N4; CPA.7.003 .vhCDRl, CPA.7.003.vhCDR2, CPA.7.003 ,vhCDR3, CPA.7.003 .vlCDRl, CPA.7.003 ,vlCDR2 and CPA.7.003.V1CDR3;

CPA.7.004, CPA.7.004.VH, CPA.7.004.VL, CPA.7.004.NS, CPA.7.004.LC,

SPA.7.004.N1, SPA.7.004.N2, SPA.7.004.NZ SPA.7.004.N4; CPA.7.004.vhCDRl,

CPA.7.004.vhCDR2, CPA.7.004.vhCDR3, CPA.7.004.vlCDRl, CPA.7.004.vlCDR2 and CPA.7.004.vlCDR3;

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CPA.7.006, CPA.7.006.VH, CPA.7.006.VL, CPA.7.006.HC, CPA.7.006.LC,

CPA.7.006.H1, CPA.7.006.H2, CPA.7.006.H3 CPA.7.006.H4; CPA.7.006.vhCDRl,

CPA.7.006.vhCDR2, CPA.7.006.vhCDR3, CPA.7.006.vlCDRl, CPA.7.006.vlCDR2 and CPA.7.006.V1CDR3;

CPA.7.008, CPA.7.008.VH, CPA.7.008.VL, CPA.7.008.HC, CPA.7.008.LC,

CPA.7.008.H1, CPA.7.008.H2, CPA.7.008.H3 CPA.7.008.H4; CPA.7.008.vhCDRl,

CPA.7.008.vhCDR2, CPA.7.008.vhCDR3, CPA.7.008.vlCDRl, CPA.7.008.vlCDR2 and CPA.7.008.V1CDR3;

CPA.7.009, CPA.7.009.VH, CPA.7.009.VL, CPA.7.009.HC, CPA.7.009.LC,

CPA.7.009.H1, CPA.7.009.H2, CPA.7.009.H3 CPA.7.009.H4; CPA.7.009.vhCDRl,

CPA.7.009.vhCDR2, CPA.7.009.vhCDR3, CPA.7.009.vlCDRl, CPA.7.009.vlCDR2 and CPA.7.009.vlCDR3;

CPA.7.010, CPA.7.010.VH, CPA.7.010.VL, CPA.7.010.HC,

CPA.7.010.LC, CPA.7.010.Hl, CPA.7.010.H2, CPA.7.010.H3 CPA.7.010.H4; CPA.7.010.vhCDRl, CPA.7.010.vhCDR2, CPA.7.010.vhCDR3, CPA.7.010.vlCDRl, CPA.7.010.V1CDR2 and CPA.7.010.vlCDR3;

CPA.7.011, CPA.7.01 l.VH, CPA.7.01 l.VL, CPA.7.011.HC, CPA.7.011.LC,

CPA.7.011.Hl, CPA.7.011.H2, CPA.7.011.H3 CPA.7.011.H4; CPA.7.01 l.vhCDRl,

CPA.7.01 l.vhCDR2, CPA.7.01 l.vhCDR3, CPA.7.01 l.vlCDRl, CPA.7.01 l.vlCDR2 and CPA.7.01 l.vlCDR3;

CPA.7.012, CPA.7.012.VH, CPA.7.012.VL, CPA.7.012.HC, CPA.7.012.LC,

CPA.7.012.H1, CPA.7.012.H2, CPA.7.012.H3 CPA.7.012.H4; CPA.7.012.vhCDRl,

CPA.7.012.vhCDR2, CPA.7.012.vhCDR3, CPA.7.012.vlCDRl, CPA.7.012.vlCDR2 and CPA.7.012.V1CDR3;

CPA.7.013, CPA.7.013.VH, CPA.7.013.VL, CPA.7.013.HC, CPA.7.013.LC,

CPA.7.013.H1, CPA.7.013.H2, CPA.7.013.H3 CPA.7.013.H4; CPA.7.013.vhCDRl,

CPA.7.013.vhCDR2, CPA.7.013.vhCDR3, CPA.7.013.vlCDRl, CPA.7.013.vlCDR2 and CPA.7.013.V1CDR3;

CPA.7.014, CPA.7.014.VH, CPA.7.014.VL, CPA.7.014.HC, CPA.7.014.LC,

CPA.7.014.H1, CPA.7.014.H2, CPA.7.014.H3 CPA.7.014.H4; CPA.7.014.vhCDRl,

CPA.7.014.vhCDR2, CPA.7.014.vhCDR3, CPA.7.014.vlCDRl, CPA.7.014.vlCDR2 and CPA.7.014.V1CDR3;

CPA.7.015, CPA.7.015.VH, CPA.7.015.VL, CPA.7.015.HC, CPA.7.015.LC,

CPA.7.015.H1, CPA.7.015.H2, CPA.7.015.H3 CPA.7.015.H4; CPA.7.015.vhCDRl,

CPA.7.015.vhCDR2, CPA.7.015.vhCDR3, CPA.7.015.vlCDRl, CPA.7.015.vlCDR2 and CPA.7.015.V1CDR3;

CPA.7.017, CPA.7.017.VH, CPA.7.017.VL, CPA.7.017.HC, CPA.7.017.LC,

CPA.7.017H1, CPA.7.017.H2, CPA.7.017.H3 CPA.7.017.H4; CPA.7.017.vhCDRl,

CP A.7.000171.vhCDR2, CPA.7.017.vhCDR3, CPA.7.017.vlCDRl, CPA.7.017.vlCDR2 and CPA.7.017.V1CDR3;

CPA.7.018, CPA.7.018.VH, CPA.7.018.VL, CPA.7.018.HC, CPA.7.018.LC,

CPA.7.018.H1, CPA.7.018.H2, CPA.7.018.H3 CPA.7.018.H4; CPA.7.017.vhCDRl,

CPA.7.017.vhCDR2, CPA.7.017.vhCDR3, CPA.7.017.vlCDRl, CPA.7.017.vlCDR2 and CPA.7.017.V1CDR3;

CPA.7.019, CPA.7.019.VH, CPA.7.019.VL, CPA.7.019.HC, CPA.7.019.LC,

CPA.7.019.H1, CPA.7.019.H2, CPA.7.019.H3 CPA.7.019.H4; CPA.7.019.vhCDRl,

CPA.7.019.vhCDR2, CPA.7.019.vhCDR3, CPA.7.019.vlCDRl, CPA.7.019.vlCDR2 and

- 44 044486

CPA.7.019.vlCDR3;

CPA.7.021, CPA.7.021.VH, CPA.7.021.VL, CPA.7.021.HC, CPA.7.021.LC,

CPA.7.021.Hl, CPA.7.021.H2, CPA.7.021.NZ CPA.7.021.H4; CPA.7.021.vhCDRl, CPA.7.021. vhCDR2, CPA.7.021.vhCDR3, CPA.7.021. vlCDRl, CPA.7.021.vlCDR2 and

CPA.7.021.vlCDR3;

CPA.7.022, CPA.7.022.VH, CPA.7.022.VL, CPA.7.022.HC, CPA.7.022.LC,

CPA.7.022.H1, CPA.7.022.H2, CPA.7.022.H3 CPA.7.022.H4; CPA.7.022.vhCDRl,

CPA.7.022.vhCDR2, CPA.7.002201.vhCDR3, CPA.7.022.vlCDRl, CPA.7.022.vlCDR2 and CPA.7.022.V1CDR3;

CPA.7.023, CPA.7.023. VH, CPA.7.023. VL, CPA.7.023 HC, CPA.7.023.LC, CPA.7.023.H1, CPA.7.023.H2, CPA.7.023.H3 CPA.7.023.H4; CPA.7.023.vhCDRl,

CPA.7.023.vhCDR2, CPA.7.023 vhCDR3, CPA.7.023 vlCDRl, CPA.7.023 vlCDR2 and CPA.7.023.V1CDR3;

CPA.7.024, CPA.7.024.VH, CPA.7.024.VL, CPA.7.024.HC, CPA.7.024.LC,

CPA.7.024.H1, CPA.7.024.H2, CPA.7.024.H3 CPA.7.024.H4; CPA.7.024.vhCDRl,

CPA.7.024.vhCDR2, CPA.7.024.vhCDR3, CPA.7.024.vlCDRl, CPA.7.024.vlCDR2 and CPA.7.024.V1CDR3;

CPA.7.033, CPA.7.033.VH, CPA.7.033.VL, CPA.7.033.HC, CPA.7.033.LC,

CPA.7.033.H1, CPA.7.033.H2, CPA.7.033.H3 CPA.7.033.H4; CPA.7.033.vhCDRl,

CPA.7.033.vhCDR2, CPA.7.033.vhCDR3, CPA.7.033.vlCDRl, CPA.7.033.vlCDR2 and CPA.7.033.vlCDR3;

CPA.7.034, CPA.7.034.VH, CPA.7.034.VL, CPA.7.034.HC, CPA.7.034.LC,

CPA.7.034.H1, CPA.7.034.H2, CPA.7.034.H3 CPA.7.034.H4; CPA.7.034.vhCDRl,

CPA.7.034.vhCDR2, CPA.7.034.vhCDR3, CPA.7.034.vlCDRl, CPA.7.034.vlCDR2 and CPA.7.034.V1CDR3;

CPA.7.036, CPA.7.036.VH, CPA.7.036.VL, CPA.7.036.HC, CPA.7.036.LC,

CPA.7.036.H1, CPA.7.036.H2, CPA.7.036.H3 CPA.7.036.H4; CPA.7.036.vhCDRl,

CPA.7.036.vhCDR2, CPA.7.036.vhCDR3, CPA.7.036.vlCDRl, CPA.7.036.vlCDR2 and CPA.7.036.V1CDR3;

CPA.7.040, CPA.7.040.VH, CPA.7.040.VL, CPA.7.040.HC, CPA.7.040.LC,

CPA.7.040.H1, CPA.7.040.H2, CPA.7.040.H3 and CPA.7.040. H4; CPA.7.040.vhCDRl, CPA.7.040.vhCDR2, CPA.7.040.vhCDR3, CPA.7.040.vlCDRl, CPA.7.040.vlCDR2 and CPA.7.040.V1CDR3;

CPA.7.046, CPA.7.046.VH, CPA.7.046.VL, CPA.7.046.HC, CPA.7.046.LC,

CPA.7.046.H1, CPA.7.046.H2, CPA.7.046.H3 CPA.7.046.H4; CPA.7.046.vhCDRl,

- 45 044486

CPA.7.046.vhCDR2, CPA.7.046.vhCDR3, CPA.7.046.vlCDRl, CPA.7.046.vlCDR2 and

CPA.7.046.vlCDR3;

CPA.7.047, CPA.7.047.VH, CPA.7.047.VL, CPA.7.047.HC, CPA.7.047.LC,

CPA.7.047.H1, CPA.7.047.H2, CPA.7.047.H3 CPA.7.047.H4; CPA.7.047.vhCDRl,

CPA.7.047.vhCDR2, CPA.7.047.vhCDR3, CPA.7.047.vlCDRl, CPA.7.004701.vlCDR2 and CPA.7.047.V1CDR3;

CPA.7.049, CPA.7.049.VH, CPA.7.049.VL, CPA.7.049.HC, CPA.7.049.LC,

CPA.7.049.H1, CPA.7.049.H2, CPA.7.049.H3 CPA.7.049.H4; CPA.7.049.vhCDRl,

CPA.7.049.vhCDR2, CPA.7.049.vhCDR3, CPA.7.049.vlCDRl, CPA.7.049.vlCDR2 and

CPA.7.049.vlCDR3; and

CPA.7.050, CPA.7.050.VH, CPA.7.050.VL, CPA.7.050.HC, CPA.7.050.LC,

CPA.7.050.H1, CPA.7.050.H2, CPA.7.050.H3 CPA.7.050.H4, CPA.7.050.vhCDRl,

CPA.7.050. vhCDR2, CPA.7.050.vhCDR3, CPA.7.050.vlCDRl, CPA.7.050.vlCDR2 and

CPA.7.050.vlCDR3.

In addition, there are a number of anti-CPA antibodies generated in this invention that bind to PVRIG but do not block the interaction of PVRIG and PVLR2. Anti-PVRIG antibodies for use in the present invention may contain an anti-PVRIG antibody and/or antigen binding domain sequence capable of binding but not blocking receptor-ligand interaction. An anti-PVRIG antibody for use in the present invention may comprise a CDR from an anti-PVRIG antibody sequence capable of binding but not blocking receptor-ligand interaction. The anti-CPA antibodies, as well as the CDR sequences that bind but do not block the receptor-ligand interaction, are as follows, with their components indicated, the sequences for which are shown in FIG. 63:

CPA.7.028, CPA.7.028.VH, CPA.7.028.VL, CPA.7.028.NS, CPA.7.028.LC,

SPA.7.028.N1, SPA.7.028.N2, SPA.7.028.NZ and SPA.7.028. H4; CPA.7.028.vhCDRl,

CPA.7.028.vhCDR2, CPA.7.028.vhCDR3, CPA.7.028.vlCDRl, CPA.7.028.vlCDR2 and

CPA.7.028. vlCDR3.

CPA.7.030, CPA.7.030.VH, CPA.7.030.VL, CPA.7.030.HC, CPA.7.030.LC,

CPA.7.030.H1, CPA.7.030.H2, CPA.7.030.H3 and CPA.7.030. H4; CPA.7.030.vhCDRl,

CPA.7.030.vhCDR2, CPA.7.030.vhCDR3, CPA.7.030.vlCDRl, CPA.7.030.vlCDR2 and

CPA.7.030. vlCDR3.

CPA.7.041, CPA.7.041.VH, CPA.7.041.VL, CPA.7.041.HC, CPA.7.041.LC,

CPA.7.041.Hl, CPA.7.041.H2, CPA.7.041.H3 and CPA.7.041. H4; CPA.7.041.vhCDRl,

CPA.7.041.vhCDR2, CPA.7.041.vhCDR3, CPA.7.041. vlCDRl, CPA.7.041.vlCDR2 and

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CPA.7.041.vlCDR3.

CPA.7.016, CPA.7.016.VH, CPA.7.016.VL, CPA.7.016.HC, CPA.7.016.LC,

CPA.7.016.Hl, CPA.7.016.H2, CPA.7.016.H3 and CPA.7.016. H4; CPA.7.016.vhCDRl, CPA.7.016.vhCDR2, CPA.7.016.vhCDR3, CPA.7.016.vlCDRl, CPA.7.016.vlCDR2 and CPA.7.016.V1CDR3.

CPA.7.020, CPA.7.020.VH, CPA.7.020.VL, CPA.7.020.HC, CPA.7.020.LC,

CPA.7.020.H1, CPA.7.020.H2, CPA.7.020.H3 and CPA.7.020. H4; CPA.7.020.vhCDRl, CPA.7.020.vhCDR2, CPA.7.020.vhCDR3, CPA.7.020.vlCDRl, CPA.7.020.vlCDR2 and CPA.7.020.V1CDR3.

CPA.7.038, CPA.7.038.VH, CPA.7.038.VL, CPA.7.038.HC, CPA.7.038.LC,

CPA.7.038.H1, CPA.7.038.H2, CPA.7.038.H3 and CPA.7.038. H4; CPA.7.038.vhCDRl, CPA.7.038.vhCDR2, CPA.7.038.vhCDR3, CPA.7.038.vlCDRl, CPA.7.038.vlCDR2 and CPA.7.038.V1CDR3.

CPA.7.044, CPA.7.044.VH, CPA.7.044.VL, CPA.7.044.HC, CPA.7.044.LC,

CPA.7.044.H1, CPA.7.044.H2, CPA.7.044.H3 and CPA.7.044. H4; CPA.7.044.vhCDRl, CPA.7.044.vhCDR2, CPA.7.044.vhCDR3, CPA.7.044.vlCDRl, CPA.7.044.vlCDR2 and

CPA.7.044.V1CDR3.

CPA.7.045, CPA.7.045.VH, CPA.7.045.VL, CPA.7.045.HC, CPA.7.045.LC,

CPA.7.045.H1, CPA.7.045.H2, CPA.7.045.H3 and CPA.7.045. H4; CPA.7.045.vhCDRl, CPA.7.045.vhCDR2, CPA.7.045.vhCDR3, CPA.7.045.vlCDRl, CPA.7.045.vlCDR2 and CPA.7.045.V1CDR3.

As described herein, the invention further provides variants of the above components, including variants in the CDR as described above. In addition, the variable heavy chains may be 80, 90, 95, 98 or 99% identical to the VH sequences of the present invention and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids substitutions or more when Fc variants are used. Variable light chains are provided which may be 80, 90, 95, 98 or 99% identical to the VL sequences of the present invention and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids substitutions or more when Fc variants are used. Likewise, heavy and light chains are provided that are 80, 90, 95, 98 or 99% identical to the HC and LC sequences of the present invention and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions or more when Fc variants are used. Anti-PVRIG antibodies for use in this invention may contain any of these anti-PVRIG antibody and/or antigen binding domain sequences.

In addition, the present invention provides a series of anti-CHA antibodies, which are murine antibodies derived from hybridomas. As is well known in the art, the six CDRs are useful when placed in either a human heavy and light chain variable region framework or when the heavy and light chain variable regions are humanized. See, for example, FIG. 5 and 63.

An anti-PVRIG antibody for use in the present invention may include any of the following CHA CDR sets of anti-PVRIG antibody sequences. Accordingly, the present invention provides the use of an anti-PVRIG antibody that contains the following CHA sets of CDRs, the sequences of which are shown in FIG. 5 and/or in FIG. 63:

- 47 044486

CHA.7.502.vhCDRl, CHA.7.502.vhCDR2, CHA.7.502.vhCDR3, CHA.7.502. vlCDRl, CHA.7.502.V1CDR2 and CHA.7.502.vlCDR3.

CHA.7.503.vhCDRl, CHA.7.503.vhCDR2, CHA.7.503.vhCDR3, CHA.7.503 vlCDRl, CHA.7.503.vlCDR2 and CHA.7.503.vlCDR3.

CHA.7.506.vhCDRl, CHA.7.506.vhCDR2, CHA.7.506.vhCDR3, CHA.7.506.vlCDRl, CHA.7.506.vlCDR2 and CHA.7.506.vlCDR3.

CHA.7.5O8.vhCDRl, CHA.7.508.vhCDR2, CHA.7.508.vhCDR3, CHA.7.508.vlCDRl, CHA.7.508.vlCDR2 and CHA.7.508.vlCDR3.

CHA.7.510.vhCDRl, CHA.7.510.vhCDR2, CHA.7.510.vhCDR3, CHA.7.510.vlCDRl, CHA.7.510.V1CDR2 and CHA.7.510.vlCDR3.

CHA.7.512.vhCDRl, CHA.7.512.vhCDR2, CHA.7.512.vhCDR3, CHA.7.512.vlCDRl, CHA.7.512.V1CDR2 and CHA.7.512.vlCDR3.

CHA.7.514.vhCDRl, CHA.7.514.vhCDR2, CHA.7.514.vhCDR3, CHA.7.514.vlCDRl, CHA.7.514.V1CDR2 and CHA.7.514.vlCDR3.

CHA.7.516.vhCDRl, CHA.7.516.vhCDR2, CHA.7.516.vhCDR3, CHA.7.516.vlCDRl, CHA.7.516.V1CDR2 and CHA.7.516.vlCDR3.

CHA.7.518.vhCDRl, CHA.7.518.vhCDR2, CHA.7.518.vhCDR3, CHA.7.518.vlCDRl,

CHA.7.518.V1CDR2 and CHA.7.518.vlCDR3.

CHA.7.520_l.vhCDRl, CHA.7.520_l.vhCDR2, CHA.7.520_l.vhCDR3,

CHA.7.520_l.vlCDRl, CHA.7.520_l.vlCDR2 and CHA.7.520_l.vlCDR3.

CHA.7.520_2.vhCDRl, CHA.7.520_2.vhCDR2, CHA.7.520_2.vhCDR3,

CHA.7.520_2.vlCDRl, CHA.7.520_2.vlCDR2 and CHA.7.520_2.vlCDR3.

CHA.7.522.vhCDRl, CHA.7.522.vhCDR2, CHA.7.522.vhCDR3, CHA.7.522.vlCDRl, CHA.7.522.vlCDR2 and CHA.7.522.vlCDR3.

- 48 044486

CHA.7.524.vhCDRl,

CHA.7.524.vhCDR2, CHA.7.524.vhCDR3,

CHA.7.524.vlCDRl, CHA.7.524.vlCDR2 and CHA.7.524.vlCDR3

CHA.7.526.vhCDRl, CHA.7.526.vhCDR2, CHA.7.526.vhCDR3, CHA.7.526.vlCDRl,

CHA.7.526.V1CDR2 and CHA.7.526.vlCDR3.

CHA.7.527.vhCDRl, CHA.7.527.vhCDR2, CHA.7.527.vhCDR3, CHA.7.527.vlCDRl, CHA.7.527.V1CDR2 and CHA.7.527.vlCDR3.

CHA.7.528.vhCDRl, CHA.7.528.vhCDR2, CHA.7.528.vhCDR3, CHA.7.528.vlCDRl, CHA.7.528.V1CDR2 and CHA.7.528.vlCDR3.

CHA.7.530.vhCDRl, CHA.7.530.vhCDR2, CHA.7.530.vhCDR3, CHA.7.530.vlCDRl,

CHA.7.530.V1CDR2 and CHA.7.530.vlCDR3.

CHA.7.534.vhCDRl, CHA.7.534.vhCDR2, CHA.7.534.vhCDR3, CHA.7.534.vlCDRl,

CHA.7.534.V1CDR2 and CHA.7.534.vlCDR3.

CHA.7.535.vhCDRl, CHA.7.535.vhCDR2, CHA.7.535.vhCDR3, CHA.7.535.vlCDRl,

CHA.7.535.V1CDR2 and CHA.7.535.vlCDR3.

CHA.7.537.vhCDRl, CHA.7.537.vhCDR2,

CHA.7.537.vhCDR3, CHA.7.537.vlCDRl,

CHA.7.537.V1CDR2 and CHA.7.537.vlCDR3

CHA.7.538_l.vhCDRl, CHA.7.538_l.vhCDR2, CHA.7.538_l.vhCDR3,

CHA.7.538_l.vlCDRl, CHA.7.538_l.vlCDR2 and CHA.7.538_l.vlCDR3.

CHA.7.538_2.vhCDRl, CHA.7.538_2.vhCDR2, CHA.7.538_2.vhCDR3,

CHA.7.538 2.V1CDR1, CHA.7.538_2.vlCDR2 and CHA.7.538_2.vlCDR3.

CHA.7.543.vhCDRl, CHA.7.543.vhCDR2, CHA.7.543.vhCDR3, CHA.7.543.vlCDRl, CHA.7.543.V1CDR2 and CHA.7.543.vlCDR3.

CHA.7.544.vhCDRl, CHA.7.544.vhCDR2, CHA.7.544.vhCDR3, CHA.7.544.vlCDRl, CHA.7.544.V1CDR2 and CHA.7.544.vlCDR3.

CHA.7.545.vhCDRl, CHA.7.545.vhCDR2, CHA.7.545.vhCDR3, CHA.7.545.vlCDRl, CHA.7.545.V1CDR2 and CHA.7.545.vlCDR3.

CHA.7.546.vhCDRl, CHA.7.546.vhCDR2, CHA.7.546.vhCDR3, CHA.7.546.vlCDRl, CHA.7.546.V1CDR2 and CHA.7.546.vlCDR3.

CHA.7.547.vhCDRl, CHA.7.547.vhCDR2, CHA.7.547.vhCDR3, CHA.7.547.vlCDRl, CHA.7.547.V1CDR2 and CHA.7.547.vlCDR3.

CHA.7.548.vhCDRl, CHA.7.548.vhCDR2, CHA.7.548.vhCDR3, CHA.7.548.vlCDRl, CHA.7.548.V1CDR2 and CHA.7.548.vlCDR3.

CHA.7.549.vhCDRl, CHA.7.549.vhCDR2, CHA.7.549.vhCDR3, CHA.7.549.vlCDRl, CHA.7.549.V1CDR2 and CHA.7.549.vlCDR3.

CHA.7.550.vhCDRl, CHA.7.550.vhCDR2, CHA.7.550.vhCDR3, CHA.7.550.vlCDRl, CHA.7.550.V1CDR2 and CHA.7.550.vlCDR3.

As noted above, these sets of CDRs may also be amino acid variants as described above.

In addition, the framework regions of the variable heavy and variable light chains can be humanized as is known in the art (random variants are generated in the CDR if necessary), and thus humanized variants of the VH and VL chains shown in FIG. 63. In addition, the humanized heavy and light chain variable domains can then be fused to human constant regions, such as the constant regions from IgG1, IgG2, IgG3 and IgG4.

In particular, mouse VH and VL chains can be humanized as known in the art, for example, using the IgBLAST program on the NCBI website, as described in Ye et al. Nucleic Acids Res. 41: W34-W40 (2013), incorporated herein by reference in its entirety for humanization methods. IgBLAST analyzes the mouse VH and/or mouse VL sequence and compares it to a library of known human germline sequences. As demonstrated herein, for the humanized sequences generated in this invention, human IMGT VH genes (F+ORF, 273 germline sequences) and VL kappa genes were used as databases

- 49 044486 human IMGT (F+ORF, 74 germline sequences). The typical five CHA sequences were selected: CHA.7.518, CHA.7.530, CHA.7.538_1, CHA.7.538_2 and CHA.7.524 (see FIG. 5 and FIG. 63 for VH and VL sequences). For this humanization option, human germline IGHV1-46 (allele 1) was selected for all 5 as the acceptor sequence and heavy chain attachment region of human IGHJ4 (allele 1) (J gene). For three of the four (CHA.7.518, CHA.7.530, CHA.7.538_1 and CHA.7.5382), human germline IGKV1-39 (allele 1) was selected as the acceptor sequence and human IGKJ2 (allele 1) light chain (J gene ). The J gene was selected from human attachment region sequences collected in IMGT®, the international information system ImMunoGeneTics, at www.imgt.org. CDRs were defined according to the AbM definition (see www.bioinfo.org.uk/abs/). Fig. 63 also illustrates humanized sequences as well as some potential changes to optimize binding to PVRIG. Anti-PVRIG antibodies for use in this invention may contain any of these humanized anti-PVRIG antibody or antigen binding domain sequences.

Specific humanized anti-CHA antibodies include, for example, those demonstrated in FIG. 5 and fig. 63. Anti-PVRIG antibodies for use in the present invention may contain PVRIG CHA antibody sequences, as demonstrated in FIG. 5 and fig. 63. As will be appreciated by those skilled in the art, each of the humanized variable heavy chain (HH) and variable light chain (Humanized Light) sequences can be combined with human IgG1, IgG2, IgG3 and IgG4 constant regions. That is, CHA.7.518.HH1 is the first humanized variable heavy chain, and CHA.7.518.HH1.1 is a full-length heavy chain containing a humanized HH1 sequence with an IgG 1 constant region (CHA.7.518.HH1.2 is CHA. 7.518.НН1 with IgG2, etc.).

In some embodiments of the present invention, anti-PVRIG antibodies for use in the present invention include anti-PVRIG antibodies, and the V _H and V _L sequences of different anti-PVRIG antibodies can be mixed and matched to create other anti-PVRIG antibodies. The binding of PVRIG to such mixed and matched antibodies can be tested using the binding assays described above, such as ELISA). In some embodiments of the present invention, when the V _H and V _L chains are mixed and matched, the V _H sequence from a particular V _H /V _L pairing is replaced with a structurally similar V _H sequence. Likewise, in some embodiments of the present invention, the V _L sequence from a particular V _H /V _L pairing is replaced with a structurally similar V _L sequence. For example, the V _H and V L sequences of homologous antibodies are particularly suitable for mixing and matching. An anti-PVRIG antibody for use in this invention may contain the PVRIG V _H and V _L sequences from different anti-PVRIG antibodies that have been mixed and matched.

Accordingly, the antibodies of this invention may contain CDR amino acid sequences selected from the group consisting of (a) the sequences listed herein; (b) sequences that differ from the CDR amino acid sequences specified in (a) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; (c) amino acid sequences having 90% or more, 95% or more, 98% or more, or 99% or more identity with the sequences specified in (a) or (b); (d) a polypeptide having an amino acid sequence encoded by a polynucleotide having a nucleic acid sequence encoding amino acids as defined herein. An anti-PVRIG antibody for use in the present invention may contain CDR sequences of a variant of PVRIG.

In addition, the definition of anti-PVRIG antibodies includes antibodies that are identical to the anti-PVRIG antibodies listed herein. That is, in certain embodiments of the present invention, the anti-PVRIG antibody of the present invention contains heavy and light chain variable regions containing amino acid sequences that are homologous to the isolated anti-PVRIG amino acid sequences of the preferred anti-PVRIG immune molecules, respectively, wherein said antibodies retain the desired functional properties of parental anti-PVRIG antibodies. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology = number of identical positions/total number of positions X 100), taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below. Anti-PVRIG antibodies for use in this invention may contain heavy and light chain variable regions containing amino acid sequences that are homologous to the isolated anti-PVRIG amino acid sequences as described herein.

The percentage identity between two amino acid sequences can be determined with

- 50 044486 using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which is included in the ALIGN program (version 2.0), using the table of weights of RAM120 residues, gap length 12 and the gap penalty in sequence 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444453 (1970)), which is included in the GAP program in the software package GCG (commercially available), using either a Blossum 62 or a PAM250 matrix, gap weight of 16, 14, 12, 10, 8, 6 or 4, and gap extension penalty of 1, 2, 3, 4, 5 or 6 .

In general, the percent identity for comparison between anti-PVRIG antibodies is at least 75%, at least 80%, at least 90%, with a percent identity of at least about 95, 96, 97, 98, or 99% preferred. . The percentage identity may be along the entire amino acid sequence, for example, the entire heavy or light chain or along a portion of the chains. For example, the definitions of anti-PVRIG antibodies of the present invention include antibodies that have identity along the entire variable region (eg, when the identity is 95% or 98% identity along the variable regions), or along the entire constant region, or along only the Fc region .

F. Antibodies to TIGIT with antitumor antibodies.

In some embodiments of the present invention, the anti-TIGIT antibodies of the present invention are co-administered with antibodies that, unlike immuno-oncology/checkpoint inhibitors that typically target the immune system to enhance the patient's native immune response, are instead directed against a specific tumor antigen -targets (TTA). There are a large number of anti-TTA antibodies that are either approved or in clinical development that can be combined with the anti-TIGIT antibodies of the present invention. Currently approved antibodies include, but are not limited to, cetuximab, panitumumab, nimotuzumab (all anti-EGFR antibodies), rituximab (CD20), trastuzumab and pertuzumab (HER2), alemtuzumab (CD52), bevacizumab (VEGF), ofatumumab (CD20) , denosumab (RANK ligand), brentuximab (CD30), daratumumab (CD38), ibritumomab (CD20) and ipilimumab (CTLA-4). Specific targeted oncology antibodies in clinical trials that can be combined with anti-TIGIT antibodies include, but are not limited to, anti-CTLA4 mAbs such as ipilimumab, tremelimumab (see, for example, US Patent Publication No. 2017/0306025); anti-PD-1, such as nivolumab BMS-936558/ MDX-1106/ONO-4538, AMP224, CT-011, MK-3475, anti-PD-L1 antagonists, such as atezolizumab (IMpowerl33), BMS-936559/ MDX-1105 , MEDI4736, RG7446/MPDL3280A, as well as antibodies described in US Patent Publication No. 2017/0281764); antiLAG-3 such as IMP-321, anti-TIM-3, anti-BTLA, anti-B7-H4, anti-B7-I3, anti-VISTA; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs such as CP870,893, lucatumumab, dacetuzumab; anti-CD137 mAb such as urelumab BMS-663513 (anti-4-1BB; see, for example, US Pat. Nos. 7,288,638 and 8,962,804, incorporated herein by reference in their entirety); PF-05082566 utomilumab (see, for example, US Patent Nos. 8821867; 8337850; and 9468678, as well as International Patent Application Publication No. W0 2012/032433, incorporated herein by reference in its entirety); anti-OX40 mAb, such as anti-OX40 (see, for example, WO 2006/029879 or WO 2010096418, incorporated herein by reference in their entirety); anti-GITR mAb such as TRX518 (see, for example, US Pat. No. 7,812,135, incorporated herein by reference in its entirety); anti-CD27 mAb such as varlilumab CDX-1127 (see, for example, WO 2016/145085 and US Patent Publications No. US 2011/0274685 and US 2012/0213771, incorporated herein by reference in their entirety); anti-ICOS mAbs (e.g., MEDI-570, JTX-2011, and anti-TIM3 antibodies (see, e.g., WO 2013/006490 or US Patent Publication No. US 2016/0257758, incorporated herein by reference in their entirety), as well as monoclonal antibodies to antigens for prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, including small cell lung cancer, kidney cancer, colorectal cancer, pancreatic cancer, stomach cancer, brain cancer ( see generally www.clinicaltrials.gov).

G. Anti-PD-1 specific antibodies.

In another embodiment, the present invention provides combinations of anti-TIGIT antibodies of the present invention and anti-PD-1 antibodies. There are two approved anti-PD-1 antibodies, pembrolizumab (Keytruda®; MK-3475-033), cemiplimab (REGN2810; see US 20170174779), and nivolumab (Opdivo®; CheckMate078) and many others. are in clinical trials that can be used in combination with the anti-TIGIT antibodies of this invention. In other options for the implementation of this invention, Anti-PD-1 anti-PD-1 may include, for example, SHR-1210 (CTR20160175 and CTR20170090), ShR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31, BGB-A31 7 (CTR20160872) and/or anti-PD-1 antibody as described in US Patent Publication No. 2017/0081409. Representative anti-PD-1 antibody sequences are shown in FIG. 7, and any of them can be used in combination therapy methods described

- 51 044486 listed in this document.

In some embodiments of the present invention, the anti-TIGIT antibodies of the present invention are combined with anti-PVRIG antibodies, as described herein, as well as αntu-PD-1 antibodies, as described herein, or with other anti-PD-1 antibodies. 1, known in the art, in the form of a three-component combination therapy.

H. Specific anti-PD-L1 antibodies.

In another embodiment, the present invention provides combinations of anti-TIGIT antibodies of the present invention and anti-PD-L1 antibodies. Three approved αntu-PD-L1 antibodies are in clinical development: atezolizumab (TECENTRIQ®; MPDL3280A), avelumab (BAVENCIO®; MSB001071 8C), and durvalumab (MEDI4736), as well as other anti-PD antibodies -L1. Numerous anti-PD-1 antibodies are available and many others are in clinical development that can be used in combination with the anti-TIGIT antibodies of this invention. In embodiments of the present invention, the anti-PD-L1 antibody is described in US Patent Publication No. 2017/0281764, as well as International Patent Publication No. WO 2013/079174 (avelumab) and WO 2010/077634 (or US Patent Application No. 20160222117 or US Patent No. 8217149; atezolizumab). In some embodiments of the present invention, the anti-PD-L1 antibody comprises the heavy chain sequence of SEQ ID NO: 34 and the light chain sequence of SEQ ID NO: 36 (from US 2017/281764). In some embodiments of the present invention, the anti-PD-L1 antibody is atezolizumab (TECENTRIQ®; MPDL3280A; IMpower110). In some embodiments of the present invention, the anti-PD-L1 antibody is avelumab (BAVENCIO®; MSB001071 8C). In some embodiments of the present invention, the anti-PD-L1 antibody is durvalumab (MEDI4736). In some embodiments of the present invention, the anti-PD-L1 antibody includes, for example, atezolizumab (IMpower133), BMS-936559/MDX-1105 and/or RG-7446/MPDL3280A and/or YW243.55.S70, as well as any of the antibodies which are presented herein in FIGS. 62.

In some embodiments of the present invention, the anti-TIGIT antibodies of the present invention are combined with anti-PVRIG antibodies, as described herein, as well as αntu-PD-L1 antibodies, as described herein, or with other anti-PD-L1 antibodies. L1, known in the art, in the form of a three-component combination therapy.

I. Optional antibody construction.

Antibodies of this invention can be modified or engineered to alter amino acid sequences by amino acid substitutions. As discussed herein, amino acid substitutions can be made to change the affinity of the CDR for an antigen (including both increased and decreased binding), as well as to change additional functional properties of the antibodies. For example, antibodies can be engineered to include modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibody according to at least some embodiments of the present invention can be chemically modified (e.g., one or more chemical groups can be attached to the antibody) or can be modified to change its glycosylation, again to change one or more functional properties antibodies. Such embodiments of the present invention are described below. The numbering of residues in the Fc region corresponds to the EU index according to Kabat.

In one embodiment of the present invention, the C _H1 hinge region is modified such that the number of cysteine residues in the hinge region is changed, for example, increased or decreased. This approach is further described in US Pat. No. 5,677,425 to Bodmer et al. The number of cysteine residues in the CH1 hinge region is varied, for example, to facilitate the assembly of light and heavy chains or to increase or decrease the stability of the antibody.

In yet another embodiment of the present invention, the antibody can be modified to abolish Fab arm turnover in vivo, particularly when IgG4 constant domains are used. Specifically, this process involves the exchange of IgG4 hemimolecules (one heavy chain plus one light chain) between other IgG4 antibodies, resulting in the efficient formation of antibodies that are functionally monovalent. Mutations in the hinge region and heavy chain constant domains can abolish this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105: 9-19). As stated herein, the mutation that finds particular use in this invention is S241P in the context of the IgG4 constant domain. IgG4 is useful in this invention because it does not have significant effector function and is thus used to block receptor-ligand binding without depleting cells.

In some embodiments of the present invention, amino acid substitutions can be made in the Fc region, primarily to alter binding to FcyR receptors. The term Fc gamma receptor, FcyR or Fc gamma R in the context of this description means any precursor

- 52 044486 member of a family of proteins that bind the Fc region of the IgG antibody and are encoded by the FcyR gene. In humans, this family includes, but is not limited to, FcyRI (CD64), including the FcyRIa, FcyRIb, and FcyRIc isoforms; FcyRII (CD32), including isoforms FcyRIIa (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-1 and FcyRIIb-2) and FcyRIIc; and FcyRIII (CD16), including isoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NA1 and FcyRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82: 57-65, which is included in its entirety herein by reference) as well as any unknown isoforms or allotypes of FcyRs or human FcyRs. FcyR can originate from any organism, including, but not limited to, humans, mice, rats, rabbits and monkeys. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII-1 (CD16), and FcyRIII-2 (CD16-2), as well as any unknown isoforms or allotypes of mouse FcyR or FcyR.

There are a number of useful Fc substitutions that can be made to alter binding to one or more FcyR receptors. Substitutions that result in increased binding as well as decreased binding may be useful. For example, it is known that increased binding to FcyRIIIa typically leads to increased ADCC (antibody-dependent cell-mediated cytotoxicity; a cell-mediated reaction in which nonspecific cytotoxic cells that express FcyR recognize bound antibody on a target cell and subsequently cause lysis of the target cell Likewise, in some cases, reducing binding to FcyRIIb (inhibitory receptor) may also be beneficial.Amino acid substitutions that find use in this invention include those listed in US Ser. No. 11/124620 (in particular, Fig. 41 ) and US Patent Publication No. 6,737,056, both of which are expressly incorporated herein by reference in their entirety and specifically to the embodiments described therein.

In other embodiments of the present invention, the Fc region is altered by replacing at least one amino acid residue with another amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with another amino acid residue such that the antibody has altered affinity for the effector ligand but retains the antigen-binding ability of the parent antibodies. The effector ligand for which the affinity is changed may be, for example, an Fc receptor or a complement component C1. This approach is described in more detail in US patents No. 5624821 and 5648260, the authors of which are Winter et al.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with another amino acid residue such that the antibody exhibits altered C1q binding and/or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in more detail by Idusogie et al. in US Patent No. 6194551.

In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described by Bodmer et al. in PCT publication WO 94/29351.

In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for the Fcy receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290 292, 293, 294, 295, 296, 29 8, 301 , 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382 , 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is further described by Presta in PCT publication WO 00/42072. In addition, the binding sites on human IgG1 for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been demonstrated to improve binding to FcyRIII. In addition, the following combinations of mutants have been demonstrated to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A, and S298A/E333A/K334A. In addition, mutations such as M252Y/S254T/T256E or M428L/N434S improve FcRn binding and increase the half-life of circulating antibodies (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301316).

In addition, the antibodies of this invention are modified to increase their biological half-life. There are different approaches. For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in Ward US Pat. No. 6,277,375. Alternatively, to increase the biological half-life, the antibody may be altered in the C _H1 or C _L region such that that it would contain a salvage receptor binding epitope derived from the two loops of the CH2 domain of the IgG Fc region, as described by Presta et al. in US Patent Nos. 5,869,046 and 6,121,022. Additional mutations to increase serum half-life are described in

- 53 044486 US patents No. 8883973, 6737056 and 7371826 and include 428L, 434A, 434S and 428L/434S.

In yet another embodiment of the present invention, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (ie, an antibody without glycosylation) can be produced. Glycosylation can be altered, for example, to increase antibody affinity for an antigen or decrease effector function such as ADCC. Such carbohydrate modifications can be accomplished, for example, by changing one or more glycosylation sites in the antibody sequence, for example N297. For example, one or more amino acid substitutions can be made that result in the removal of one or more glycosylation sites of the variable region backbone, thereby removing glycosylation at that site using an alanine substitution in some embodiments of the present invention.

In an additional or alternative embodiment, an antibody with an altered glycosylation pattern can be generated, such as a hypofucosylated antibody with a reduced number of fucosyl residues, or an antibody with an increased number of GlcNac bisection structures. These altered glycosylation characteristics have been demonstrated to enhance the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies according to at least some embodiments of the present invention are expressed, resulting in an antibody with altered glycosylation. For example, the Ms704, Ms705 and Ms709 cell lines lack the fucosyltransferase gene, FUT8 (α(1,6) fucosyltransferase), such that the antibodies expressed in the Ms704, Ms705 and Ms709 cell lines lack fucose on their carbohydrates. Fs8 cell lines Ms704, Ms705 and Ms709 are created by targeting the FUT8 gene in CHO/DG44 cells using two replacement vectors (see US Patent Publication No. 20040110704 Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87: 614-22). As another example, Hanai et al. EP 1176195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, whereby antibodies expressed in such a cell line exhibit hypofucosylation by reducing the activity or eliminating the α 1,6-linked enzyme. Hanai et al. also describe cell lines that have low enzymatic activity when fucose is added to N-acetylglucosamine, which binds to the Fc region of the antibody, or have no enzymatic activity, for example, the rat myeloma cell line YB2/0 (ATCC CRL 1662), In publication PCT WO 03/035835, Presta, describes a variant of the CHO cell line, Lec 13 cells, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also leads to hypofucosylation of antibodies expressed in this host cell (see also, Shields, R. L. et al (2002) J Biol Chem 277:26733-26740). PCT publication WO 99/54342, Umana et al., describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (eg, β(1,4)-T-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in engineered cell lines show an increase in the number of GlcNac bisection structures, which leads to an increase in ADCC antibody activity (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, the fucose residues of the antibody can be cleaved using the enzyme fucosidase. For example, fucosidase a-L-fucosidase removes fucosyl residues from antibodies (Tarentino, AL et al. (1975) Biochem. 14: 5516-23).

Another modification of the antibodies herein that is proposed in this invention is PEGylation or the addition of other water-soluble moieties, typically polymers, for example, to increase half-life. An antibody may be pegylated, for example, to increase the biological (eg, serum) half-life of a particular antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions where one or more PEG groups are attached to the antibody or antibody fragment. Preferably, PEGylation is carried out through an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol refers to any form of PEG that has been used to derivatize other proteins, such as mono(Ci-Cio)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments of the present invention, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to antibodies in accordance with at least some embodiments of the present invention. See, for example, publication EP 0 154 316, Nishimura et al., and publication EP 0401384, Ishikawa et al.

In addition to substitutions made to change the binding affinity to FcyRs and/or FcRn, and/or to increase serum half-life in vivo, additional

- 54 044486 new modifications of antibodies, as described in more detail below.

In some cases, affinity maturation is achieved. Amino acid modifications in CDRs are sometimes called affinity maturation. An affinity matured antibody is an antibody that has one or more changes in one or more CDRs that results in improved affinity of the antibody for an antigen compared to a parent antibody that does not have these changes. In some cases, it may be desirable to reduce the affinity of an antibody for its antigen.

In some embodiments of the present invention, one or more amino acid modifications are introduced into one or more CDRs of the antibodies of the present invention. Typically, only 1, 2, or 3 amino acid substitutions are made in any one CDR, and typically no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 changes are made in a set of CDRs. However, it should be understood that any combination of no substitutions, 1, 2 or 3 substitutions in any CDR can be independently and optionally combined with any other substitution.

Affinity maturation can be performed to increase the binding affinity of an antibody to an antigen from at least about 10% to 50-100-150% or more or 1 to 5 times that of the parent antibody. Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the antigen. Affinity matured antibodies are prepared using known methods. See, for example, Marks et al., 1992, Biotechnology 10: 779783, which describes affinity maturation using variable heavy chain (VH) and variable light chain (VL) domain shuffling. Random mutagenesis of CDR and/or framework residues is described in the publications: Barbas, et al. 1994, Proc. Nat. Acad. Sci USA 91:3809-3813; Shier et al. 1995 Gene 169:147155; Yelton et al., 1995, J. Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154 (7): 3310-9; and Hawkins et al., 1992, J. Mol. Biol. 226:889-896, for example.

Alternatively, amino acid modifications can be made to one or more CDRs of the antibodies of this invention that are silent, for example, that do not significantly change the affinity of the antibody for the antigen. This may be done for a number of reasons, including optimization of expression (as may be accomplished for nucleic acids encoding antibodies of the present invention).

Thus, CDR and antibody variants are included in the definition of CDRs and antibodies of this invention; that is, the antibodies of this invention may include amino acid modifications in one or more CDRs of the listed antibodies of this invention. In addition, as discussed below, amino acid modifications can also be independently and optionally carried out in any region outside the CDR, including framework and constant regions.

VI. Anti-TIGIT antibodies in combination therapy

The anti-TIGIT and anti-PVRIG antibodies of this invention find particular use in the treatment of cancer when used in combination with, for example, a checkpoint inhibitor such as an anti-PD-1 antibody as described herein. In general, the antibodies of the present invention are immunomodulatory because the ani-TIGIT and anti-PVRIG antibodies of the present invention, instead of directly attacking cancer cells, stimulate the immune system, generally inhibiting the action of TIGIT and PVRIG, respectively. Thus, unlike tumor-directed therapies, which rely on inhibiting molecular pathways critical for tumor growth and development and/or depleting tumor cells, cancer immunotherapy aims to stimulate the patient's own immune system to eliminate cancer cells, providing prolonged destruction of the tumor. Various approaches can be used for cancer immunotherapy, including therapeutic cancer vaccines to induce tumor-specific T-cell responses and immunostimulatory antibodies (ie, inhibitory receptor = immune checkpoint antagonists) to remove immunosuppressive pathways.

Clinical responses with targeted therapy or conventional cancer therapy tend to be short-lived as cancer cells develop resistance and tumor relapse occurs. However, the clinical application of anticancer immunotherapy in the past few years has shown that this therapy can be characterized by durable clinical responses, which has a significant impact on long-term survival. However, although responses are long-lasting, only a small number of patients respond to immunotherapy (unlike conventional or targeted therapy, where a large number of patients respond, but the responses are not sustained).

By the time a tumor is detected clinically, it has already bypassed the immune defense system, acquiring immunoresistant and immunosuppressive properties and creating an immunosuppressive tumor microenvironment through various mechanisms and a variety of immune cells.

Accordingly, combinations of anti-TIGIT and anti-PVRIG antibodies of the present invention are useful for the treatment of cancer. Due to the nature of the immuno-oncology mechanism of action, TIGIT and/or PVRIG are not necessarily overexpressed or correlated with a specific cancer type; that is, the goal is for anti-TIGIT antibodies to suppress the activation of T cells and NK cells so that the cancer is attacked by the immune system.

VII. Compositions of nucleic acids.

Also provided are nucleic acid compositions encoding the anti-TIGIT, anti-PVRIG and anti-PD-1 antibodies of the present invention, as well as expression vectors containing nucleic acids and host cells transformed with the nucleic acid compositions and/or expression vectors. As will be appreciated by those skilled in the art, the protein sequences described herein may be encoded by any number of possible nucleic acid sequences due to the degeneracy of the genetic code.

The nucleic acid compositions that encode antibodies will depend on the antibody format. Traditional tetrameric antibodies, containing two heavy chains and two light chains, are encoded by two different nucleic acids, one encoding the heavy chain and the other encoding the light chain. They can be placed in one expression vector or two expression vectors, as is known in the art, transformed into host cells where they are expressed to form the antibodies of this invention. In some embodiments of the present invention, for example, when scFv constructs are used, a single nucleic acid encoding a variable heavy chain-linker-variable light chain complex is typically used, which can be inserted into an expression vector for transformation into host cells. Nucleic acids can be introduced into expression vectors that contain appropriate transcriptional and translational control sequences, including, but not limited to, signal and secretory sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.

Preferred mammalian host cells for expression of recombinant antibodies in at least some embodiments of the present invention include Chinese hamster ovary cells (CHO cells), PER.C6, HEK293 and others, as known in the art.

The nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. A nucleic acid is isolated or isolated substantially pure if it is purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard methods including alkali/SDS treatment, CsCl separation, column chromatography, electrophoresis agarose gel and other well known methods in the art.

To create the scFv gene, the VH and VL encoding DNA fragments are operably linked to another flexible linker encoding fragment, for example encoding the amino acid sequence (Gly ₄ -Ser) ₃ , so that the V _H and V _L sequences can be expressed as a contiguous single strand protein with V _L and V H regions connected by a flexible linker (see, for example, Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 -5883; McCafferty et al., (1990) Nature 348:552-554).

VIII. Antibody compositions of this invention.

Therapeutic compositions used in the practice of the above methods may be formulated into pharmaceutical compositions containing a carrier suitable for the desired mode of delivery. Suitable carriers include any substance that, in combination with a therapeutic composition, preserves the antitumor function of the therapeutic composition and generally does not cause a response from the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers, such as sterile phosphate-buffered saline solutions, bacteriostatic water, and the like. (See generally Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations used and may include buffers.

In a preferred embodiment of the present invention, the pharmaceutical composition that contains the antibodies of the present invention may be in a water-soluble form, such as present in the form of pharmaceutically acceptable salts, which should include both an acid addition salt and a base addition salt. The term pharmaceutically acceptable acid addition salt refers to those salts which retain the biological effectiveness of the free bases and which are not biologically or otherwise undesirable formed with inorganic acids and the like. Pharmaceutically acceptable base addition salts include salts derived from inorganic bases and the like.

Administration of a pharmaceutical composition containing antibodies of this invention, preferably in the form of a sterile aqueous solution, can be accomplished by a variety of routes, including, but not limited to, subcutaneous and intravenous.

The dosage amounts and frequency of administration in a preferred embodiment of the present invention are selected to be therapeutically or prophylactically effective. As is known in the art, adjustments may need to be made for protein degradation, systemic and local delivery and rate of new protease synthesis, as well as age, weight, general health, gender,

- 56 044486 diet, time of administration, interaction with the drug and the severity of the pathological condition, while those skilled in the art can determine the above correction through routine experimentation.

A therapeutically effective dose of the Fc variant of the present invention may be administered to treat a patient. The term therapeutically effective dose as used herein means a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of treatment and will be determined by one skilled in the art using known techniques.

A. Combined formulations.

The antibodies of this invention (either as a three-part combination of anti-TIGIT, anti-PVRIG and anti-PD-1 antibodies, or as a two-part combination of anti-TIGIT and anti-PVRIG antibodies) can be produced using various methods, as known to those skilled in the art. . In some cases, the antibodies are administered simultaneously, for example, as separate infusions (eg, each IV bag contains one antibody) or as a single infusion of a mixture of antibodies. Alternatively, the antibodies can be administered sequentially, for example, over several hours or days.

In some cases, the antibodies are provided in an administration kit containing dosage units of each antibody, again packaged either separately in different dosage units or together as a mixture of antibodies in a single dosage unit.

IX. Combined methods of therapy and their use.

A. Methods of cancer therapy.

As used herein, the term cancer broadly refers to any tumor disease (invasive or metastatic) characterized by abnormal and uncontrolled cell division causing malignant growth or tumor development (eg, unregulated cell growth). The term cancer or cancerous as used herein should be understood to cover any neoplastic disease (whether invasive, non-invasive or metastatic) that is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor development, non-limiting examples of which are described herein. The term includes any physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer are given in the working examples and are also described herein.

Non-limiting examples of cancers that can be treated with anti-TIGIT antibodies, anti-PVRIG antibodies, and combinations of anti-TIGIT antibodies and other antibodies such as any of anti-TIGIT, anti-PVRIG, anti-PD-1 and/or or anti-PD-L1 antibodies are provided herein. Such cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of such cancers include squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung cancer), peritoneal cancer, hepatocellular cancer, stomach cancer (including gastrointestinal cancer tract), esophageal cancer, melanoma, mesothelioma, Merkel cell cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterus, salivary gland carcinoma, kidney cancer, prostate cancer, vaginal cancer, thyroid cancer, liver carcinoma and various types of head and neck cancer, laryngeal cancer, oral cancer, urothelial cancer, KRAS-mutant tumors, myelodysplastic syndromes (MDS ), as well as B-cell malignancies, B-cell lymphoma (including low-grade/follicular non-Hodgkin lymphoma (NHL), small cell lymphocytic lymphoma (SCL) NHL, moderately differentiated/follicular NHL, moderately differentiated diffuse NHL, well-differentiated immunoblastic NHL, well-differentiated lymphoblastic NHL, highly differentiated NHL from small non-split cells, NHL with massive damage to the lymph nodes, mantle cell lymphoma; AIDS-associated lymphoma and Waldenström's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; Adult T-cell leukemia/lymphoma; myeloma; multiple myeloma and post-transplant lymphoproliferative disorder (PTLD), lymphoid malignancies, abnormal vascular proliferation associated with phakomatoses, edema (eg, associated with brain tumors) and Meigs syndrome, rectal cancer, renal cell carcinoma, soft tissue sarcoma, sarcoma Kaposi, ovarian carcinoid carcinoma in early or late stages (including metastatic). Cancers treatable by the present invention include cancers that do or do not express TIGIT, PVRIG, PVRL, PD-1 and/or PD-L1, and further include non-metastatic or non-invasive, as well as invasive or metastatic cancers. diseases in which expression of TIGIT, PVRIG, PVRL, PD-1 and/or PD-L1 by immune, stromal or disease cells suppresses anti-tumor and anti-invasive immune responses. The method of this invention is particularly suitable for the treatment of vascular diseases

- 57 044486 bath tumors. In some embodiments of the present invention, the cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, cell carcinoma Merkel, MSI-high cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS). In some embodiments of the method, the cancer is selected from the group consisting of triple negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merkel cell cancer, high MSI cancer, KRAS- mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

The term cancer therapy as used herein refers to any method that prevents or treats cancer or alleviates one or more symptoms of cancer. Typically, such treatments involve administering anti-TIGIT and anti-PVRIG immunostimulatory antibodies (including antigen-binding moieties) in combination with chemotherapy or radiation therapy or other biological agents and to enhance their activity, that is, in individuals who express TIGIT and/or PVRIG suppresses antitumor responses and the effectiveness of chemotherapy or radiation therapy, or biological effectiveness.

The present invention provides combination treatment methods and the use of anti-TIGIT antibodies and anti-PVRIG antibodies, sometimes with the addition of anti-PD-1 antibodies, for triple combination therapy. This invention provides methods for combination treatment and use of anti-TIGIT antibodies and anti-PVRIG antibodies, sometimes with the addition of anti-PD-LL antibodies. Any of the anti-PVRIG antibodies listed above or in the figures can be used for triple combination therapy. Any of the anti-TIGIT antibodies listed above or in the figures can be used for triple combination therapy. Any of the anti-PD-1 antibodies listed above or in the figures can be used for triple combination therapy. Any of the anti-PD-L1 antibodies listed above or in the figures can be used for triple combination therapy. In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3. In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63. In some embodiments of the present invention, the anti-PD-1 antibody is an antibody selected from any of the anti-PD-1 antibodies described herein, including any of the antibodies described in FIG. 7.

In some embodiments of the present invention, the anti-PD-1 antibody is selected from pembrolizumab (Keytruda®; MK-3475-033), nivolumab (Opdivo®; CheckMate078), cemplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872) and/or anti-PD-1 antibodies as specified in US Patent Publication No. 2017/0 081409 .

In some embodiments of the present invention, the anti-PD-L1 antibody is selected from the antibodies described in US Patent Publication No. 2017/0281764, as well as in International Patent Publication No. WO 2013/079174 (avelumab) and WO 2010/077634 (or in the application US Patent No. 20160222117 or US Patent No. 8217149; atezolizumab). In some embodiments of the present invention, the anti-PD-L1 antibody comprises the heavy chain sequence of SEQ ID NO: 34 and the light chain sequence of SEQ ID NO: 36 (from US 2017/281764). In some embodiments of the present invention, the anti-PD-L1 antibody is atezolizumab (TECENTRIQ®; MPDL3280A; IMpower110). In some embodiments of the present invention, the anti-PD-L1 antibody is avelumab (BAVENCIO®; MSB001071 8C). In some embodiments of the present invention, the anti-PD-L1 antibody is durvalumab (MEDI4736). In some embodiments of the present invention, the anti-PD-L1 antibody includes, for example, atezolizumab (IMpower133), BMS-936559/MDX-1105 and/or RG-7446/MPDL3280A and/or YW243.55.S70, as well as any of the antibodies which are presented herein in FIGS. 62.

In some embodiments of the present invention, the anti-PVRIG antibody is selected from the antibody whose sequences are shown in FIG. 5 and/or in FIG. 63:

- 58 044486

CPA.7.001, CPA.7.001.VH, CPA.7.001.VL, CPA.7.001.HC, CPA.7.001.LC and CPA.7.001.H1, CPA.7.001.H2, CPA.7.001.H3, CPA.7.001. H4; CPA.7.001.vhCDRl,

CPA.7.001.vhCDR2, CPA.7.001.vhCDR3, CPA.7.001.vlCDRl, CPA.7.001.vlCDR2 and CPA.7.001.vlCDR3;

CPA.7.003, CPA.7.003.VH, CPA.7.003.VL, CPA.7.003.HC, CPA.7.003.LC,

CPA.7.003.H1, CPA.7.003.H2, CPA.7.003 H3, CPA.7.003.H4; CPA.7.003.vhCDRl,

CPA.7.003.vhCDR2, CPA.7.003.vhCDR3, CPA.7.003.vlCDRl, CPA.7.003,vlCDR2 and

CPA.7.003.vlCDR3; CPA.7.004, CPA.7.004.VH, CPA.7.004.VL, CPA.7.004.HC, CPA.7.004.LC,

CPA.7.004.H1, CPA.7.004.H2, CPA.7.004.H3 CPA.7.004.H4; CPA.7.004.vhCDRl,

CPA. 7.004. vhCDR2, CPA.7.004.vhCDR3, CPA.7.004.vlCDRl, CPA.7.004.vlCDR2 and CPA. 7.004. vlCDR3; CPA.7.006, CPA.7.006.VH, CPA.7.006.VL, CPA.7.006.HC, CPA.7.006.LC,

CPA.7.006.H1, CPA.7.006.H2, CPA.7.006.H3 CPA.7.006.H4; CPA.7.006.vhCDRl,

CPA. 7.006. vhCDR2, CPA.7.006.vhCDR3, CPA.7.006.vlCDRl, CPA.7.006.vlCDR2 and CPA.7.006.V1CDR3; CPA.7.008, CPA.7.008.VH, CPA.7.008.VL, CPA.7.008.HC, CPA.7.008.LC,

CPA.7.008.H1, CPA.7.008.H2, CPA.7.008.H3 CPA.7.008.H4; CPA.7.008.vhCDRl,

CPA.7.008.vhCDR2, CPA.7.008.vhCDR3, CPA.7.008.vlCDRl, CPA.7.008.vlCDR2 and CPA.7.008.V1CDR3; CPA.7.009, CPA.7.009.VH, CPA.7.009.VL, CPA.7.009.HC, CPA.7.009.LC,

CPA.7.009.H1, CPA.7.009.H2, CPA.7.009.H3 CPA.7.009.H4; CPA.7.009.vhCDRl,

CPA.7.009.vhCDR2, CPA.7.009.vhCDR3, CPA.7.009.vlCDRl, CPA.7.009.vlCDR2 and CPA.7.009.V1CDR3; CPA.7.010, CPA.7.010.VH, CPA.7.010.VL, CPA.7.010.HC, CPA.7.010.LC,

CPA.7.010.H1, CPA.7.010.H2, CPA.7.010.H3 CPA.7.010.H4; CPA.7.010.vhCDRl,

CPA.7.010.vhCDR2, CPA.7.010.vhCDR3, CPA.7.010.vlCDRl, CPA.7.010.vlCDR2 and CPA.7.010.vlCDR3;

CPA.7.011, CPA.7.011.VH, CPA.7.011.VL, CPA.7.011.HC, CPA.7.011.LC,

CPA.7.011.Hl, CPA.7.011.H2, CPA.7.011.H3 CPA.7.011.H4; CPA.7.01 l.vhCDRl, CPA.7.011. vhCDR2, CPA.7.01 l.vhCDR3, CPA.7.011.vlCDRl, CPA.7.01 l.vlCDR2 and CPA.7.011.V1CDR3;

CPA.7.012, CPA.7.012.VH, CPA.7.012.VL, CPA.7.012.HC, CPA.7.012.LC,

CPA.7.012.H1, CPA.7.012.H2, CPA.7.012.H3 CPA.7.012.H4; CPA.7.012.vhCDRl,

CPA.7.012.vhCDR2, CPA.7.012.vhCDR3, CPA.7.012.vlCDRl, CPA.7.012.vlCDR2 and CPA.7.012.V1CDR3;

CPA.7.013, CPA.7.013.VH, CPA.7.013.VL, CPA.7.013.HC, CPA.7.013.LC,

CPA.7.013.H1, CPA.7.013.H2, CPA.7.013.H3 CPA.7.013.H4; CPA.7.013.vhCDRl,

CPA.7.013.vhCDR2, CPA.7.013.vhCDR3, CPA.7.013.vlCDRl, CPA.7.013.vlCDR2 and CPA.7.013.V1CDR3;

- 59 044486

CPA.7.014, CPA.7.014.VH, CPA.7.014.VL, CPA.7.014.HC, CPA.7.014.LC,

CPA.7.014.Hl, CPA.7.014.H2, CPA.7.014.H3 CPA.7.014.H4; CPA.7.014.vhCDRl, CPA.7.014.vhCDR2, CPA.7.014.vhCDR3, CPA.7.014.vlCDRl, CPA.7.014.vlCDR2 and CPA.7.014.vlCDR3;

CPA.7.015, CPA.7.015.VH, CPA.7.015.VL, CPA.7.015.HC, CPA.7.015.LC,

CPA.7.015.H1, CPA.7.015.H2, CPA.7.015.H3 CPA.7.015.H4; CPA.7.015.vhCDRl,

CPA.7.015.vhCDR2, CPA.7.015.vhCDR3, CPA.7.015.vlCDRl, CPA.7.015.vlCDR2 and CPA.7.015.vlCDR3;

CPA.7.017, CPA.7.017.VH, CPA.7.017.VL, CPA.7.017.HC, CPA.7.017.LC,

CPA.7.017H1, CPA.7.017.H2, CPA.7.017.H3 CPA.7.017.H4; CPA.7.017.vhCDRl,

CPA.7.000171.vhCDR2, CPA.7.017.vhCDR3, CPA.7.017.vlCDRl,

CPA.7.017.V1CDR2 and CPA.7.017.vlCDR3;

CPA.7.018, CPA. 7.018. VH, CPA.7.018.VL, CPA.7.018.HC, CPA.7.018.LC, CPA.7.018.Hl, CPA.7.018.H2, CPA.7.018.H3 CPA.7.018.H4; CPA.7.017.vhCDRl,

CPA.7.017.vhCDR2, CPA.7.017.vhCDR3, CPA.7.017.vlCDRl, CPA.7.017.vlCDR2 and CPA.7.017.vlCDR3;

CPA.7.019, CPA.7.019.VH, CPA.7.019.VL, CPA.7.019.HC, CPA.7.019.LC,

CPA.7.019.Hl, CPA.7.019.H2, CPA.7.019.H3 CPA.7.019.H4; CPA.7.019.vhCDRl,

CPA.7.019.vhCDR2, CPA.7.019.vhCDR3, CPA.7.019.vlCDRl, CPA.7.019.vlCDR2 and CPA.7.019.vlCDR3;

CPA.7.021, CPA.7.021.VH, CPA.7.021.VL, CPA.7.021.HC, CPA.7.021.LC, CPA.7.021.Hl, CPA.7.021.H2, CPA.7.021.H3 CPA.7.021. H4; CPA.7.021. vhCDRl, CPA.7.021. vhCDR2, CPA.7.021.vhCDR3, CPA.7.021.vlCDRl, CPA.7.021.vlCDR2 and CPA.7.021.vlCDR3;

CPA.7.022, CPA. 7.022. VH, CPA.7.022.VL, CPA.7.022.HC, CPA.7.022.LC, CPA.7.022.H1, CPA.7.022.H2, CPA.7.022.H3 CPA.7.022.H4; CPA.7.022.vhCDRl,

CPA.7.022.vhCDR2, CPA.7.002201.vhCDR3, CPA.7.022.vlCDRl, CPA.7.022.vlCDR2 and CPA.7.022.vlCDR3;

CPA.7.023, CPA. 7.023. VH, CPA.7.023.VL, CPA.7.023.HC, CPA.7.023 LC,

CPA.7.023.Hl, CPA.7.023.H2, CPA.7.023.H3 CPA.7.023.H4; CPA.7.023 vhCDRl,

CPA.7.023. vhCDR2, CPA.7.023 vhCDR3, CPA.7.023 vlCDRl, CPA.7.023 vlCDR2 and CPA.7.023.vlCDR3;

CPA.7.024, CPA.7.024.VH, CPA.7.024.VL, CPA.7.024.HC, CPA.7.024.LC,

CPA.7.024.H1, CPA.7.024.H2, CPA.7.024.H3 CPA.7.024.H4; CPA.7.024.vhCDRl,

CPA.7.024.vhCDR2, CPA.7.024.vhCDR3, CPA.7.024.vlCDRl, CPA.7.024.vlCDR2 and CPA.7.024. V1CDR3;

CPA.7.033, CPA. 7.033. VH, CPA.7.033.VL, CPA.7.033.HC, CPA.7.033.LC, CPA.7.033.H1, CPA.7.033.H2, CPA.7.033.H3 CPA.7.033.H4; CPA.7.033.vhCDRl,

CPA.7.033. vhCDR2, CPA.7.033.vhCDR3, CPA.7.033.vlCDRl, CPA.7.033.vlCDR2 and CPA.7.033.vlCDR3;

CPA.7.034, CPA.7.034.VH, CPA.7.034.VL, CPA.7.034.HC, CPA.7.034.LC,

CPA.7.034.H1, CPA.7.034.H2, CPA.7.034.H3 CPA.7.034.H4; CPA.7.034.vhCDRl,

CPA.7.034.vhCDR2, CPA.7.034.vhCDR3, CPA.7.034.vlCDRl, CPA.7.034.vlCDR2 and CPA.7.034.vlCDR3;

CPA.7.036, CPA.7.036.VH, CPA.7.036.VL, CPA.7.036.HC, CPA.7.036.LC,

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CPA.7.036.H1, CPA.7.036.H2, CPA.7.036.H3 CPA.7.036.H4;

CPA.7.036.vhCDRl, CPA.7.036.vhCDR2, CPA.7.036.vhCDR3, CPA.7.036.vlCDRl,

CPA.7.036.V1CDR2 and CPA.7.036.vlCDR3;

CPA.7.040, CPA.7.040.VH, CPA.7.040.VL, CPA.7.040.HC, CPA.7.040.LC,

CPA.7.040.H1, CPA.7.040.H2, CPA.7.040.H3 and CPA.7.040. H4; CPA.7.040.vhCDRl, CPA.7.040.vhCDR2, CPA.7.040.vhCDR3, CPA.7.040.vlCDRl, CPA.7.040.vlCDR2 and CPA.7.040.V1CDR3;

CPA.7.046, CPA.7.046.VH, CPA.7.046.VL, CPA.7.046.HC, CPA.7.046.LC,

CPA.7.046.H1, CPA.7.046.H2, CPA.7.046.H3 CPA.7.046.H4; CPA.7.046.vhCDRl,

CPA.7.046.vhCDR2, CPA.7.046.vhCDR3, CPA.7.046.vlCDRl, CPA.7.046.vlCDR2 and CPA.7.046.V1CDR3;

CPA.7.047, CPA.7.047.VH, CPA.7.047.VL, CPA.7.047.HC, CPA.7.047.LC,

CPA.7.047.H1, CPA.7.047.H2, CPA.7.047.H3 CPA.7.047.H4; CPA.7.047.vhCDRl,

CPA.7.047.vhCDR2, CPA.7.047.vhCDR3, CPA.7.047.vlCDRl, CPA.7.004701.vlCDR2 and CPA.7.047.V1CDR3;

CPA.7.049, CPA.7.049.VH, CPA.7.049.VL, CPA.7.049.HC, CPA.7.049.LC,

CPA.7.049.H1, CPA.7.049.H2, CPA.7.049.H3 CPA.7.049.H4; CPA.7.049.vhCDRl,

CPA.7.049.vhCDR2, CPA.7.049.vhCDR3, CPA.7.049.vlCDRl, CPA.7.049.vlCDR2 and

CPA.7.049.vlCDR3; and

CPA.7.050, CPA.7.050.VH, CPA.7.050.VL, CPA.7.050.HC, CPA.7.050.LC,

CPA.7.050.H1, CPA.7.050.H2, CPA.7.050.H3 CPA.7.050.H4, CPA.7.050.vhCDRl,

CPA.7.050.vhCDR2, CPA.7.050.vhCDR3, CPA.7.050.vlCDRl, CPA.7.050.vlCDR2 and CPA.7.050.V1CDR3.

CPA.7.028, CPA.7.028.VH, CPA.7.028.VL, CPA.7.028.HC, CPA.7.028.LC,

CPA.7.028.H1, CPA.7.028.H2, CPA.7.028.H3 and CPA.7.028. H4; CPA.7.028.vhCDRl, CPA.7.028.vhCDR2, CPA.7.028.vhCDR3, CPA.7.028.vlCDRl, CPA.7.028.vlCDR2 and CPA.7.028.V1CDR3.

CPA.7.030, CPA.7.030.VH, CPA.7.030.VL, CPA.7.030.HC, CPA.7.030.LC,

CPA.7.030.H1, CPA.7.030.H2, CPA.7.030.H3 and CPA.7.030. H4; CPA.7.030.vhCDRl, CPA.7.030.vhCDR2, CPA.7.030.vhCDR3, CPA.7.030.vlCDRl, CPA.7.030.vlCDR2 and CPA.7.030.V1CDR3.

CPA.7.041, CPA.7.041.VH, CPA.7.041.VL, CPA.7.041.HC, CPA.7.041.LC, CPA.7.041.H1, CPA.7.041.H2, CPA.7.041.NZ and CPA.7.041. H4; CPA.7.041.vhCDRl, CPA.7.041. vhCDR2, CPA.7.041.vhCDR3, CPA.7.041. vlCDRl, CPA.7.041.vlCDR2 and CPA.7.041.vlCDR3.

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CPA.7.016, CPA.7.016.VH, CPA.7.016.VL, CPA.7.016.HC,

CPA.7.016.LC, CPA.7.016.Hl, CPA.7.016.H2, CPA.7.016.H3 and CPA.7.016. H4;

CPA.7.016.vhCDRl, CPA.7.016.vhCDR2, CPA.7.016.vhCDR3, CPA.7.016.vlCDRl, CPA.7.016.V1CDR2 and CPA.7.016.vlCDR3.

CPA.7.020, CPA.7.020.VH, CPA.7.020.VL, CPA.7.020.HC, CPA.7.020.LC,

CPA.7.020.H1, CPA.7.020.H2, CPA.7.020.H3 and CPA.7.020. H4; CPA.7.020.vhCDRl, CPA.7.020.vhCDR2, CPA.7.020.vhCDR3, CPA.7.020.vlCDRl, CPA.7.020.vlCDR2 and CPA.7.020.V1CDR3. CPA.7.038, CPA.7.038.VH, CPA.7.038.VL, CPA.7.038.HC, CPA.7.038.LC,

CPA.7.038.H1, CPA.7.038.H2, CPA.7.038.H3 and CPA.7.038. H4; CPA.7.038.vhCDRl, CPA.7.038.vhCDR2, CPA.7.038.vhCDR3, CPA.7.038.vlCDRl, CPA.7.038.vlCDR2 and CPA.7.038.V1CDR3. CPA.7.044, CPA.7.044.VH, CPA.7.044.VL, CPA.7.044.HC, CPA.7.044.LC,

CPA.7.044.H1, CPA.7.044.H2, CPA.7.044.H3 and CPA.7.044. H4; CPA.7.044.vhCDRl, CPA.7.044.vhCDR2, CPA.7.044.vhCDR3, CPA.7.044.vlCDRl, CPA.7.044.vlCDR2 and CPA.7.044.V1CDR3. CPA.7.045, CPA.7.045.VH, CPA.7.045.VL, CPA.7.045.HC, CPA.7.045.LC,

CPA.7.045.H1, CPA.7.045.H2, CPA.7.045.H3 and CPA.7.045. H4; CPA.7.045.vhCDRl, CPA.7.045.vhCDR2, CPA.7.045.vhCDR3, CPA.7.045.vlCDRl, CPA.7.045.vlCDR2 and CPA.7.045.vlCDR3.

In some embodiments of the present invention, the anti-TIGIT antibody is selected from the antibody whose sequences are shown in FIG. 3: CPA.9.018, CPA.9.018.VH, CPA.9.018.VL, CPA.9.018.NS, CPA.9.018.LC,

SRA.9.018.N1, SRA.9.018.N2, SRA.9.018.NZ, SRA.9.018.N4; CPA.9.018.H4(S241P);

CPA.9.018.vhCDRl, CPA.9.018.vhCDR2, CPA.9.018.vhCDR3, CPA.9.018.vlCDRl,

CPA.9.018.V1CDR2, CPA.9.018.vlCDR3 and scFv-CPA.9.018;

CPA.9.027, CPA.9.027.VH, CPA.9.027.VL, CPA.9.027.HC, CPA.9.027.LC,

CPA.9.027.H1, CPA.9.027.H2, CPA.9.027.H3, CPA.9.027.H4; CPA.9.018.H4(S241P); CPA.9.027.vhCDRl, CPA.9.027.vhCDR2, CPA.9.027.vhCDR3, CPA.9.027.vlCDRl,

CPA.9.027.vlCDR2, CPA.9.027.vlCDR3 and scFv-CPA.9.027;

CPA.9.049, CPA.9.049.VH, CPA.9.049.VL, CPA.9.049.HC, CPA.9.049.LC,

CPA.9.049.H1, CPA.9.049.H2, CPA.9.049.H3; CPA.9.049.H4; CPA.9.049.H4(S241P); CPA.9.049.vhCDRl, CPA.9.049.vhCDR2, CPA.9.049.vhCDR3, CPA.9.049.vlCDRl,

CPA.9.049.V1CDR2, CPA.9.049.vlCDR3 and scFv-CPA.9.049;

CPA.9.057, CPA.9.057.VH, CPA.9.057.VL, CPA.9.057.HC, CPA.9.057.LC,

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CPA.9.057.H1, CPA.9.057.H2, CPA.9.057.H3; CPA.9.057.H4;

CPA.9.057.H4(S241P); CPA.9.057.vhCDRl, CPA.9.057.vhCDR2, CPA.9.057.vhCDR3,

CPA.9.057.vlCDRl, CPA.9.057.vlCDR2, CPA.9.057.vlCDR3 and scFv-CPA.9.057;

CPA.9.059, CPA.9.059.VH, CPA.9.059.VL, CPA.9.059.HC, CPA.9.059.LC,

CPA.9.059.H1, CPA.9.059.H2, CPA.9.059.H3; CPA.9.059.H4; CPA.9.059.H4(S241P); CPA.9.059.vhCDRl, CPA.9.059.vhCDR2, CPA.9.059.vhCDR3, CPA.9.059.vlCDRl,

CPA.9.059.vlCDR2, CPA.9.059.vlCDR3 and scFv-CPA.9.059;

CPA.9.083, CPA.9.083.VH, CPA.9.083.VL, CPA.9.083.HC, CPA.9.083.LC,

CPA.9.083.H1, CPA.9.083.H2, CPA.9.083.H3; CPA.9.083.H4; CPA.9.083.H4(S241P); CPA.9.083.vhCDRl, CPA.9.083.vhCDR2, CPA.9.083.vhCDR3, CPA.9.083.vlCDRl, CPA.9.083.vlCDR2, CPA.9.083.vlCDR3 and scFv-CPA.9.083;

CPA.9.086, CPA.9.086.VH, CPA.9.086.VL, CPA.9.086.HC, CPA.9.086.LC,

CPA.9.086.H1, CPA.9.086.H2, CPA.9.086.H3; CPA.9.086.H4; CPA.9.086.H4(S241P); CPA.9.086.vhCDRl, CPA.9.086.vhCDR2, CPA.9.086.vhCDR3, CPA.9.086.vlCDRl,

CPA.9.086.V1CDR2, CPA.9.086.vlCDR3 and scFv-CPA.9.086;

CPA.9.089, CPA.9.089.VH, CPA.9.089.VL, CPA.9.089.HC, CPA.9.089.LC,

CPA.9.089.H1, CPA.9.089.H2, CPA.9.089.H3; CPA.9.089.H4; CPA.9.089.H4(S241P); CPA.9.089.vhCDRl, CPA.9.089.vhCDR2, CPA.9.089.vhCDR3, CPA.9.089.vlCDRl,

CPA.9.089.V1CDR2, CPA.9.089.vlCDR3 and scFv-CPA.9.089;

CPA.9.093, CPA.9.093. VH, CPA.9.093.VL, CPA.9.093.HC, CPA.9.093.LC, CPA.9.093.H1, CPA.9.093.H2, CPA.9.093 NZ; CPA.9.093.H4; CPA.9.093.H4(S241P); CPA.9.093.vhCDRl, CPA.9.093.vhCDR2, CPA.9.093,vhCDR3, CPA.9.093.vlCDRl, CPA.9.093.vlCDR2, CPA.9.093,vlCDR3 and scFv-CPA.9.093;

CPA.9.101, CPA.9.101.VH, CPA.9.101.VL, CPA.9.101.HC, CPA.9.101.LC,

CPA.9.101.H1, CPA.9.101.H2, CPA.9.101.H3; CPA.9.101.H4; CPA.9.101.H4(S241P); CPA.9.101.vhCDRl, CPA.9.101.vhCDR2, CPA.9.101.vhCDR3, CPA.9.101.vlCDRl, CPA.9.101.vlCDR2, CPA.9.101.vlCDR3 and scFv-CPA.9.101; and

CPA.9.103, CPA.9.103.VH, CPA.9.103.VL, CPA.9.103.HC, CPA.9.103.LC,

CPA.9.103.Hl, CPA.9.103.H2, CPA.9.103 NZ; CPA.9.103.H4; CPA.9.103.H4(S241P); CPA.9.103.vhCDRl, CPA.9.103.vhCDR2, CPA.9.103.vhCDR3, CPA.9.103.vlCDRl, CPA.9.103.V1CDR2, CPA.9.103.vlCDR3 and scFv-CPA.9.103.

CHA.9.536.1, CHA.9.536.1. VH, CHA.9.536.1. VL, CHA.9.536.1.HC, CHA.9.536.1.LC,

CHA.9.536.1.Hl, CHA.9.536.1. H2, CHA.9.536.1. H3; CHA.9.536.1.H4,

CHA.9.536.1.H4(S241P), CHA.9.536.1.vhCDRl, CHA.9.536.1. vhCDR2,

CHA.9.536.1.vhCDR3, CHA.9.536.1.vlCDRl, CHA.9.536.1.vlCDR2 and CHA.9.536.1.vhCDR3;

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CHA.9.536.3, CHA.9.536.3 VH, CHA.9.536.3.VL, CHA.9.536.3.HC,

CHA.9.536.3.LC, CHA.9.536.3.H1, CHA.9.536.3.H2, CHA.9.536.3.H3; CHA.9.536.3.H4, CHA.9.536.3.H4(S241P); CHA.9.536.3.vhCDRl, CHA.9.536.3.vhCDR2,

CHA.9.536.3.vhCDR3, CHA.9.536.3.vlCDRl, CHA.9.536.3.vlCDR2 and CHA.9.536.3.vhCDR3;

CHA.9.536.4, CHA.9.536.4.VH, CHA.9.536.4.VL, CHA.9.536.4.HC, CHA.9.536.4.LC,

CHA.9.536.4.H1, CHA.9.536.4.H2, CHA.9.536.4.H3; CHA.9.536.4.H4,

CHA.9.536.4.H4(S241P), CHA.9.536.4.vhCDRl, CHA.9.536.4.vhCDR2,

CHA.9.536.4.vhCDR3, CHA.9.536.4.vlCDRl, CHA.9.536.4.vlCDR2 and CHA.9.536.4.vhCDR3;

CHA.9.536.5, CHA.9.536.5.VH, CHA.9.536.5.VL, CHA.9.536.5.HC, CHA.9.536.5.LC,

CHA.9.536.5.H1, CHA.9.536.5.H2, CHA.9.536.5.H3; CHA.9.536.5.H4,

CHA.9.536.5.H4(S241P), CHA.9.536.5.vhCDRl, CHA.9.536.5.vhCDR2,

CHA.9.536.5.vhCDR3, CHA.9.536.5.vlCDRl, CHA.9.536.5.vlCDR2 and CHA.9.536.5.vhCDR3;

CHA.9.536.6, CHA.9.536.6.VH, CHA.9.536.6.VL, CHA.9.536.6.HC, CHA.9.536.6.LC, CHA.9.536.6.H1, CHA.9.536. 6.H2, CHA.9.536.6.H3; CHA.9.536.6.H4, CHA.9.536.6.vhCDRl, CHA.9.536.6.vhCDR2, CHA.9.536.6.vhCDR3, CHA.9.536.6.vlCDRl, CHA.9.536.6.vlCDR2 and CHA. 9.536.6.vhCDR3;

CHA.9.536.7, CHA.9.536.7.VH, CHA.9.536.7.VL, CHA.9.536.7.HC, CHA.9.536.7.LC,

CHA.9.536.7.H1, CHA.9.536.7.H2, CHA.9.536.7.H3; CHA.9.536.7.H4,

CHA.9.536.5.H4(S241P); CHA.9.536.7.vhCDRl, CHA.9.536.7.vhCDR2,

CHA.9.536.7.vhCDR3, CHA.9.536.7.vlCDRl, CHA.9.536.7.vlCDR2 and CHA.9.536.7.vhCDR3;

CHA.9.536.8, CHA.9.536.8.VH, CHA.9.536.8.VL, CHA.9.536.8.HC, CHA.9.536.8.LC,

CHA.9.536.8.H1, CHA.9.536.8.H2, CHA.9.536.8.H3; CHA.9.536.8.H4,

CHA.9.536.8.H4(S241P), CHA.9.536.8.vhCDRl, CHA.9.536.8.vhCDR2,

CHA.9.536.8.vhCDR3, CHA.9.536.8.vlCDRl, CHA.9.536.8.vlCDR2 and CHA.9.536.8.vhCDR3;

CHA.9.560.1, CHA. 9,560.1VH, CHA. 9.560. l.VL, CHA. 9.560. l.HC, CHA. 9.560.l.LC, CHA. 9.560.1.Hl, CHA. 9.560.1.H2, CHA. 9.560.1.H3; CHA. 9.560.1.H4, CHA. 9.560.1.H4 (S241P), CHA. 9.560.1.vhCDRl, CHA. 9.560.l.vhCDR2, CHA. 9.560.l.vhCDR3, CHA. 9.560.l.vlCDRl, CHA. 9.560.l.vlCDR2 and CHA. 9.560.1.vhCDR3;

CHA.9.560.3, CHA. 9.560. 3VH, CHA. 9.560. 3.VL, CHA. 9.560. 3.HC, CHA. 9.560. 3.LC, CHA. 9.560. 3.H1, CHA. 9.560. 3.H2, CHA. 9.560. Z.NZ; CHA.9.560.3.H4, CHA.9.560.3.H4 (S241P); CHA. 9.560. 3.vhCDRl, CHA. 9.560. 3.vhCDR2, CHA. 9.560. 3.vhCDR3, CHA. 9.560. 3.vlCDRl, CHA. 9.560. 3.vlCDR2 and CHA. 9.560. 3.vhCDR3;

CHA.9.560.4, CHA. 9.560. 4VH, CHA. 9.560. 4.VL, CHA. 9.560. 4.HC, CHA. 9.560. 4.LC, CHA. 9.560. 4.H1, CHA. 9.560. 4.H2, CHA. 9.560. 4.H3; CHA.9.560.4.H4, CHA.9.560.4.H4 (S241P), CHA. 9.560. 4.vhCDRl, CHA. 9.560. 4.vhCDR2, CHA. 9.560.

-64044486

4.vhCDR3, SNA. 9.560. 4.vlCDRl, SNA. 9.560. 4.vlCDR2 and SNA. 9.560. 4.vhCDR3;

SNA.9.560.5, SNA. 9.560. 5VH, SNA. 9.560. 5 VL, SNA. 9.560. 5.NS, SNA. 9.560. 5.LC, SNA. 9.560. 5.H1, SNA. 9.560. 5.H2, SNA. 9.560. 5.NZ; SNA. 9.560. 5.H4, SNA. 9.560. 5.vhCDRl, SNA. 9.560. 5.vhCDR2, SNA. 9.560. 5.vhCDR3, SNA. 9.560. 5.V1CDR1, SNA. 9.560. 5.V1CDR2 and SNA. 9.560. 5.vhCDR3;

SNA.9.560.6, SNA. 9.560. 6VH, SNA. 9.560. 6 VL, SNA. 9.560. 6.NS, SNA. 9.560. 6.LC, SNA. 9.560. 6.H1, SNA. 9.560. 6.H2, SNA. 9.560. 6.NZ; SNA.9.560.6.N4, SNA.9.560.6.N4 (S241P), SNA. 9.560. 6.vhCDRl, SNA. 9.560. 6.vhCDR2, SNA. 9.560. 6.vhCDR3, SNA. 9.560. 6.V1CDR1, SNA. 9.560. 6.V1CDR2 and SNA. 9.560. 6.vhCDR3;

SNA.9.560.7, SNA. 9.560. 7VH, SNA. 9.560. 7.VL, SNA. 9.560. 7.NS, SNA. 9.560. 7.LC, SNA. 9.560. 7.H1, SNA. 9.560. 7.H2, SNA. 9.560. 7.NZ; SNA.9.560.7.N4; SNA.9.560.7.N4 (S241P); SNA. 9.560. 7.vhCDRl, SNA. 9.560. 7.vhCDR2, SNA. 9.560. 7.vhCDR3, SNA. 9.560. 7.V1CDR1, SNA. 9.560. 7.V1CDR2 and SNA. 9.560. 7.vhCDR3;

SNA.9.560.8, SNA. 9.560. 8VH, SNA. 9.560. 8 VL, SNA. 9.560. 8.NS, SNA. 9.560. 8.LC, SNA. 9.560. 8.H1, SNA. 9.560. 8.H2, SNA. 9.560. 8.NZ; SNA.9.560.8.N4, SNA.9.560.8.N4 (S241P); SNA. 9.560. 8.vhCDRl, SNA. 9.560. 8.vhCDR2, SNA. 9.560. 8.vhCDR3, SNA. 9.560. 8.V1CDR1, SNA. 9.560. 8.V1CDR2, as well as SNA. 9.560. 8.vhCDR3;

SNA.9.546.1, SNA. 9. 546.1VH, SNA. 9. 546.1.VL, SNA. 9. 546.1.NS, SNA. 9. 546.1.LC, SNA. 9. 546.1.N1, SNA. 9. 546.1.N2, SNA. 9. 546.1.NZ; SNA.9.546.1.N4, SNA.9.546.1.N4 (S241P), SNA. 9. 546.1.vhCDRl, SNA. 9. 546.1.vhCDR2, SNA. 9. 546.1.vhCDR3, SNA. 9. 546.1.vlCDRl, SNA. 9. 546.1.V1CDR2 and SNA. 9.546.1.vhCDR3;

SNA.9.547.1, SNA. 9. 547.1VH, SNA. 9. 547.1.VL, SNA. 9. 547.1.NS, SNA. 9. 547.1.LC, SNA. 9. 547.1.N1, SNA. 9. 547.1.N2, SNA. 9. 547.1.NZ; SNA.9.547.1.N4, SNA.9.547.1.N4 (S241P), SNA. 9. 547.1.vhCDRl, SNA. 9. 547.1.vhCDR2, SNA. 9. 547.1.vhCDR3, SNA. 9. 547.1.vlCDRl, SNA. 9. 547.1.vlCDR2 and SNA. 9.547.1.vhCDR3;

SNA.9.547.2, SNA. 9. 547. 2VH, SNA. 9. 547. 2.VL, SNA. 9. 547. 2.NS, SNA. 9. 547. 2.LC, SNA. 9. 547. 2.N1, SNA. 9. 547. 2.H2, SNA. 9. 547. 2.NZ; SNA.9.547.2.N4, SNA.9.547.2.N4 (S241P), SNA. 9. 547. 2.vhCDRl, SNA. 9. 547. 2.vhCDR2, SNA. 9. 547. 2.vhCDR3, SNA. 9. 547. 2.V1CDR1, SNA. 9. 547. 2.V1CDR2 and SNA. 9.547.2.vhCDR3;

SNA.9.547.3, SNA. 9. 547. 3VH, SNA. 9. 547. 3 VL, SNA. 9. 547. Z.NS, SNA. 9. 547. 3.LC, SNA. 9. 547. Z.N1, SNA. 9. 547. Z.N2, SNA. 9. 547. Z.NZ; SNA.9.547.3.N4, SNA.9.547.3.N4 (S241P), SNA. 9. 547. 3.vhCDRl, SNA. 9.547. 3.vhCDR2, SNA. 9. 547. 3.vhCDR3, SNA. 9. 547. 3.V1CDR1, SNA. 9. 547. 3.V1CDR2 and SNA. 9.547.3.vhCDR3;

SNA.9.547.4, SNA. 9. 547. 4VH, SNA. 9. 547. 4.VL, SNA. 9. 547. 4.NS, SNA. 9.547. 4.LC, SNA. 9. 547. 4.N1, SNA. 9. 547. 4.H2, SNA. 9. 547. 4.NZ; SNA.9.547.4.N4,

- 65 044486

CHA.9.547.4.H4 (S241P), SHA. 9. 547. 4.vhCDRl, SNA. 9. 547. 4.vhCDR2, SNA. 9. 547. 4.vhCDR3, SNA. 9. 547. 4.vlCDRl, SNA. 9. 547. 4.vlCDR2 and SNA. 9.547.4.vhCDR3;

SNA.9.547.6, SNA. 9. 547. 6 VH, SNA. 9. 547. 6.VL, SNA. 9. 547. 6.NS, SNA. 9. 547. 6.LC, SNA. 9. 547. 6.N1, SNA. 9. 547. 6.H2, SNA. 9. 547. 6.NZ; SNA.9.547.6.N4, SNA.9.547.6.N4 (S241P), SNA. 9. 547. 6.vhCDRl, SNA. 9. 547. 6.vhCDR2, SNA. 9. 547. 6.vhCDR3, SNA. 9. 547. 6.vlCDRl, SNA. 9. 547. 6.V1CDR2 and SNA. 9.547.6.vhCDR3;

SNA.9.547.7, SNA. 9.547.7VH, SNA. 9. 547. 7.VL, SNA. 9. 547. 7.NS, SNA. 9. 547. 7.LC, SNA. 9. 547. 7.N1, SNA. 9. 547. 7.N2, SNA. 9. 547. 7.NZ; SNA.9.547.7.N4, SNA.9.547.7.N4 (S241P), SNA. 9. 547. 7.vhCDRl, SNA. 9. 547. 7.vhCDR2, SNA. 9. 547. 7.vhCDR3, SNA. 9. 547. 7.vlCDRl, SNA. 9. 547. 7.V1CDR2 and SNA. 9.547.7.vhCDR3;

SNA.9.547.8, SNA. 9.547.8VH, SNA. 9. 547. 8.VL, SNA. 9. 547. 8.NS, CHA.9.547.8.LC, SNA. 9. 547. 8.N1, SNA. 9. 547. 8.H2, SNA. 9. 547. 8.NZ; SNA.9.547.8.N4, SNA.9.547.8.N4 (S241P), SNA. 9. 547. 8.vhCDRl, SNA. 9. 547. 8.vhCDR2, SNA. 9. 547. 8.vhCDR3, SNA. 9. 547. 8.vlCDRl, SNA. 9. 547. 8.vlCDR2, as well as SNA. 9.547.8.vhCDR3;

SNA.9.547.9, SNA.9.547.9, CHA.9.547.9VH, CHA.9.547.9.VL, SNA.9. 547.9.NS, CHA.9.547.9.LC, SNA.9.547.9.N1, SNA.9.547.9.N2, SNA.9.547.9.NZ; SNA.9.547.9.H4, SNA.9.547.9.H4, CHA.9.547.9.H4(S241P), CHA.9.547.9.H4(S241P), SNA.9.547.9.vhCDRl, SNA.9.547. 9.vhCDR2, CHA.9.547.9.vhCDR3, CHA.9.547.9. vlCDRl, SNA.9.547.9. vlCDR2 and CHA.9.547.9.vhCDR3;

SNA.9.547.13, SNA.9.547.13, SNA.9.547. 13VH, SNA.9. 547.13.VL, SNA.9. 547.13.NS, SNA. 9.547.13.LC, SNA. 9.547.13.N1, SNA.9.547.13.N2, SNA.9. 547.13.H3; SNA.9.547.13.N4, SNA.9.547.13.N4, SNA.9.547.13.N4 (S241P), SNA.9.547.13.N4 (S241P), SNA. 9.547.13. vhCDRl, SNA.9.547.13. vhCDR2, SNA.9.547. 13.vhCDR3, SNA. 9. 547.13.vlCDRl, SNA. 9. 547.13.vlCDR2 and SNA. 9.547.13.vhCDR3;

SNA.9.541.1, SNA. 9. 541.1.VH, SNA. 9. 541.1.VL, SNA. 9. 541.1.NS, SNA. 9. 541.1.LC, SNA. 9. 541.1.N1, SNA. 9. 541.1.N2, SNA. 9. 541.1.NZ; SNA.9.541.1.N4, SNA.9.541.1.N4 (S241P), SNA. 9. 541.1.vhCDRl, SNA. 9. 541.1.vhCDR2, SNA. 9. 541.1.vhCDR3, SNA. 9. 541.1.vlCDRl, SNA. 9. 541.1.vlCDR2 and SNA. 9.541.l.vhCDR3;

SNA.9.541.3, SNA. 9. 541. 3.VH, SNA. 9. 541. 3.VL, SNA. 9. 541. Z.NS, SNA. 9. 541. 3.LC, SNA. 9. 541. Z.N1, SNA. 9. 541. Z.N2, SNA. 9. 541. Z.NZ; SNA.9.541.3.N4, SNA.9.541.3.N4 (S241P), SNA. 9. 541. 3.vhCDRl, SNA. 9. 541. 3.vhCDR2, SNA. 9. 541. 3.vhCDR3, SNA. 9. 541. 3.vlCDRl, SNA. 9. 541. 3.vlCDR2 and SNA. 9.541. 3.vhCDR3;

SNA.9.541.4, SNA. 9. 541.4.VH, SNA. 9. 541. 4.VL, SNA. 9. 541. 4.NS, SNA. 9. 541. 4.LC, SNA. 9. 541. 4.N1, SNA. 9. 541. 4.H2, SNA. 9. 541. 4.NZ; SNA.9.541.4.N4, SNA.9.541.4.N4 (S241P), SNA. 9. 541. 4.vhCDRl, SNA. 9. 541. 4.vhCDR2, SNA. 9.541.

- 66 044486 .vhCDR3, SNA. 9. 541. 4.vlCDRl, SNA. 9. 541. 4.vlCDR2 and SNA. 9.541. 4.vhCDR3;

SNA.9.541.5, SNA. 9. 541. 5.VH, SNA. 9. 541. 5.VL, SNA. 9. 541. 5.NS, SNA. 9.541.

.LC, SNA. 9. 541. 5.N1, SNA. 9. 541. 5.H2, SNA. 9. 541. 5.NZ; SNA.9.541.5.N4, SNA.9.541.5.N4 (S241P), SNA. 9. 541. 5.vhCDRl, SNA. 9. 541. 5.vhCDR2, SNA. 9.541.

.vhCDR3, SNA. 9. 541. 5.vlCDRl, SNA. 9. 541. 5.vlCDR2 and SNA. 9.541. 5.vhCDR3;

SNA.9.541.6, SNA. 9. 541. 6.VH, SNA. 9. 541. 6.VL, SNA. 9. 541. 6.NS, SNA. 9.541.

.LC, SNA. 9. 541. 6.N1, SNA. 9. 541. 6.H2, SNA. 9. 541.6.NZ; SNA.9.541.6.N4, SNA.9.541.6.N4 (S241P), SNA. 9. 541. 6.vhCDRl, SNA. 9. 541. 6.vhCDR2, SNA. 9.541.

.vhCDR3, SNA. 9. 541. 6.V1CDR1, SNA. 9. 541. 6.V1CDR2 and SNA. 9.541. 6.vhCDR3;

SNA.9.541.7, SNA. 9. 541. 7.VH, SNA. 9. 541. 7.VL, SNA. 9. 541. 7.NS, SNA. 9.541.

.LC, SNA. 9. 541. 7.N1, SNA. 9. 541. 7.N2, SNA. 9. 541. 7.NZ; SNA.9.541.7.N4, SNA.9.541.7.N4 (S241P), SNA. 9. 541. 7.vhCDRl, SNA. 9. 541. 7.vhCDR2, SNA. 9.541.

.vhCDR3, SNA. 9. 541. 7.V1CDR1, SNA. 9. 541. 7.V1CDR2 and SNA. 9.541. 7.vhCDR3; And

SNA.9.541.8, SNA. 9. 541. 8.VH, SNA. 9. 541. 8.VL, SNA. 9. 541. 8.NS, SNA. 9.541.

.LC, SNA. 9. 541. 8.N1, SNA. 9. 541. 8.H2, SNA. 9. 541. 8.NZ; SNA.9.541.8.N4, SNA.9.541.8.N4 (S241P); SNA. 9. 541. 8vhCDRl, SNA. 9. 541. 8.vhCDR2, SNA. 9.541.

.vhCDR3, SNA. 9. 541. 8.V1CDR1, SNA. 9. 541. 8.V1CDR2, as well as SNA. 9.541. 8.vhCDR3.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PD-1 antibody is pembrolizumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PD-1 antibody is pembrolizumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), an anti-PD-! is pembrolizumab, and the anti-PVRIG antibody is one of the antibodies mentioned above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PD-1 antibody is pembrolizumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PD-1 antibody is nivolumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PD-1 antibody is nivolumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PD-1 antibody is nivolumab, and the anti-PVRIG antibody is one of the antibodies listed above and/or at fig. 5 or FIG. 63.

In some embodiments of the invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PD-1 antibody is nivolumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. . 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PD-1 antibody is cemiplimab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PD-1 antibody is cemiplimab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PD-1 antibody is cemiplimab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PD-1 antibody is cemiplimab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. . 5 or FIG. 63.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-1 antibody is pembrolizumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is

- 67 044486 is CHA.7.538.1.2.H4 (S241P), the anti-PD-l antibody is pembrolizumab, and the anti-TIGIT antibody is one of the antibodies mentioned above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-1 antibody is nivolumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), the anti-PD-1 antibody is nivolumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-1 antibody is cemiplimab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), the anti-PD-1 antibody is cemiplimab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is pembrolizumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is pembrolizumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is pembrolizumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is pembrolizumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is pembrolizumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is pembrolizumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is pembrolizumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is pembrolizumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is SHR-1210 .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is SHR-1210 .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is SHR-1210 .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is SHR-1210 .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is SHR -1210.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is SHR -1210.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is SHR -1210.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is SHR -1210.

- 68 044486

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is IBI308.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is IBI308.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is IBI308.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is IBI308.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is IBI308 .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is IBI308 .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is IBI308 .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is IBI308 .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is BGB-A317 .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is BGB-A317 .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is BGB-A317 .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is BGB-A317 .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.h4 (S241P), and the anti-PD-1 antibody is BGB -A317.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is BGB -A317.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is BGB -A317.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is BGB -A317.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is anti- PD-1 as stated in US Patent Publication No. 2017/0081409.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is anti- PD-1 as stated in US Patent Publication No. 2017/0081409.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is anti- PD-1 as stated in US Patent Publication No. 2017/0081409.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the

- 69 044486 lo anti-PD-l is an anti-PD-l antibody as described in US Patent Publication No. 2017/0081409.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is αntu-PD-1, as described in US Patent Publication No. 2017/0081409.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is αntu-PD-1, as described in US Patent Publication No. 2017/0081409.

In some embodiments of the invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is anti-PD-1 as specified in US Patent Publication No. 2017/0081409.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is αntu-PD-1, as described in US Patent Publication No. 2017/0081409.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is cemiplimab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is cemiplimab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is cemiplimab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is cemiplimab.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is cemiplimab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is cemiplimab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.h4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-1 antibody is cemiplimab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-1 antibody is cemiplimab .

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PD-L1 antibody is atezolizumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PD-L1 antibody is atezolizumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PD-L1 antibody is atezolizumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PD-L1 antibody is atezolizumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PD-L1 antibody is avelumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PD-L1 antibody is avelumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PD-L1 antibody is avelumab, and the anti-PVRIG antibody is

- 70 044486 is one of the antibodies mentioned above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PD-L1 antibody is avelumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PD-L1 antibody is durvalumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PD-L1 antibody is durvalumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PD-L1 antibody is durvalumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PD-L1 antibody is durvalumab, and the anti-PVRIG antibody is one of the antibodies identified above and/or in FIG. 5 or FIG. 63.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-L1 antibody is atezolizumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), the anti-PD-L1 antibody is atezolizumab, and the anti-TIGIT antibody is one of the antibodies listed above and/or at fig. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-L1 antibody is atezolizumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), the anti-PD-L1 antibody is atezolizumab, and the anti-TIGIT antibody is one of the antibodies listed above and/or at fig. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-L1 antibody is avelumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), the anti-PD-L1 antibody is avelumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-L1 antibody is avelumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), the anti-PD-L1 antibody is avelumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-L1 antibody is durvalumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), the anti-PD-L1 antibody is durvalumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), the anti-PD-L1 antibody is durvalumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments of the present invention, the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), the anti-PD-L1 antibody is durvalumab, and the anti-TIGIT antibody is one of the antibodies identified above and/or in FIG. 3.

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is atezolizumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is atezolizumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is atezolizumab.

In some embodiments of the present invention, the anti-TIGIT antibody is

- 71 044486 fight CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is atezolizumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is atezolizumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is atezolizumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is atezolizumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is atezolizumab .

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is avelumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is avelumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is avelumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is avelumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is avelumab .

In some embodiments of the invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is avelumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is avelumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is avelumab .

In some embodiments, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is durvalumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.083.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is durvalumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is durvalumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CPA.9.086.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is durvalumab.

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is durvalumab .

In some embodiments of the invention, the anti-TIGIT antibody is CHA.9.547.7.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is durvalumab .

In some embodiments of the present invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.538.1.2.H4 (S241P), and the anti-PD-L1 antibody is durvalumab .

In some embodiments of the invention, the anti-TIGIT antibody is CHA.9.547.13.H4 (S241P), the anti-PVRIG antibody is CHA.7.518.1.H4 (S241P), and the anti-PD-L1 antibody is durvalumab .

- 72 044486

B. Biomarker analysis.

As demonstrated herein, the selection of combination therapy to administer can be made using assessment of the expression of specific biomarkers from tumor biopsies. That is, by taking a biopsy from a patient's tumor sample and checking the presence and content of certain proteins through protein staining and sorting, the appropriate therapy can be selected. As demonstrated in Example 2, tumor cells can be screened to identify immune and non-immune cell populations, and then the immune cell populations can be assessed for levels of a number of biomarkers, including PD-1, PD-L1, PVRIG, PVR, PVRL2 and TIGIT, including by analyzing both ligand and antigen levels.

Thus, for example, to identify immune cell populations, antibodies to one or more of the following markers: CD45, CD3, CD8, CD33, CD25, CD127, CD14, CD4, and CD56 can be assessed to classify cell populations in a tumor sample as shown in table. 1.

Table 1

Name Gating markers CD4 ⁺ T cell subsets CD45 ⁺ CD3 ⁺ CD14' CD4 ⁺ CD8 ⁺ T cells CD45 ⁺ CD3 ⁺ CD14'CD8 ⁺ CD4 CD8 T cells CD45 ⁺ CD3 ⁺ CD14' CD4 CD8 NK cells CD45 ⁺ CD3'CD14' CD56 ⁺ Monocytes CD45 ⁺ CD3' CD14 ⁺ mDC CD45 ⁺ CD3'CD14'CD56' pDC CD33 ^BbIC CD45 ⁺ CD3'CD14'CD56' CD45 cells CD33 ^cpe « ^H CD45'

Several of these cell types are then assessed for expression of one or more of the following markers: PD-1, PD-L1, PVRIG, PVR, PVRL2 and TIGIT, typically using labeled antibodies, and counted. If the percentage of PD-L1-positive tumor cells or immune cells exceeds 1% (> 1%) compared with the same tumor cells stained with an antibody corresponding to the isotype control antibody for the antibodies used, then a three-component combination of anti-TIGIT antibodies should be administered, anti-PVRIG and anti-PD-1. Whereas, patients with PD-L1-positive tumor cells or immune cells in abundance below 1% (<1%) of isotype controls should be treated with a two-component combination of anti-TIGIT and anti-PVRIG antibodies.

1. Combination Therapy with Anti-TIGIT, Anti-PVRIG, and Anti-PD-1 Antibodies In some embodiments of the present invention, after tumor immune cells are optionally analyzed for expression of at least one cell surface marker selected from PD- 1, PD-L1, PVRIG, PVR, PVRL2 and TIGIT, decisions regarding the route of therapy can be made. When the percentage of PD-L1 positive tumor cells or immune cells expressing >1%, the patient can be administered a triple combination of anti-TIGIT, anti-PVRIG and anti-PD-1 antibodies as described herein.

Accordingly, in one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumab. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and pembrolizumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and pembrolizumab. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and pembrolizumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumab. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and pembrolizumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and pembrolizumab. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and pembrolizumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumab. In a specific embodiment of the present invention, CHA.9.547.7H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and pembrolizumab.

In one embodiment of the present invention, antibodies containing sets of CDRs from an

- 73 044486 ti-TIGIT antibody CHA.9.547.7, combined with antibodies containing sets of CDRs from the anti-PVRIG antibody CHA.7.538.1.2, and pembrolizumab. In a specific embodiment of the present invention, CHA.9.547.7.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and pembrolizumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and pembrolizumab. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and pembrolizumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and pembrolizumab. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and pembrolizumab.

Accordingly, in one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and nivolumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumab. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and nivolumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and nivolumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumab. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and nivolumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab. In a specific embodiment of the present invention, CHA.9.547.7H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and nivolumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumab. In a specific embodiment of the present invention, CHA.9.547.7.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and nivolumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and nivolumab. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and nivolumab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and nivolumab. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and nivolumab.

Accordingly, in one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and cemiplimab. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and cemiplimab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and cemiplimab. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and cemiplimab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and cemiplimab. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and cemiplimab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and cemiplimab. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and cemiplimab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and cemiplimab. In a specific embodiment of this invention

- 74 044486

CHA.9.547.7H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and cemiplimab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and cemiplimab. In a specific embodiment of the present invention, CHA.9.547.7.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and cemiplimab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1 and cemiplimab. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and cemiplimab.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2 and cemiplimab. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and cemiplimab.

2. Combination therapy with anti-TIGIT and anti-PVRIG antibodies.

Likewise, once tumor immune cells have been analyzed for expression of at least one cell surface marker selected from PD-1, PD-L1, PVRIG, PVR, PVRL2 and TIGIT, decisions regarding the mode of therapy can be made. When the percentage of PD-L1-positive tumor cells or immune cells expressing is <1%, the patient can be administered a two-component combination of anti-TIGIT and anti-PVRIG antibodies as described herein.

Accordingly, in one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P).

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P).

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P).

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P).

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a specific embodiment of the present invention, CHA.9.547.7H4 (S241P) is combined with CHA.7.518.1.H4 (S241P).

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2. In a specific embodiment of the present invention, CHA.9.547.7.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P).

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P).

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P).

In one embodiment of the present invention, combinations of anti-TIGIT antibodies of the present invention and anti-PD-1 antibodies are provided. In one embodiment, the present invention provides combinations of anti-TIGIT antibodies of the present invention and anti-PD-L1 antibodies.

In one embodiment of the present invention, a biopsy is obtained from a tumor in a patient with cancer and dissected, as is known in the art, for FACS analysis. Cells are stained with labeled antibodies to (1) TIGIT (eg, using any of the antibodies described herein or others known in the art, such as MBSA43); (2) PD-1 (eg, using antibodies known in the art, including EH12.2H7, Keytruda®, Opdivo®, cemiplimab, etc.); (3) PD-L1 (for example, using known in the art

- 75 044486 technology of antibodies, such as BM-1, atezolizumab, avelumab and durvalumab described herein) and (4) PVR (for example, using antibodies known in the art, such as SKII.4); and (5) the appropriate isotype control antibody for the antibodies used. FACS analysis is performed and the percentage of cells expressing the receptor relative to the control antibody is calculated for each receptor. If the percentage of positive cells for TIGIT, PD-1, PD-1, and PVR is >1% for all 4 receptors, then the patient is treated with antibodies to TIGIT and PD-1 as described herein.

In one embodiment of the present invention, a biopsy is obtained from a tumor in a patient with cancer and dissected, as is known in the art, for FACS analysis. Cells are stained with labeled antibodies to (1) PVRIG (usually using, for example, CHA.7.518.Sh4 (S241P), although any antibody described in WO 2016/134333 can be used (especially including any antibody that binds, even if not blocks the target), or any antibody described in WO 2017/041004); (2) PD-1 (eg, using antibodies known in the art, including EH12.2H7, Keytruda®, Opdivo®, cemiplimab, etc.); (3) PD-L1 (for example, using antibodies known in the art, such as BM-1, atezolizumab, avelumab and durvalumab described herein) and (4) PVRL2 (for example, using antibodies known in the art antibodies such as TX11); and (5) the appropriate isotype control antibody for the antibodies used. FACS analysis is performed and the percentage of cells expressing the receptor relative to the control antibody is calculated for each receptor. If the percentage of positive cells for PVRIG, PD-1, PD-1, and PVRL2 is >1% for all 4 receptors, then the patient is treated with antibodies to PVRIG and PD-1 as described herein.

In one embodiment of the present invention, a biopsy is obtained from a tumor in a patient with cancer and dissected, as is known in the art, for FACS analysis. Cells are stained with labeled antibodies to (1) PVRIG (usually using, for example, CHA.7.518.Sh4 (S241P), although any antibody described in WO 2016/134333 can be used (especially including any antibody that binds, even if not blocks the target), or any antibody described in WO 2017/041004); (2) TIGIT (eg, using any of the antibodies described herein or others known in the art, such as MBSA43); (3) PVR (eg, using any antibodies known in the art, such as SKII.4) and (4) PVRL2 (eg, using antibodies known in the art, such as TX11); and (5) the appropriate isotype control antibody for the antibodies used. FACS analysis is performed and the percentage of cells expressing the receptor relative to the control antibody is calculated for each receptor. If the percentage of positive cells for PVRIG, TIGIT, PVR and PVRL2 is >1% for all 4 receptors, then the patient is treated with antibodies to PVRIG and TIGIT. The preferred combinations in this regard are CHA.7.518.1.H4 (S241P) and CPA.9.086.

In one embodiment of the present invention, a biopsy is obtained from a tumor in a patient with cancer and dissected, as is known in the art, for FACS analysis. Cells are stained with labeled antibodies to (1) PVRIG (usually using, for example, CHA.7.518.Sh4 (S241P), although any antibody described in WO 2016/134333 can be used (especially including any antibody that binds, even if not blocks the target), or any antibody described in WO 2017/041004); (2) TIGIT (eg, using any of the antibodies described herein or others known in the art, such as MBSA43); (3) PVR (eg, using any antibodies known in the art, such as SKII.4) and (4) PVRL2 (eg, using antibodies known in the art, such as TX11); (5) PD-1 (eg, using antibodies known in the art, including EH12.2H7, Keytruda®, Opdivo®, citiplimab, etc.); and (6) the appropriate isotype control antibody for the antibodies used. FACS analysis is performed and the percentage of cells expressing the receptor relative to the control antibody is calculated for each receptor. If the percentage of positive cells for PVRIG, TIGIT, PVR, PVRL2 and PD-1 is >1% for all 5 receptors, then the patient is treated with antibodies to PVRIG, TIGIT and PD-1. Preferred combinations in this regard are CHA.7.518.1.H4 (S241P), CPA.9.086 and EH12.2H7. Other preferred combinations in this regard are CHA.7.518.1.H4 (S241P), CPA.9.086 and Keytruda®. Other preferred combinations in this regard are CHA.7.518.1.H4 (S241P), CPA.9.086 and Opdivo®.

In one embodiment of the present invention, a biopsy is obtained from a tumor in a patient with cancer and dissected, as is known in the art, for FACS analysis. Cells are stained with labeled antibodies to (1) PVRIG (usually using, for example, CHA.7.518.1H4 (S241P), although any antibody described in WO 2016/134333 can be used (especially including any antibody that binds, even if not blocks the target), or any antibody described in WO 2017/041004); (2) TIGIT (eg, using any of the antibodies described herein or others known in the art, such as MBSA43); (3) PD-L1 (for example, using any antibodies known in the art, such as BM-1, atezolizumab, avelumab and durvalumab, described in

- 76 044486 herein) and (4) PVR (for example, using antibodies known in the art, such as SKII.4); (5) PD-1 (eg, using antibodies known in the art, including EH12.2H7, Keytruda®, Opdivo®, citiplimab, etc.); and (6) the appropriate isotype control antibody for the antibodies used. FACS analysis is performed and the percentage of cells expressing the receptor relative to the control antibody is calculated for each receptor. If the percentage of positive cells for PVRIG, TIGIT, PD-L1, PVR and PD-1 is >1% for all 5 receptors, then the patient is treated with antibodies to PVRIG, TIGIT and PD-1. Preferred combinations in this regard are CHA.7.518.1.H4 (S241P), CPA.9.086 and EH12.2H7. Other preferred combinations in this regard are CHA.7.518.1.H4 (S241P), CPA.9.086 and Keytruda®. Other preferred combinations in this regard are CHA.7.518.1.H4 (S241P), CPA.9.086 and Opdivo®.

3. Combination therapy with anti-TIGIT and anti-PVRIG antibodies with anti-PD-1 antibodies for refractory patients.

In some embodiments of the present invention, the treatment includes a combination of anti-TIGIT antibodies, anti-PVRIG antibodies and anti-PD-1 antibodies to target tumor cells with high expression of PD-L1. In some embodiments of the present invention, the treatment includes a combination of anti-TIGIT antibodies, anti-PVRIG antibodies and anti-PD-1 antibodies for use in a patient whose tumors express PD-L1. In some embodiments of the present invention, the treatment includes a combination of anti-TIGIT antibodies, anti-PVRIG antibodies and anti-PD-1 antibodies for use in a patient with cancer whose tumor expresses PD-L1 and/or who is refractory to anti-PD-L1 antibody drugs. PD-1. In some embodiments, the treatment includes a combination of anti-TIGIT antibodies, anti-PVRIG antibodies, and anti-PD-1 antibodies for use in a patient with cancer whose tumor expresses PD-L1 and who is refractory to anti-PD antibody drugs -1.

Accordingly, in one embodiment of the present invention, antibodies comprising the CDR sets of the anti-TIGIT antibody CPA.9.083 are combined with antibodies comprising the CDR sets of the anti-PVRIG antibody CHA.7.518.1, and pembrolizumab, for use in a patient with cancer , whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1 and/or who is drug refractory anti-PD-1 antibodies.

In one embodiment of the present invention, antibodies containing CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2, and pembrolizumab, for use in a patient with cancer, tumor which expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1 and/or which is refractory to anti-PD-1 antibody drugs.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1, and pembrolizumab, for use in a patient with cancer, tumor which expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs.

In one embodiment of the present invention, antibodies containing CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2, and pembrolizumab, for use in a patient with cancer, tumor which expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs.

In one embodiment of the present invention, antibodies comprising the CDR sets of the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies comprising the CDR sets of the anti-PVRIG antibody CHA.7.518.1, and pembrolizumab, for use in a patient with cancer , whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CHA.9.547.7H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs.

In one embodiment of the present invention, antibodies containing CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from anti-PVRIG

- 77 044486 antibody CHA.7.538.1.2, and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1 and/or who is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CHA.9.547.7.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is drug-resistant anti-PD-1 antibodies.

In one embodiment of the present invention, antibodies comprising the CDR sets of the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies comprising the CDR sets of the anti-PVRIG antibody CHA.7.518.1, and pembrolizumab, for use in a patient with cancer. , whose tumor expresses PD-L1, and/or which is resistant to αntu-PD-1 antibody drugs. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is drug-resistant anti-PD-1 antibodies.

In one embodiment of the present invention, antibodies comprising the CDR sets of the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies comprising the CDR sets of the anti-PVRIG antibody CHA.7.538.1.2, and pembrolizumab, for use in a patient with cancer , whose tumor expresses PD-L1, and/or which is resistant to αntu-PD-1 antibody drugs. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and pembrolizumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is drug-resistant anti-PD-1 antibodies.

Accordingly, in one embodiment of the present invention, antibodies comprising the CDR sets of the anti-TIGIT antibody CPA.9.083 are combined with antibodies comprising the CDR sets of the anti-PVRIG antibody CHA.7.518.1, and nivolumab, for use in a patient with cancer , whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1 and/or which is refractory to anti-PD-1 antibody drugs.

In one embodiment of the present invention, antibodies containing CDR sets from the anti-TIGIT antibody CPA.9.083 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2, and nivolumab, for use in a patient with cancer, tumor which expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CPA.9.083.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs.

In one embodiment of the present invention, antibodies containing CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1, and nivolumab, for use in a patient with cancer, tumor which expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1 and/or which is refractory to αntu-PD-1 antibody preparations.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CPA.9.086 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2, and nivolumab, for use in a patient with cancer, tumor which expresses PD-L1, and/or which is resistant to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CPA.9.086.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1, and nivolumab, for use in a patient with cancer , whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CHA.9.547.7H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs.

In one embodiment of the present invention, antibodies comprising CDR sets from the anti-TIGIT antibody CHA.9.547.7 are combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.538.1.2, and nivolumab, for use in a patient with cancer , whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CHA.9.547.7.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is drug-resistant anti-PD-1 antibodies.

In one embodiment of the present invention, antibodies containing sets of CDRs from an

- 78 044486 ti-TIGIT antibody CHA.9.547.13, combined with antibodies containing CDR sets from the anti-PVRIG antibody CHA.7.518.1, and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1, and/ or who is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.518.1.H4 (S241P) and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is drug-resistant anti-PD-1 antibodies.

In one embodiment of the present invention, antibodies comprising the CDR sets of the anti-TIGIT antibody CHA.9.547.13 are combined with antibodies comprising the CDR sets of the anti-PVRIG antibody CHA.7.538.1.2, and nivolumab, for use in a patient with cancer , whose tumor expresses PD-L1, and/or which is refractory to anti-PD-1 antibody drugs. In a specific embodiment of the present invention, CHA.9.547.13.H4 (S241P) is combined with CHA.7.538.1.2.H4 (S241P) and nivolumab, for use in a patient with cancer whose tumor expresses PD-L1, and/or which is drug-resistant anti-PD-1 antibodies.

In some embodiments of the present invention, the anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3. In some embodiments of the present invention, the anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63. In some embodiments of the present invention, the anti-PD-1 antibody is an antibody selected from any αntu-PD-1 antibody described herein, including any of the antibodies described in FIG. 7.

4. Treatment evaluation.

Typically, the antibodies of this invention, alone or in combination (PVRIG with PD-1, TIGIT with PD-1, or TIGIT with PVRIG and/or PVRIG with TIGIT and PD-1) are administered to patients with cancer, and their effectiveness is assessed in several ways , as described in this document. Thus, along with standard efficacy tests such as cancer load, tumor size, presence or extent of metastasis, etc., the effectiveness of treatment with immuno-oncology drugs can also be assessed based on immune status parameters. This can be done using a number of methods, including both in vitro and in vivo assays. For example, by assessing changes in immune status (eg, presence of ICOS+ CD4+ T cells after ipi treatment) along with traditional measurements such as tumor burden, tumor size, invasiveness, lymph node involvement, metastasis, etc. Thus, any or all of the following parameters can be assessed: inhibitory effects of PVRIG on CD4+ T cell activation or proliferation, CD8+ T (CTL) cell activation or proliferation, CD8+ T cell-mediated cytotoxic activity, and/or CTL-mediated cell depletion. NK cell activity and NK-mediated cell depletion, potentiating effects of PVRIG on Treg cell differentiation and proliferation, and immunosuppression or immunotolerance mediated by Treg or myeloid-derived suppressor cells (MDSCs), and/or effects of PVRIG on anti-inflammatory cytokine production by immune cells, e.g. , production of IL-2, IFN-γ or TNF-α by T cells or other immune cells.

In some embodiments of the present invention, treatment evaluation is performed by analyzing immune cell proliferation using, for example, the CFSE dilution method, intracellular Ki67 immune effector cell staining, and the 3H-thymidine incorporation method.

In some embodiments of the present invention, treatment is assessed by analyzing increased gene expression or increased protein levels of markers associated with activation, including one or more of the following: CD25, CD69, CD137, ICOS, PD1, GITR, OX40, as well as measurements of degranulation cells by surface expression of CD1O7A.

In some embodiments of the present invention, treatment evaluation is performed by analyzing the amount of T cell proliferation without treatment, for example, before administration of antibodies of the present invention. If, after administration, the patient experiences an increase in T cell proliferation, for example, a subset of the patient's T cells proliferates, this indicates that the T cells have been activated.

Likewise, evaluation of antibody treatment of the present invention can be performed by measuring the patient's IFN-γ levels before and after administration to assess the effectiveness of the treatment. This can be accomplished within a few hours or days.

Typically, gene expression assays are performed as is known in the art. See, for example, Goodkind et al., Computers and Chem. Eng. 29(3):589 (2005), Han et al., Bioinform. Biol. Insights 11/15/15 9(Suppl. 1):29-46, Campo et al., Nod. Pathol. Jan 2013; 26 suppl. 1: S97-S110, techniques for measuring gene expression in which are expressly incorporated herein by reference.

In general, protein expression measurements are also performed similarly as known in the art, see, for example, Wang et al., Recent Advances in Capillary Electrophoresis-Based Pro

- 79 044486 teomic Techniques for Biomarker Discovery, Methods. Mol. Biol. 2013:984:1-12; Taylor et al, BioMed Res. Volume 2014, Article ID 361590, 8 pages, Becerk et al., Mutat. Res 2011 June 17: 722 (2): 171-182, expression measurement techniques of which are expressly incorporated herein by reference.

In some embodiments of the present invention, treatment evaluation is performed by analyzing cytotoxic activity, measured by determining the viability of target cells, by assessing multiple cellular parameters such as enzyme activity (including protease activity), cell membrane permeability, cell adhesion, ATP production, coenzyme production and nucleotide uptake activity. Specific examples of such assays include, but are not limited to, trypan blue or PI staining method, ⁵¹ Cr or ³⁵ S release method, LDH activity assay, MTT and/or WST assays, calcein-AM assay, luminescence-based assay and others.

In some embodiments of the present invention, treatment evaluation is performed by analyzing T cell activity as measured by cytokine production, wherein the culture supernatant is examined for intracellular production of cytokines, including, but not limited to, IFNγ, TNF-α, GM-CSF, IL -2, IL-6, IL-4, IL-5, IL-10 and/or IL-13 using well known techniques.

Accordingly, treatment evaluation can be performed using assays that evaluate one or more of the following mechanisms: (i) increased immune response, (ii) increased αβ and/or γδ T cell activation, (iii) increased cytotoxic T cell activity , (iv) increased activity of NK and/or NKT cells, (v) decreased suppression of αβ and/or γδ T cells, (vi) increased secretion of anti-inflammatory cytokines, (vii) increased secretion of IL-2; (viii) increased production of interferon-γ, (ix) increased Th1 response, (x) decreased Th2 response, (xi) decreased number or elimination of cells and/or activity of at least one of the regulatory T cells (Treg).

Assays to measure effectiveness.

In some embodiments of the present invention, T cell activation is assessed using a mixed lymphocyte response (MLR) assay as described in the Examples. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in the immune response, as measured, for example, by the phosphorylation or dephosphorylation of various factors or by measuring other post-translational modifications. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in activation of αβ and/or γδ T cells, as measured, for example, by cytokine secretion or proliferation, or by changes in the expression of activation markers, such as, for example, CD137, CD107a, PD1, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in cytotoxic T cell activity, as measured, for example, by direct killing of target cells, such as cancer cells, or by cytokine secretion, or proliferation, or by changes in the expression of activation markers, such as, for example, CD137, CD107a, PD1, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in NK and/or NKT cell activity, as measured, for example, by direct killing of target cells, such as, for example, cancer cells, or by the secretion of cytokines, or by changes in the expression of activation markers, such as, for example, CD107a, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in αβ and/or γδ T cell suppression, as measured, for example, by cytokine secretion or proliferation, or by changes in the expression of activation markers, such as CD137. , CD107a, PD1, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in the secretion of anti-inflammatory cytokines, as measured, for example, by ELISA, or by Luminex or Multiplex bead-based methods, or by intracellular staining and FACS analysis, or by using Alispot, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

- 80 044486

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in IL-2 secretion, as measured, for example, by ELISA, or by Luminex or Multiplex bead-based methods, or by intracellular staining and FACS analysis, or using Alispot, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in interferon-γ production as measured, for example, by ELISA, or by Luminex or Multiplex bead-based methods, or by intracellular staining and FACS analysis, or using Alispot, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in the Th1 response, as measured, for example, by cytokine secretion or changes in the expression of activation markers. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in the Th2 response, as measured, for example, by cytokine secretion or changes in the expression of activation markers. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in cell number and/or activity of at least one of the regulatory T cells (Tregs), as measured, for example, by flow cytometry or IHC. Decreased response indicates immunostimulatory activity. The corresponding reductions are the same as for the increases described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in the number of M2 macrophage cells, as measured, for example, by flow cytometry or IHC. Decreased response indicates immunostimulatory activity. The corresponding reductions are the same as for the increases described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in protumorigenic activity of M2 macrophages, as measured, for example, by cytokine secretion or changes in the expression of activation markers. Decreased response indicates immunostimulatory activity. The corresponding reductions are the same as for the increases described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in the number of neutrophils N2, as measured, for example, by flow cytometry or IHC. Decreased response indicates immunostimulatory activity. The corresponding reductions are the same as for the increases described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in protumorigenic activity of N2 neutrophils, as measured, for example, by cytokine secretion or changes in the expression of activation markers. Decreased response indicates immunostimulatory activity. The corresponding reductions are the same as for the increases described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in inhibition of T cell activation, as measured, for example, by cytokine secretion or proliferation, or by changes in the expression of activation markers, such as, for example, CD137, CD107a, PD1 etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in inhibition of CTL activation, as measured, for example, by direct killing of target cells, such as, for example, cancer cells, or by cytokine secretion, or by proliferation, or by changes in the expression of activation markers, such as, for example, CD137, CD107a, PD1, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures increases or decreases in αβ and/or γδ T cell exhaustion, as measured, for example, by changes in the expression of activation markers. Decreased response indicates immunostimulatory activity. The corresponding reductions are the same as for the increases described below.

In one embodiment of the present invention, the signaling pathway assay measures increases or decreases in αβ and/or γδ T cell responses, as measured, for example, by cytokine secretion or proliferation, or by changes in the expression of activation markers, such as, for example, CD137, CD107a, PD1, etc.

Increased activity indicates immunostimulating activity.

- 81 044486

The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in stimulation of antigen-specific memory responses, as measured, for example, by cytokine secretion or proliferation, or by changes in the expression of activation markers, such as, for example, CD45RA, CCR7 etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in apoptosis or lysis of cancer cells, as measured, for example, by cytotoxicity assays such as, for example, the MTT assay, Cr release assay, calcein AM assay, or by flow cytometry-based assays, such as CFSE dilution or propidium iodide staining, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in stimulation of a cytotoxic or cytostatic effect on cancer cells, as measured, for example, by cytotoxicity assays such as, for example, the MTT assay, Cr release assay, calcein AM assay, or by flow cytometry-based assays, as in the example of CFSE dilution or propidium iodide staining, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in direct killing of cancer cells, as measured, for example, by cytotoxicity assays such as, for example, the MTT assay, Cr release assay, calcein AM assay, or by based on flow cytometry, as in the example of CFSE dilution or propidium iodide staining, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in Th17 activity, as measured, for example, by cytokine secretion or proliferation or changes in the expression of activation markers. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, the signaling pathway assay measures the increase or decrease in induction of complement-dependent cytotoxicity and/or antibody-dependent cell-mediated cytotoxicity, as measured, for example, by cytotoxicity assays, such as, for example, the MTT assay, the Cr release assay , calcein AM assay, or by flow cytometry-based assays, as in the example of CFSE dilution or propidium iodide staining, etc. Increased activity indicates immunostimulating activity. The corresponding increases in activity are described below.

In one embodiment of the present invention, T cell activation is measured, for example, by direct killing of target cells, such as, for example, cancer cells, or by the secretion of cytokines, or by proliferation, or by changes in the expression of activation markers, such as, for example, , CD137, CD107a, PD1, etc. For T cells, increased proliferation, cell surface activation markers (e.g., CD25, CD69, CD137, PD1), cytotoxicity (ability to kill target cells), and cytokine production (e.g., IL-2, IL-4, IL-6, IFNy, TNF-α, IL-10, IL-17A) would indicate immune modulation, which is consistent with enhanced killing of cancer cells.

In one embodiment of the present invention, NK cell activation is measured, for example, by direct killing of target cells, such as, for example, cancer cells, or by the secretion of cytokines, or by changes in the expression of activation markers, such as, for example, CD107a, etc. .d. For NK cells, increased proliferation, cytotoxicity (the ability to kill target cells, and increased expression of CD107a, granzyme, and perforin), cytokine production (eg, IFNy and TNF), and expression of cell surface receptors (eg, CD25) may indicate immunomodulation, which consistent with enhanced killing of cancer cells.

In one embodiment of the present invention, activation of γδ T cells is measured, for example, by cytokine secretion, or proliferation, or changes in the expression of activation markers.

In one embodiment of the present invention, Th1 cell activation is measured, for example, by the secretion of cytokines, or by changes in the expression of activation markers.

Relevant increases in activity or response (or decreases, as appropriate, as above) are increases of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 90%, 95% or 98 to 99% compared to the signal in either the reference sample or control samples, for example, test samples that do not contain the anti-PVRIG antibodies of the present invention. Specific indicators of increased activity are depicted in the accompanying figures. For example, with regard to increasing T cell proliferation, CHA.7.518.1.H4 (S241P) demonstrated

- 82 044486 shows an increase of about 60%, and SNA.7.538.1.2.H4 (S241P) shows an increase of 47%; corresponding increases of about 10 to 70% are observed with either T cell proliferation or IFN-γ, with increases of about 20 to 60% also being used.

Likewise, an increase of at least 1, 2, 3, 4, or 5 times over reference or control samples indicates effectiveness.

X. Exemplary Embodiments of the Invention

1. A method of treating cancer in a patient, comprising:

a) obtaining a biopsy from the specified patient containing tumor cells;

b) measuring the number of PD-L1 positive tumor cells or immune system cells in said biopsy;

c) if the indicated number of PD-L1-positive tumor cells or immune cells exceeds 1% compared to the same tumor cells stained with an antibody corresponding to the isotype control antibody for the antibodies used, administration of a triple combination therapy comprising an anti-TIGIT antibody and an anti-TIGIT antibody. -PVRIG and anti-PD-1 antibody; and

d) if the indicated number of PD-L1-positive tumor cells or immune cells is less than 1% compared to the same tumor cells stained with an antibody corresponding to the isotype control antibody for the antibodies used, administration of a two-component combination therapy comprising an anti-TIGIT antibody and an antibody anti-PD-1.

2. The method of claim 1, wherein said anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

3. The method of claim 1, wherein said anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

4. The method of claim 1, wherein said anti-PD-1 antibody is an antibody selected from any of the anti-PD-1 antibodies described herein, including any of the antibodies described in FIG. 7.

5. The method according to any one of claims 1 to 4, characterized in that said anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 ( S241P), SNA.9.547.7.N4 (S241P) and SNA.9.547.13.N4 (S241P).

6. The method according to any one of claims 1 to 5, characterized in that said anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA7.518.1.H4 (S241P) and CHA.7.538.1.2. H4 (S241P).

The method according to any one of claims 1 to 6, characterized in that said anti-PD-1 antibody is an antibody selected from at least one of the following antibodies: pembrolizumab, cemiplimab and nivolumab.

8. The method according to any one of claims 1-7, characterized in that the specified two-component combination therapy is selected from the introduction of CPA.9.083.H4 (S241P) and CHA.7.518.1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SNA.9.547.13.N4 (S241P) and SNA.7.538.1.2.N4 (S241P).

9. The method according to any one of claims 1 to 8, characterized in that said two-component combination therapy is selected from the administration of CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); CHA.9.547.7.H4 (S241P, nivolumab and CHA.7.538.1.2.H4 (S241P); and CHA.9.547.13.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P).

10. Method according to any one of claims 1 to 9, characterized in that said antibodies are administered simultaneously.

11. Method according to any one of claims 1-10, characterized in that said antibodies are administered sequentially.

12. The method according to any one of claims 1 to 11, characterized in that said cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative cancer breast, pancreatic cancer, stomach cancer (gastric cancer), cervical cancer, head and neck cancer, thyroid cancer

- 83 044486 zy, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or CL ), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel cell cancer, high-MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

13. The method according to any one of claims 1 to 12, characterized in that said cancer is selected from the group consisting of triple negative breast cancer, stomach cancer (gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), and Merkel cell cancers, high-MSI cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS).

14. A method of treating cancer in a patient, comprising administering a three-component combination therapy containing an anti-TIGIT antibody, an anti-PVRIG antibody and an anti-PD-1 antibody.

15. The method according to claim 14, characterized in that said anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA .9.547.7.H4 (S241P) and CHA.9.547.13.H4 (S241P).

16. The method according to claim 14 or 15, characterized in that said anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA7.518.1.H4 (S241P) and CHA.7.538.1.2.H4 ( S241P).

17. The method according to any one of claims 14, 15 or 16, characterized in that said anti-PD-1 antibody is an antibody selected from the group consisting of pembrolizumab and nivolumab.

18. The method according to any one of claims 14-17, characterized in that said three-component combination therapy includes the administration of an anti-PD-1 antibody in combination with a two-component combination therapy selected from the administration of CPA.9.083.H4 (S241P) and CHA.7.518 .1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SHA.9.547.13.H4 (S241P) and CHA.7.538.1.2.H4 (S241P).

19. The method according to any one of claims 14-18, characterized in that said three-component combination therapy is selected from the administration of CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), pembrolizumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), pembrolizumab and CHA7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), pembrolizumab and SHA.7.538.1.2.H4 (S241P); CHA9.547.13.H4 (S241P), pembrolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), nivolumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), nivolumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P, nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), nivolumab and SHA.7.538.1.2.H4 (S241P); SHA.7.538 .1.2.H4 (S241P); CPA.9.083.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CNA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), cemiplimab and CHA.7.518.1.H4 (S241P); CHA.9.547.13.H4 (S241P), cemiplimab and

SNA.7.518.1.N4 (S241P); CPA.9.083.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), cemiplimab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), cemiplimab and

SNA.7.538.1.2.N4 (S241P); as well as SHA.9.547.13.H4 (S241P), cemiplimab and SHA.7.538.1.2.H4 (S241P).

20. Method according to any one of claims 14-19, characterized in that said antibodies are administered simultaneously.

21. Method according to any one of claims 14-20, characterized in that said antibodies are administered sequentially.

22. The method according to any one of claims 14-21, characterized in that said cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative cancer breast, pancreatic cancer, stomach cancer (stomach cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, germ cell testicular tumors, mesothelioma, esophageal cancer, Merkel cell cancer, high-MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

- 84 044486

23. The method according to any one of claims 14-22, characterized in that said cancer is selected from the group consisting of triple-negative breast cancer, stomach cancer (gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), and Merkel cell cancers, high-MSI cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS).

24. A set of pharmaceutical doses containing:

a) a container containing a unit dose of anti-TIGIT antibody; and

b) a container containing a unit dose of anti-PVRIG antibody.

25. A set of pharmaceutical doses containing:

a) a container containing a unit dose of anti-TIGIT antibody;

b) a container containing a unit dose of anti-PVRIG antibody; and

c) container containing αntu-PD-1 antibody.

26. The method of claim 24 or 25, wherein said anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

27. The method of claim 24 or 25, wherein said anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

28. The method of claim 24 or 25, wherein said anti-PD-1 antibody is an antibody selected from any of the anti-PD-1 antibodies described herein, including any of the antibodies described in FIG. 7.

29. A method comprising:

a) obtaining a population of cells from a tumor sample from the patient;

b) staining said population with labeled antibodies that bind:

i) TIGIT protein;

ii) PVRIG protein;

iii) PVR protein;

iv) PD-1 protein;

v) PD-L1 protein;

vi) PVRL2; and vii) appropriate isotype control for antibodies in i)-vi);

c) performing fluorescence activated cell sorting (FACS);

d) for each of TIGIT, PVRIG, PVR, PD-1, PVRL2 and PD-L1, determining the percentage of cells in the specified population that express the protein, relative to the specified isotype control antibody;

however, if the percentage of positive cells is > 1% for TIGIT or PVR, and for PVRIG or PVRL2, as well as for PD-1 or PD-L1, proceed to step e); and e) administering antibodies to TIGIT, PVRIG and PD-1 to the specified patient.

30. The method of claim 27, wherein said anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

31. The method of claim 27, wherein said anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

32. The method of claim 27, wherein said anti-PD-1 antibody is an antibody selected from any of the anti-PD-1 antibodies described herein, including any of the antibodies described in FIG. 7.

33. The method according to claims 29, 31 or 32, characterized in that said anti-TIGIT antibody is CPA.9.086.

34. The method according to claims 29, 30 or 31, characterized in that said anti-PD-1 antibody is selected from pembrolizumab, cemliplimab and nivolumab.

35. The method according to claim 29, 30 or 32, characterized in that said antibody to PVRIG is CHA.7.518.1.H4 (S241P).

36. A method of treating cancer in a patient, comprising:

a) obtaining a biopsy from the specified patient containing tumor cells;

b) measuring the number of PD-L1 positive tumor cells or immune system cells in said biopsy;

c) if the indicated number of PD-L1-positive tumor cells or immune cells exceeds 1% compared to the same tumor cells stained with an antibody corresponding to the isotype control antibody for the antibodies used, administration of a triple combination therapy comprising an anti-TIGIT antibody and an anti-TIGIT antibody. -PVRIG and αntu-PD-L1 antibody; and

- 85 044486

d) if the indicated number of PD-Ll-positive tumor cells or immune cells is less than 1% compared to the same tumor cells stained with an antibody corresponding to the isotype control antibody for the antibodies used, administration of a two-component combination therapy comprising an anti-TIGIT antibody and an anti-TIGIT antibody anti-PVRIG.

37. The method of claim 36, wherein said anti-TIGIT antibody is an antibody selected from any of the anti-TIGIT antibodies described herein, including any of the antibodies described in FIG. 3.

38. The method of claim 36, wherein said anti-PVRIG antibody is an antibody selected from any of the anti-PVRIG antibodies described herein, including any of the antibodies described in FIG. 5 and/or in FIG. 63.

39. The method of claim 36, wherein said anti-PD-1 antibody is an antibody selected from any of the anti-PD-L1 antibodies described herein, including any of the antibodies described in FIG. 62.

40. The method according to any one of claims 36-39, characterized in that said anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 ( S241P), SNA.9.547.7.N4 (S241P) and SNA.9.547.13.N4 (S241P).

41. The method according to any one of claims 36-39, characterized in that said anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA.7.518.1.H4 (S241P) and CHA.7.538. 1.2.Н4 (S241P).

42. The method according to any one of claims 36-41, characterized in that said anti-PD-L1 antibody is an antibody selected from at least one of the following antibodies: atezolizumab, avelumab and durvalumab.

43. The method according to any one of claims 36-42, characterized in that said two-component combination therapy is selected from the introduction of CPA.9.083.H4 (S241P) and CHA.7.518.1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SHA.9.547.13.H4 (S241P) and CHA.7.538.1.2.H4 (S241P).

44. The method according to any one of claims 36-43, characterized in that said three-component combination therapy is selected from the administration of CPA.9.083.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), atezolizumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), atezolizumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), atezolizumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), atezolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); CHA9.547.7.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), avelumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), avelumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), avelumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P, avelumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), avelumab and SHA.7.538.1.2.H4 (S241P); SHA7.538.1 .2.H4 (S241P); CPA.9.083.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CHA.9.547.13.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), durvalumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), durvalumab and CHA.7.538.1.2.H4 (S241P); CHA.9.547.7. H4 (S241P), durvalumab and SHA.7.538.1.2.H4 (S241P), and SHA.9.547.13.H4 (S241P), durvalumab and SHA.7.538.1.2.H4 (S241P).

45. The method according to any one of claims 36-44, characterized in that said antibodies are administered simultaneously.

46. The method according to any one of claims 36-45, characterized in that said antibodies are administered sequentially.

47. The method according to any one of claims 36-46, characterized in that said cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative cancer breast, pancreatic cancer, stomach cancer (stomach cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, germ cell testicular tumors, mesothelioma, esophageal cancer, Merkel cell cancer, high-MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

48. The method according to any one of claims 36-47, characterized in that said cancer is selected from the group with

- 86 044486 from triple-negative breast cancer, stomach cancer (gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), as well as Merkel cell cancer, cancer with a high level of MSI, KRAS-mutant tumors, T-cell adult leukemia/lymphoma and myelodysplastic syndromes (MDS).

49. A method of treating cancer in a patient, comprising administering a three-component combination therapy containing an anti-TIGIT antibody, an anti-PVRIG antibody and an αntu-PD-L1 antibody.

50. The method according to claim 49, characterized in that said anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P), CPA.9.086.H4 (S241P), CHA .9.547.7.H4 (S241P) and CHA.9.547.13.H4 (S241P).

51. The method according to claim 49 or 50, characterized in that said anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA.7.518.1.H4 (S241P) and CHA.7.538.1.2 .H4 (S241P).

52. The method according to any one of claims 49, 50 or 51, characterized in that said anti-PD-L1 antibody is an antibody selected from the group consisting of atezolizumab, avelumab and durvalumab.

53. The method according to any one of claims 49 to 52, characterized in that said three-component combination therapy includes the administration of an anti-PD-L1 antibody in combination with a two-component combination therapy selected from the administration of CPA.9.083.H4 (S241P) and CHA.7.518 .1.H4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.518.1.N4 (S241P); SNA.9.547.13.N4 (S241P) and SNA.7.518.1.N4 (S241P); SPA.9.083.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SPA.9.086.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); SNA.9.547.7.N4 (S241P) and SNA.7.538.1.2.N4 (S241P); as well as SHA.9.547.13.H4 (S241P) and CHA.7.538.1.2.H4 (S241P).

54. The method according to any one of claims 49-53, characterized in that said three-component combination therapy is selected from the administration of CPA.9.083.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); CHA.9.547.7.H4 (S241P), atezolizumab and CHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), atezolizumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), atezolizumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P), atezolizumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), atezolizumab and SHA.7.538.1.2.H4 (S241P); CPA.9.083.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), avelumab and CHA.7.518.1.H4 (S241P); SHA.9.547.7.H4 (S241P), avelumab and SHA.7.518.1.H4 (S241P); SHA.9.547.13.H4 (S241P), avelumab and SHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), avelumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), avelumab and CHA.7.538.1.2.H4 (S241P); SHA.9.547.7.H4 (S241P, avelumab and SHA.7.538.1.2.H4 (S241P); SHA.9.547.13.H4 (S241P), avelumab and SHA.7.538.1.2.H4 (S241P); SHA7.538.1 .2.H4 (S241P); CPA.9.083.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CPA.9.086.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P ); CHA.9.547.7.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CHA.9.547.13.H4 (S241P), durvalumab and CHA.7.518.1.H4 (S241P); CPA.9.083.H4 (S241P), durvalumab and CHA.7.538.1.2.H4 (S241P); CPA.9.086.H4 (S241P), durvalumab and CHA.7.538.1.2.H4 (S241P); CHA.9.547.7. H4 (S241P), durvalumab and SHA.7.538.1.2.H4 (S241P), and SHA.9.547.13.H4 (S241P), durvalumab and SHA.7.538.1.2.H4 (S241P).

55. Method according to any one of claims 49-54, characterized in that said antibodies are administered simultaneously.

56. The method according to any one of claims 49-54, characterized in that said antibodies are administered sequentially.

57. The method according to any one of claims 49-56, characterized in that said cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative cancer breast, pancreatic cancer, stomach cancer (stomach cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, germ cell testicular tumors, mesothelioma, esophageal cancer, Merkel cell cancer, high-MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS).

58. The method according to any one of claims 49-57, characterized in that said cancer is selected from the group consisting of triple-negative breast cancer, stomach cancer (gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), and Merkel cell cancers, high-MSI cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS).

- 87 044486

Examples

Example 1: Functional tests.

The purpose of this study was to characterize the functional activity of CHA.7.518.1.H4 (S241P) on human T cell function, either alone or in combination with anti-TIGIT and/or anti-PD-1 antibody in primary in vitro cell assays. We demonstrated that CHA.7.518.1.H4 (S241P) enhances cytokine production by viral antigen-specific CD8 T cells, used as a model surrogate antigen to study CD8 T cell responses. The combination of CHA.7.518.1.H4 (S241P) with anti-TIGIT antibody results in an additive or, under some conditions, synergistic enhancement of T cell function. The inventors used a three-component combination of CHA.7.518.1.H4 (S241P), anti-TIGIT and anti-PD-1 and observed the greatest increase in the function of T cells co-cultured with PD-L1high target tumor cells when using a three-component combination of CHA .7.518.1.H4 (S241P) versus two-component combination or single antibody. In co-culture with PD-L1hu3k target tumor cells, the three-component combination of CHA.7.518.1.H4 (S241P), anti-TIGIT, anti-PD-1 did not further enhance T-cell function compared to the two-component combination CHA.7.518.1 .H4 (S241P) and anti-TIGIT, suggesting that treatment with SHA.7.518.1.H4 (S241P) and anti-TIGIT may be effective in patients with low or negative PD-L1 expression. Taken together, the authors demonstrated the effect of CHA.7.518.1.H4 (S241P) on enhancing human CD8+ T cell function, alone or in combination with anti-TIGIT or anti-PD-1.

This report describes the characterization of CHA.7.518.1.H4 (S241P), a fully humanized anti-PVRIG IgG4 antibody, in cell-based assays. CHA.7.518.1.H4 (S241P) binds to PVRIG with high affinity and specificity and blocks the interaction of PVRIG with PVRL2. To understand the effect of CHA.7.518.1.H4 (S241P) on T cell function, we examined the effect of CHA.7.518.1.H4 (S241P) on cytokine production in an in vitro assay. This assay was developed based on the 2-signal hypothesis of T cell activation: signal 1 comes from T cell receptor activation; Signal 2 are immunomodulatory receptors that help enhance or inhibit T cell responses. The design of these assays consists of co-cultivating human T cells with a target cell line that has been injected with a viral antigen (CMV)-derived antigenic peptide. This signal provides T cell activation signal 1 through the T cell receptor. These target cell lines express endogenous PVRL2, and in this context, PVRL2 provides signal 2 to the T cell.

CMV: tumor cell line assay.

CMVpp65-reactive T cells were expanded by thawing CMV-reactive donor cells according to the CTL protocol. Thaw cryopreserved PBMCs, and 2e6 cells/ml were resuspended in medium (Gibco) supplemented with 1% glutamax (Gibco), 1% NEAA, penicillin/streptomycin (Gibco), 10% human AB serum (Corning), 1 μg/ml CMV peptide, 2 ng/ml IL2 (R&D), and 10 ng/ml IL-7 (R&D). PBMCs were cultured for eight days with the addition of IL-2 and IL-7 on the third and sixth days. On day 8, cells were harvested and replicated with low dose IL-2 (100 U/ml) at 2e6/ml in complete RPMI medium for 5 days. On day nine, cells were phenotyped for CD8+ T cell purity and CMV tetramer reactivity. Cells were stained with 0.25 μl CD3 (clone: OCT)-allophycocyanin seven (APC-Cy7; Biolegend), 0.25 μl CD8 (clone: H1T8a)-Alexafluor 488 (AF488; Biolegend), 0.125 μl CD14 (clone: HCD14) , 0.5 µl CD19 (clone: HIBCD14), 0.5 µl CD56 (clone: HCD56) peridinin-chlorophyll protein (PerCP; Biolegend), 1.25 µl TIGIT (clone: MBSA43)-allophycocyanin (APC; e-Bioscience ) or 1.25 µl isotype control IgG4 (in-lab) (APC: Biolegend), 1.25 µl CHA.7.518.1.H4 (S241P)-allophycocyanin (in-lab) or 1.25 µl isotype control IgG4-APC (in-lab) and 0.5 μl of PD-1 (clone: EH12.2H7)-brilliant violet 421 (BV421; Biolegend) or 1.25 μl of IgG1 (clone: MOPC21)-brilliant violet 421 (BV421; Biolegend). To assess the number of tetramer-reactive CD8+ T cells, unlabeled PBMCs were stained after culture with 10 μl iTAg tetramer-HLA-A*02:01 CMV pp65 (NLVPMVATV)-phycoerythrin (PE, MBL-BION) for 30 min at room temperature. Cells were washed with PBS/BSA/azide solution and resuspended in buffer). Data were acquired using Fortessa and analyzed using FlowJo (Treestar) and Prism (Graphpad).

The target cells used in the co-culture assay were Panc.05.4 and Colo205 (ATCC) cell lines. These cell lines were stained with 1.25 μl PVR (SKII.4)-phycoerythrin (PE, Biolegend), 1.25 μl PVRL2 (TX31)-peridinin-chlorophyll protein (PerCP5.5; Biolegend), 2.5 μl PD-L1 (29E.2A3)-brilliant violet 785 (BV785; Biolegend) and 1.25 μl of HLA-A2 (BB7.2) allophycocyanin (APC; Biolegend). 1.25 μl of the corresponding isotype for each fluorophore (MORS-21) was also evaluated.

For co-culture, tumor cell lines were harvested from culture and tumor cell lines were injected with CMV peptide (Anaspec) for 1 hour at 37°C with occasional agitation. After incubation, target cells were washed thoroughly, counted, and resuspended in complete RPMI medium. Assays were performed with a ratio of T cells (100,000) to cells

- 88 044486 targets (100000) 1:1. Target cells, T cells, and 10 μg/ml of each antibody were co-added to a 96-well U-bottom plate (Costar) and incubated for 24 h at 37°C. Antibody treatments included CHA.7.518.1.H4 (S241P) hIgG4, anti-TIGIT hIgG4 (Reference 26, Compugen), anti-PD-1 hIgG4 (Reference 3, Compugen), and human IgG4 isotype control (Compugen). To match total antibody concentrations across all single and combination groups, an additional human IgG4 isotype control was added in single or double combinations to a final total antibody concentration of 30 μg/mL. After a 24-hour incubation period, coculture supernatants were assayed for secreted cytokines including IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F , IL-21, IL-22, TNF-α and/or IFN-γ, using the Human Cytokine Thl/Th2/Th17 Microsphere Array (CBA) Cytometry Kit (BD Biosciences) or using the Cytometry Kit human Th cytokines LEGENDplex™ (BioLegend). Data were acquired using Fortessa and analyzed using FlowJo (Treestar) and Prism (Graphpad).

1. Results and discussion.

A. CMV T cell assay: CMVpp65 reactive T cells express PVRIG, TIGIT and PD1.

Human cytomegalovirus (CMV) is a widespread, persistent Herpesvirus that affects a large percentage of the population, with slightly lower seroprevalence in Western Europe and the United States (Cannon MJ et al. 2010). The immune system of patients with chronic viral infections or cancer is often functionally impaired and unable to mount an effective response against the virus or recognize and eliminate malignant cells. In these patients, the expression of inhibitory receptors is increased and this has been found to be associated with T cell dysfunction. Thus, activation of negative checkpoint receptors may serve as a potential target for reversing T cell exhaustion. CD8 T cells specific for the CMV pp65 protein have been well characterized, and these CMV-specific T cells can be used to study the role of modulatory receptors on T cells.

Stimulation of HLA-A2 + donor PBMCs with CMV pp65 peptide, IL-2, and IL-7 resulted in robust expansion of CMV pp65-specific T cells to a purity ranging from 50 to 90% as determined by tetramer staining. In fig. 8A shows the percentage of CMV pp65-specific T cells from multiple donors after expansion. Surface expression of PVRIG, TIGIT, and PD-1 on T cells was assessed from CMV+ donors and compared to isotype-matched receptor expression using flow cytometry. CMV pp65-specific T cells expressed PVRIG (median gMFI ratio: 7), TIGIT (median gMFI ratio: 37), and PD1 (median gMFI ratio: 2) on day 9 of activation (in Fig. 8B).

In addition, the authors assessed the kinetics of PVRIG, TIGIT, and PD1 expression relative to the expansion of CMV pp65-specific T cells over time. For each donor, the number of CMVpp65-reactive CD8+ T cells was plotted over time (in Fig. 9A). A significant increase in CMVpp65-reactive T cells (range: 50-97%) was observed in all donors, with donor 198 showing initial expansion on day three (Day 3 CMV + percentage: 85.7%). However, donor 198 had loss of CMV tetramer expression on day 6 (CMV Day 6 + percentage: 50.4%). The expression of PVRIG, TIGIT and PD-1 on CMV-specific CD8+ T cells was assessed by flow cytometry over a course of twelve days. In donor 4, donor 198, and donor 210, TIGIT expression among CMVpp65-specific CD8+ T cells increased over the twelve-day expansion period (average gMFIr expansion in three donors, TIGIT expression in three donors, Day 0 gMFIr: 1.2, Day 12 gMFIr : 47) (in Fig. 9B). PVRIG expression on CMV+ T cells was also increased (average gMFI PVRIG for three donors, Day 0 gMFIr: 0.92, Day 12 gMFIr: 8.6) (Fig. 6C). Expression of gMFI PVRIG was also assessed, with the authors observing minimal induction of expression (average gMFIr PD-1, Day 0 gMFI: 0.93, Day 12 gMFI: 2) (in Fig. 9C).

b. Antibodies CHA.7.518.1.H4 (S241P), anti-TIGIT and anti-PD1 enhanced IFN-γ secretion.

Given that increased expression of TIGIT, PVRIG, and PD-1 by CMV CD8 cells correlates with T cell dysfunction, we aimed to evaluate the effect of blockade of PVRIG, TIGIT, and PD-1 on the ability to produce anti-inflammatory cytokines. CMVpp65-reactive T cells from 2 donors were co-cultured with CMV peptide-loaded PD-L1high (Panc04.05) and PD-L1hu3k (Colo205) tumor cell lines prior to flow cytometry analysis to assess cytokine production (Fig. 10 ).

In a co-culture of Panc.04.05 (PD-L1high), the authors observed that a single anti-TIGIT blockade increased IFN-γ production compared to control IgG mAbs, whereas CHA.7.518.1.H4 (S241P) or anti-PD-1 had minimal effect (in Fig. 11). Dual blockade with antiTIGIT and CHA.7.518.1.H4 (S241P) synergistically and consistently increased cytokine production by CD8+ T cells compared with blockade with CHA.7.518.1.H4 (S241P) or anti-TIGIT alone. In donor 4, an additional increase in IFN-γ production was observed with

- 89 044486 three-component combination of CHA.7.518.1.H4 (S241P), anti-TIGIT and αntu-PD-1, indicating that when PD-L1, PVR and PVRL2 are expressed at high levels on tumor cells, the greatest increase T cell activation is achieved using a three-component combination. In Colo205 (PD-L1hu3k) co-cultures, blockade with anti-TIGIT alone increased IFN-γ secretion, whereas CHA.7.518.1.H4 (S241P) or anti-PD1 antibody had minimal effect, similar to the results obtained with co-culture Panc.04.05. Also similar to the Panc.04.05 coculture, dual blockade with anti-TIGIT and CHA.7.518.1.H4 (S241P) also synergistically increased IFN-γ production compared with anti-TIGIT, anti-PD-1, or CHA.7.518. 1.H4 (S241P) used alone, and with greater potency when using either CHA.7.518.1.H4 (S241P) or antiTIGIT in combination with αntu-PD-1. In contrast to Panc.04.05 (PD-L1hi), the condition of the three-component combination for donor 4 was no better than the two-component combination of CHA.7.518.1.H4 (S241P) and anti-TIGIT in the Colo205 co-culture (PD-Low), pushing to the suggestion that when PVR and PVRL2 are expressed at high levels on tumor cells and PD-L1 is expressed at low levels, the two-component combination of CHA.7.518.1.H4 (S241P) and anti-TIGIT results in the greatest increase in IFN-γ expression γ. These results demonstrate that blockade of TIGIT and PVRIG was sufficient to enhance CD8+ T cell responses in PD-L1 ^hu3k tumors, and that the three-component combination resulted in the greatest increase in T cell activation in PD-L1 ^b “ ^c tumors.

With. Summary.

Human anti-CMV T cell responses are used as an in vitro antigen-specific method to assess the functional capacity of a checkpoint inhibitor antibody. The authors observed that combined blockade of TIGIT and CHA.7.518.1.H4 (S241P) resulted in greater recovery of T cell function compared with blockade with antibody alone, suggesting that disruption of the TIGIT and PVRIG pathways may be more important. than disruption of the PD1 pathway in CD8 tumor cell cultures. In addition, the authors observed that triple blockade using antibodies to PD1, TIGIT, and PVRIG could result in the greatest increase in IFN-γ production when PD-L1 positive tumor cells or immune cells were >1%, equivalent to high levels of PD-L1 expression . These results demonstrate that blockade of TIGIT and PVRIG was sufficient to enhance CD8+ T cell responses in PD-L1hu3k tumors, and that the three-component combination resulted in the greatest increase in T cell activation in PD-L1high tumors.

The present invention provides methods comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) TIGIT protein; ii) PVRIG protein; iii) PVR protein; iv) PD-1 protein; v) PD-L1 protein; vi) PVRL2; and vii) appropriate isotype control for antibodies in i)-vi); c) performing fluorescence activated cell sorting (FACS); d) for each of TIGIT, PVRIG, PVR, PD-1, PVRL2 and PD-L1, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for TIGIT or PVR, and for PVRIG or PVRL2, as well as for PD-1 or PD-L1, proceed to step e); and e) administering antibodies to TIGIT, PVRIG and PD-1 to said patient.

The present invention provides a method comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) TIGIT protein; ii) PVRIG protein; iii) PVR protein; iv) PD-1 protein; v) PD-L1 protein; vi) PVRL2; and vii) appropriate isotype control for antibodies in i)-vi); c) performing fluorescence activated cell sorting (FACS); d) for each of TIGIT, PVRIG, PVR, PD-1, PVRL2 and PD-L1, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for TIGIT or PVR, and for PVRIG or PVRL2, as well as for PD-1 or PD-L1, proceed to step e); and e) administering antibodies to TIGIT, PVRIG and PD-1 to said patient.

The present invention provides a method comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) TIGIT protein; and ii) PVR protein; and iii) the appropriate isotype control for the antibodies in i)-ii); c) performing fluorescence activated cell sorting (FACS); d) for each of TIGIT and PVR, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for TIGIT or PVR, proceed to step e); and e) administering antibodies to TIGIT, PVRIG and PD-1 to said patient.

The present invention provides a method comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) PVRIG protein; and ii) PVRL2 protein; and iii) the appropriate isotype control for the antibodies in i)-ii); c) performing fluorescence activated cell sorting (FACS); d) for each of PVRIG and PVRL2, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; Moreover, if the percentage is positive

- 90 044486 cells are > 1% for PVRIG or PVRL2, go to step e); and e) administering antibodies to TIGIT, PVRIG and PD-1 to said patient.

The present invention provides a method comprising: a) obtaining a population of cells from a tumor sample from a patient; b) staining said population with labeled antibodies that bind: i) PD-1 protein; and ii) PD-1 protein; and iii) the appropriate isotype control for the antibodies in i)-ii); c) performing fluorescence activated cell sorting (FACS); d) for each of PD-1 and PD-L1, determining the percentage of cells in the specified population that express the protein relative to the specified isotype control antibody; however, if the percentage of positive cells is > 1% for PD-1 or PD-1, proceed to step e); and e) administering antibodies to TIGIT, PVRIG and PD-1 to said patient.

Example 2: Expression of PVRIG and PVRL2 in human cancerous and normal adjacent tissues.

The aim of this study was to examine the expression of PVRIG and PVRL2 in human tumor samples and normal adjacent tissues. PVRIG was found to be expressed most highly on CD8+ T cells, followed by NK cells, CD4-CD8 T cells, and CD4+ T cells. No expression was observed on monocytes, mDCs, pDCs, or tumor cells. Of the tumor types examined, endometrial, lung, and kidney tumors expressed the highest levels of PVRIG on lymphocytes. Comparison of PVRIG expression on CD4+ and CD8+ T cells from normal adjacent tissues versus tumor tissues from the same patient demonstrated a significant increase in PVRIG in tumor tissues. Correlation analysis of PVRIG expression level with TIGIT or PD-1 expression level demonstrated a positive and significant correlation on CD4 and CD8 T cells. In addition, single cell co-expression analysis of PD-1, TIGIT and PVRIG demonstrated that PVRIG is co-expressed with PD-1 and TIGIT in a subset of cells. These data support the conclusion that combined blockade of PVRIG with TIGIT and/or PD-1 will result in increased levels of T-cell responses. The ligand for PVRIG, PVRL2, was expressed on myeloid cells (monocytes, mDC, pDC) and CD45-nonimmune cells from a variety of tumors, likely consisting of tumor epithelium and stromal cells. Comparison of PVRL2 on cells derived from normal adjacent tissue and tumor tissue demonstrated a significant increase in PVRL2 expression on monocytes and CD45 non-immune cells. Correlation analysis of PVRL2 expression level with PD-L1 expression level demonstrated a positive and significant correlation with CD45-non-immune cells and monocytes. In these samples, the authors also assessed the co-expression of PVRIG and PVRL2 in the same sample to understand which tumor type has high levels of co-expression of both receptor and ligand. Among the tumor types examined, the authors observed high levels of expression of both PVRIG and PVRL2 in the majority of endometrial, kidney, and lung tumor samples. In summary, these data demonstrate that PVRIG and PVRL2 are expressed on leukocytes and tumor cells from the tumor microenvironment and suggest that this pathway may be used to regulate antitumor responses.

The authors examined the expression of PVRIG and PVRL2 using flow cytometry on cells from dissected human tumors and compared with and normal adjacent tissues from a variety of different tissues. Measures of tumor expression of immune regulators can be used to predict which tumor types or patients may be most responsive to specific therapies.

Human peripheral blood mononuclear cells from healthy donors (PBMCs) were obtained from the Stanford Blood Bank. Buffy coats or LRS products were diluted 1:1 in 1× PBS + 2% FBS, and PBMCs were isolated using a Ficoll-Paque gradient (Sigma). Purified PBMCs were washed 2 times with PBS + 2% FBS and placed in liquid nitrogen. Tumor and normal adjacent tissue (NCT) samples were provided by the United Human Tissue Network, a resource supported by the National Cancer Institute. Tumor type was determined based on review of the histopathological characteristics report for each specimen. The number of samples per tumor type that the authors examined for PVRIG and PVRL2 expression is listed below.

Target Talspya intestine, Flour gland _pr „ _Ш| stomach Endometrium and uterus Head and neck Kidneys Lungs Prostate Ovaries PVRIG J 15 24 J 14 5 5 PVRL2 : :1 22 1E And ι: K ¹

1. Tumor dissection protocol.

Tumor and NCT samples were cut into small pieces with a scalpel and transferred into GentleMAC™ C tubes (Miltenyi Biotec) containing an enzyme mixture. Samples were dissected on GentleMAC (Miltenyi Biotec) according to the manufacturer's protocol. After dissection, cells were filtered through a 100-μm filter prior to FACS staining.

- 91 044486

2. Antibodies and reagents.

In order to identify immune and non-immune cell populations, the following antibodies were used at the concentrations recommended by the manufacturer.

table 2

Antibodies have been demonstrated to be used to identify subsets of specific cells

Antibody Fluorophore Clone Provider Neuer no catalog SM5 Ales Huor 700 ISO In Legend EOKED CD3 DRSSu7 gas BoLegend 317342 SE" No. 785 RPAI8 BoLegend 300046 CD33 No. 711 WM53 EioLegend 30B424 CD25 №650 VS96 BoLegend 30204 CD127 No. 605 401905 BoLegend 351334 CD14 BUV395 MoRE EDPhamingen 50562 SEN BUV496 SK3 EDPharmngai 564651 CD56 PEtezde KD56 BoLegend 31Я348

The following antibodies were used at a concentration of 5 μg/ml in an isotype control cocktail.

Table 3

Demonstrated antibodies used as isotype controls for targets of interest

Antibody Fluorophore Clone Provider Number according to ___ catalog _ hmm DR647 mine Soggrisrp mine Ides re MINE Sogtrisrp mine hmm hell MOPZ21 Boie^nd 400158 hmm R&CP 0/5-5 MORS21 Boie^nd 40Q15D hmm geo/7 mogsa Bete^nd 400126 hmm ts MORS21 Boietpnd lggtpl

The following cocktail was used to stain targets of interest at a concentration of 5 μg/ml.

Table 4

Antibodies used to analyze targets of interest have been demonstrated

Antibody Fluorophore Clone Provider Catalog number bastard DR547 VM1(M1) CorrpiKfn mine Pu4G π sasw Bclecpnd 30000 GO1 hell. M2E17(MC Bcle^nd 32SSQD Nt RrCP 0/5-5 hall (mi lzli2 FVR2 geo/7 1X31 (Ml) Bcletpnd 337414 ΤΚΙΓ HIT ΝΕ9Μ3(Μΰ eYoeoyatsE 11-900042

All isotype control and target antibodies were used at a final concentration of 5 μg/ml.

3. FACS staining.

1x10 ⁶ PBMCs or dissected tumor cells were seeded in a 96-well plate with a V-shaped well bottom for staining. Samples were first stained with Aqua Live Dead (Thermo Scientific) to distinguish live from dead cells and with a cocktail of anti-CD16 (Biolegend), anti-CD32 (Thermo Scientific), anti-CD64 (Biolegend) Abs to block Fc receptors. Samples were washed twice with FACS buffer and stained with the appropriate isotype control for the antibodies used, or with the target antibody cocktail described in the Antibodies and Reagents section. All staining was performed for 30 min at 4°C. Samples were then washed twice and collected on a BD Fortessa flow cytometer. The analysis was performed using FlowJo, gating on specific populations as indicated in Table. 1 above (all gating was performed on living cells).

From each population containing at least 100 cells, MFI values were exported, and the ratio

- 92 044486 MFI (MFIr) was calculated by dividing the MFI of the target by the MFI of the corresponding isotype control. An MFIr value greater than one indicates positive expression detected.

4. Results and discussion.

a. PVRIG is expressed on TILS from a variety of tumor types and is coexpressed with TIGIT and PD1.

To examine PVRIG expression on tumor-derived cells using flow cytometry, tumors were dissected and stained for immune cell lineage markers to identify immune and nonimmune cell subsets, and for PVRIG, TIGIT, PD1, PVRL2, and PVR to examine expression these targets on these subsets. PVRIG expression was detected on CD4+ T cells, CD8+ T cells, CD4 CD8 T cells and NK cells from breast, colon/rectal/stomach, endometrial, head and neck, lung, kidney, prostate and ovarian tumors (in Fig. 12A). In all tumor tissues examined, PVRIG was expressed from highest to lowest levels on CD8+ T cells, NK cells, CD4 CD8 T cells, and CD4+ T cells. No PVRIG expression was observed on monocytes, mDCs, pDCs, or nonimmune cells (Fig. 1B). Since CD8+ T cells and NK cells are known to be important cytotoxic lymphocytes in the immune system, this suggests that PVRIG may directly modulate the activity of these cytotoxic lymphocytes.

We then examined the expression level of PVRIG relative to that of TIGIT and PD-1 on tumor-infiltrating T cells. For this analysis, the authors focused on endometrial samples because there were sufficient samples available to perform correlation analyses. The expression level of PVRIG was significantly and directly correlated with the expression level of TIGIT and PD1 on both CD4 and CD8 T cells, suggesting that these molecules are co-regulated within the TME (in Fig. 13).

In addition, the authors examined the co-expression of PVRIG, TIGIT and PD1 on a single-cell basis on CD8 T cells. Co-expression of PVRIG with PD-1 and TIGIT was observed in representative lung and kidney cancer cells (in Fig. 14).

b. PVRIG expression was significantly increased on T cells from the tumor compared with normal adjacent tissue.

For a subset of colon/rectal/stomach, endometrial, kidney, lung, or ovarian tumor cells, the authors were able to obtain samples of the corresponding tumor and a normal adjacent tumor (NCT) from the same donor. Using these matched samples, we compared PVRIG and PD1 expression on cells derived from NCT or tumor samples to determine whether expression was modulated in tumor compared to healthy tissue (in Fig. 15). Overall, PVRIG expression was significantly increased on CD4 and CD8 T cells derived from tumor tissue compared with the corresponding normal adjacent tissues (Fig. 15A). Among tumor types, PVRIG expression was increased at least 2-fold in 3 of 9 colon tumors, 1 of 2 endometrial tumors, 4 of 11 kidney tumors, and 4 of 5 lung tumors (Figure 15A). The authors also assessed PD-1 expression in the same samples. Correlation analysis between PVRIG fold change (between NCT and tumor) and PD-1 fold change on CD4 and CD8 T cells demonstrated a positive and significant correlation in these samples, suggesting that these molecules may be co-regulated in a similar manner in tumor .

c. PVRL2 is expressed on myeloid and CD45 (non-immune) cells from a variety of tumors.

In the same samples from which the authors examined PVRIG expression, the authors also examined the expression of PVRL2, a PVRIG ligand. PVRL2 expression was detected on 2 major cell subsets: myeloid cells, which include monocytes, mDC, and the pDC population, and CD45-nonimmune cells, likely consisting of epithelial tumor cells, stromal cells, and endothelial cells (Figure 16). ).

Expression of PVRL2 on CD45-non-immune cells was detected on tumor cells of the breast, colon/rectum/stomach, endometrium, lung, prostate and ovary (Fig. 17). The highest median expression of PVRL2 was found on endometrial and ovarian tumor cells.

On immune cells, PVRL2 was expressed on myeloid cells from breast, colon/rectal/stomach, endometrial/uterine, head and neck, lung, kidney, prostate and ovary tissue (Figure 18).

Median PVRL2 expression on myeloid cells (monocytes, mDC, pDC) was comparable across tumor types.

When comparing tumor tissue with normal adjacent tissue, PVRL2 expression was significantly increased on CD45 tumor cells or on monocytes from tumor tissues (Figure 19). PVRL2 expression on monocytes or CD45 cells was induced at least 2-fold higher in tumors compared to normal tissue: in 5 of 9 colon tumors, 1 of 2 endometrial tumors, 5 of 11 kidney tumors, 4 of 5 tumors lung and 1 of 1 ovarian tumor. These data support increased expression of PVLR2 on tumor cells and on immune cells in the tumor. Correlation

- 93 044486 analysis between PVRL2 fold change (between NCT and tumor) and PD-L1 fold change on CD45 cells and monocytes demonstrated a positive and significant correlation in these samples, suggesting that these molecules may be co-regulated in a similar manner in tumors.

d. PVRIG and PVRL2 are coexpressed in the same tumor.

The authors further assessed which tumor types had high expression of both PVRIG on T cells and PVRL2 on monocytes and tumor cells. In this set of samples, tumors from uterine, lung and kidney tissues showed high levels of expression of both PVRIG on T cells and PVRL2 on either monocytes or CD45 cells (in Fig. 20), suggesting that these tumor types may be more responsive to treatment with SHA.7.518.1.H4 (S241P).

e. Conclusion.

The results of these studies demonstrate that PVRIG is expressed on effector lymphocytes such as CD8 T cells and NK cells in the tumor microenvironment. Both PVRIG and PVRL2 were expressed in a variety of breast, colon/rectal/stomach, endometrial, lung, prostate, and ovarian tumor samples. PVRIG expression on T cells was significantly higher in tumor tissues compared with corresponding normal adjacent tissues. In addition, a significant direct correlation was observed between the expression of PVRIG and PD-1 and PVRIG and TIGIT on CD4 and CD8 T cells from endometrial tissue samples. Based on single-cell analysis, co-expression of PVRIG with PD1 or with TIGIT was observed on CD8 T cells. The ligand for PVRIG, PVRL2, is expressed on antigen-presenting cells (monocytes, mDC, pDC) as well as CD45 cells (presumably composed of epithelial, stromal, endothelial cells) from a variety of tumor tissues. Induction of PVRL2 expression was detected in tumor-derived cells compared with normal adjacent tissues. The cellular expression profile of the receptor and ligand suggests a role for this pathway in regulating lymphocyte effector responses in a variety of tumor types.

Example 3: PVRL2 and PD-L1 expression in human cancer and normal tissues analyzed by IHC.

The aim of this study was to examine the expression of PVRL2 and PD-L1 in healthy and cancerous human tissue. Two anti-PVRL2 antibodies were identified to stain PVRL2 in formalin-fixed paraffin-embedded (FFPE) specimens. PD-L1 was detected using a commercially validated antibody. Using these antibodies, the authors examined the expression of PVRL2 and PD-L1 in serial tissue sections of a tumor microarray (TMA) consisting of breast, colon, lung, ovarian, and skin tissues. Increased expression of PVRL2 was observed in breast, colon, lung, ovarian, and skin cancers compared with healthy tissues. Similar staining was observed between the two anti-PVRL2 antibodies, helping to confirm the results obtained. PD-L1 expression was also increased in breast, colon, lung, ovarian and skin cancers compared to healthy tissues. PVRL2 expression was observed in tumor epithelium as well as in infiltrating immune cells. These tumor samples showed a higher frequency of PVRL2 expression compared to PD-L1 expression. Individual tumor samples were further grouped by negative and positive PD-L1 expression, and PVRL2 expression was analyzed in these subgroups. All PD-L1-positive tumors also expressed PVRL2, providing rationale for combination tumor treatment. In PD-L1-negative tumors, PVRL2 expression was detected in a subset of these samples, providing a rationale for targeting the PVRL2 pathway in PD-L1-negative tumors. Taken together, these data demonstrate that PVRL2 expression was increased in the tumor microenvironment in breast, colon, lung, ovarian, and skin cancers and provide a rationale for monotherapy and combination treatments using agents targeting the PVRL2 pathway.

Protocols.

Antibodies.

Anti-PVRL2 (Abeam ab135246, Sigma HPA-012759) and anti-PDL1 (SpringBio Sp142) were used in this study. An isotype control antibody (rabbit IgG) was used as a negative control.

IHC staining.

Tumor microarrays were obtained from breast, colon, lung, ovary, and skin. Each microarray contains healthy tissue from 2-4 donors and tumor tissue from 30-40 donors, present in duplicate in microslides. Ahtu-PVRL2 Abeam abl35246 staining was performed at a 1:250 dilution without heat-induced antigen retrieval (HTER). AntiPVRL2 (Sigma HPA-012759) was used at a concentration of 0.1 μg/ml with HTER at pH 9.5. Anti-PD-LI (SpringBio SP142) was used at a concentration of 1 μg/ml with HTER at pH 6.2 according to the manufacturer's recommendations. The appropriate rabbit IgG isotype control was used under each of the appropriate conditions. Two separate operators qualitatively scored each nucleus according to the following scale: no staining (score 0), partially positive (score 1), positive

- 94 044486 (point 2), very positive result (point 3). In case of discrepancies in scores between two operators, the sample was re-scored by both operators to obtain a final score. The score of 2 cores obtained from the same tumor was averaged, and one score was obtained for each tumor. Results were plotted and samples were grouped according to histopathology data provided by the supplier.

Results and discussion.

PVRL2 and PD-L1 expression is increased in breast, colon, lung, ovarian and skin cancers

Two anti-PVRL2 antibodies (ab135246, HPA-012759) were tested for their ability to detect PVRL2 expression in formalin-fixed, paraffin-embedded tissue samples. PVRL2 and PD-L1 expression was assessed on serial sections using a tumor microarray for breast, colon, lung, ovarian and skin cancers. Expression of PVRL2 and PD-L1 was increased in breast, colon, lung, ovarian and skin cancers (Fig. 21).

PVRL2 is expressed in PD-L1-positive and PD-L1-negative tumors.

Because the expression analysis of PVRL2 and PD-L1 was performed on serial sections using the same TMA, we were able to examine the expression of PVRL2 and PD-L1 in each of these tumors from the same part of the tumor (Fig. 22). A subset of PD-L1-negative tumor samples, particularly lung, ovarian, and breast tumors, expressed PVRL2 (defined as at least partially positive), as detected by both anti-PVRL2 antibodies. These data demonstrate that PVRL2 can be expressed in PD-L1-negative tumors. In contrast, all PD-L1-positive tumors expressed PVRL2, as detected by both anti-PVRL2 antibodies.

PVRL2 is expressed on epithelial cells and on the immune compartment at the invasive frontal surface.

Spatial expression of immune checkpoints at the invasive tumor frontal surface is important for regulating the antitumor response. Known checkpoint targets such as PD-1 and PD-L1 are highly expressed at the invasive frontal surface. Because these TMAs are generated from targeted biopsy material and do not contain the entire tumor, the authors examined these samples for the presence of immune infiltrate on the invasive frontal surface of the tumor. The authors identified 1 sample in which PVRL2 expression was observed in the immune infiltrate and in the tumor epithelium (Fig. 23). PD-L1 expression was observed in the immune infiltrate, further suggesting that this may be an invasive frontal surface of the tumor.

Conclusion.

These results demonstrate that PVRL2 expression is increased in tumors of the breast, colon, lung, ovary, and skin compared with healthy tissue from the same organs. PVRL2 is expressed on both tumor epithelium and infiltrating leukocytes. The authors also demonstrated that all PD-L1-positive tumors express PVRL2 and suggested that agents targeting the PVRL2 pathway may be effective in combination with PD-1/PD-L1 inhibitors. Additionally, PVRL2 expression was detected in PD-L1-negative tumors, suggesting that agents targeting the PVRL2 pathway may be effective in PD-L1-negative tumors.

Example 4: antitumor responses to treatment with mono-, dual- and triple-component antibody combinations in the CT26 tumor model.

Rationale and objectives.

Objective: To investigate whether blockade of PVRIG, TIGIT and PD-L1 antibodies could enhance tumor growth inhibition and survival in a syngeneic mouse tumor model compared with single or dual antibody combinations.

Materials and methods.

In vivo tumor model.

CT26 colon carcinoma cells (ATCC) were cultured in RPMI 1640 with 10% FBS and 100 μg/ml penicillin/streptomycin. For tumor implantation, 5x105 CT26 cells were injected subcutaneously into the right flank of 8-week-old female BALB/c mice. After tumor randomization, antibodies were administered by intraperitoneal (IP) injection starting on day 7 after tumor inoculation when tumors reached a volume of 60-90 mm ³ and continued for 3 weeks for a total of 6 administrations. Tumor size was measured using an electronic caliper every 2-3 days and recorded as 0.5 x W ² x L mm ³ . Mice were sacrificed either at the end of the study or when clinical study parameters were reached, including tumor volume >3250 mm ³ , tumor ulceration, body weight loss >20%, or moribund state.

Antibodies.

Chimeric anti-mouse PVRIG antibody (clone 407, made in our laboratory), with

- 95 044486 changed in these studies, was designed as a mouse IgG1 antibody (mIgGI). This antibody was demonstrated to bind to 293HEK cells overexpressing mouse PVRIG and block binding of the ligand, mouse PVRL2. Anti-mouse PD-L1 antibody mIgG1 (clone YW243.55.S70) was generated as described in WO/2010/077634. Anti-mouse TIGIT antibody mIgG1 (clone 11A11) was generated as described in WO 2016/028656. Synagis IgG1 was used as an isotype control and was produced in-house. Antibodies were formulated in sterile low-endotoxin PBS (<0.05 IU/mg). Anti-PVRIG antibody was administered at a dose of 10 mg/kg, anti-PD-L1 at 5 mg/kg, and anti-TIGIT at 18 mg/kg.

Statistical analysis.

Two-way repeated measures ANOVA followed by two-way repeated measures ANOVA for selected pairs of groups was determined using JUMP (Statistical Discoveries™) software. Analyzes of tumor growth measurements were performed by comparing tumor volumes measured on the last day on which all study animals were alive. Statistical differences in the percentage of tumor-free mice were determined using the Mantel-Cox log-rank test. P values <0.05 were considered significant. * p < 0.05; ** p < 0.01; *** p < 0.001.

Results.

Efficacy of anti-TIGIT and anti-PVRIG antibodies in combination with anti-PD-L1 antibody in vivo

The in vivo efficacy of combination therapy to block murine PVRIG, TIGIT, and PD-L1 was assessed in the CT26.WT syngeneic ectopic subcutaneous tumor mouse model. Treatment of tumor-bearing mice with anti-PVRIG antibody in combination with αntu-PD-L1 antibody resulted in tumor growth inhibition (TGI) of 47% compared to isotype control. However, in this study, no benefit was observed with the dual combination of anti-PD-L1 and anti-PVRIG antibodies compared with treatment with αntu-PD-L1 antibody alone. TIGIT blockade using a three-component combination (anti-PVRIG, anti-TIGIT and anti-PD-L1) led to a significant improvement in TGI compared with other two-component combinations with anti-TIGIT antibody (anti-PDL-1 + anti-TIGIT, anti-TIGIT). PVRIG + anti-TIGIT, and anti-PDL-1 + anti-TIGIT, corresponding to 29%, 61% and 55% TGI, respectively) (Fig. 24A and C). The three-component combination resulted in higher response rates (55% vs. 40%) and promoted durable antitumor activity with a trend towards higher survival to Day 35 (Fig. 24B).

Example 5: PVRIG antagonism enhances T cell effector function and reduces tumor growth rates.

Summary.

Despite recent advances, most patients do not benefit long-term from checkpoint inhibitors. PVRIG is a novel immunosuppressive receptor of the DNAM/TIGIT family, and in this example, we demonstrate the role of PVRIG in regulating antitumor responses. PVRIG binds to PVRL2 and shows significantly increased expression in tumor-infiltrating lymphocytes compared with lymphocytes from normal tissues. Antagonism of PVRIG enhances human T cell activation, and combination of PVRIG inhibitors with PD-1 or TIGIT further synergistically enhances lymphocyte function. The authors then turned to the role of PVRIG in preclinical tumor models. In PVRIG ^- / ^- mice, T cell activation in vitro was significantly increased and the growth rate of MC38 tumor was reduced, which was due to increased effector function of CD8. The antagonist anti-PVRIG antibody significantly reduced tumor growth rate when combined with anti-PD-L1 or when tested in TIGIT ⁻ / ⁻ mice. In summary, the authors demonstrated that the PVRIG-PVRL2 pathway was induced in human cancer and that antagonism of PVRIG-PVRL2 interactions resulted in increased T cell function and decreased tumor growth rate.

State of significance.

These data demonstrate that PVRIG is a promising target for cancer treatment and provide a rationale for testing the PVRIG inhibitor, CHA.7.518.1.H4 (S241P), as a novel anticancer immunotherapy, either as monotherapy or in combination with TIGIT or TIGIT blockade. PD1.

Introduction.

Increasing evidence suggests that endogenous immune responses are critical in shaping cancer initiation, progression, and suppression (1)(2). The immune status of patients, as well as the abundance of tumor-infiltrating leukocytes (TILs) in the tumor microenvironment (TME), are key prognostic indicators of not only cancer survival, but also how patients will respond to therapy (3)(4). T cells are a key component of TILs that can induce antitumor responses, and most antitumor immune responses ultimately depend on the functionality of effector lymphocyte cells. CD8 T cell enrichment in the patient's tumor TME, as well as other factors that bias the immune response toward an effective CD8 T cell response, such as mutational load and Th1-polarized TME, are key prognostic indicators for a favorable antitumor immune response

- 96 044486 (5) (6).

A key observation in many solid tumors is that effector T cells have an activated or exhausted phenotype within the TME ( 7 ). This indicates that although T cells in the TME initially contacted the cognate antigen, were activated, and were recruited to the tumor, they were subsequently unable to mount an effective antitumor response. Preactivated or exhausted T cells are identified by increased surface expression of coinhibitory receptors such as PD-1 and CTLA-4 ( 8 ). Therapeutic targeting of antibodies to these coinhibitory receptors, which inhibit interactions with their cognate ligands, has demonstrated remarkable clinical efficacy in patients with multiple advanced cancers (9). Mechanistically, it has been demonstrated that targeting these coinhibitory receptors results in the expansion of tumor-reactive T cells that already exist in the TME and the formation of T cell pools with expanded T cell receptor diversity (10)(11)(12). Although checkpoint inhibitors currently in clinical use have revolutionized cancer treatment and demonstrated the power of the immune system in fighting cancer, many patients still experience relapses and/or lack of response to treatment. Therefore, a better understanding of the immune response in cancer and targeting additional immune pathways will facilitate additional therapeutic treatments.

Among these new pathways, a group of receptors and ligands in the family of nectins and nectin-like agents are currently being investigated as potential new antitumor immunotherapies. Receptors in this family include DNAM-1 (CD226), CD96 (TACTILE), TIGIT, and, most recently, PVRIG (CD112R) (13)(14)(15). Of these molecules, DNAM is an activating receptor within this subfamily, binding to 2 ligands, PVR (CD155) and PVRL2 (CD112), to deliver an activation signal to lymphocytes (16). Two receptors in this family have been demonstrated to inhibit human lymphocyte function, TIGIT, and, more recently, PVRIG (17) (18). TIGIT is reported to have high affinity for PVR, much weaker affinity for PVRL2, and has been demonstrated to inhibit both T cell and NK cell responses by delivering an inhibitory signal to lymphocytes through its ITSM motif (19) (20). More recently, PVRIG was demonstrated to bind with high affinity to PVRL2 and deliver an inhibitory signal through its ITIM motif ( 15 ). In both cases, the affinity of TIGIT for PVR and PVRIG for PVRL2 is higher than the affinity of DNAM for PVR or PVRL2, suggesting that TIGIT and PVRIG may displace PVR and PVRL2 from DNAM, providing an indirect mechanism by which TIGIT and PVRIG may impair T cell function. Among this family, PVR is also a ligand for CD96. CD96 function has been reported to inhibit mouse lymphocytes (21) but activate human lymphocytes (22). Based on these data, the authors propose that 2 receptors on human lymphocytes, TIGIT and PVRIG, bind with high affinity to PVR and PVRL2, respectively, to deliver inhibitory signals to attenuate T cell function.

Although one recent report demonstrated that human PVRIG inhibits T cell responses, the role of PVRIG and PVRL2 in cancer immune surveillance is not fully understood. In particular, the expression profile of this pathway in cancer and the role of PVRIG in regulating antitumor CD8 T cell responses have not been reported. In addition, the functional characteristics of the mouse PVRIG gene and the effect of disrupting the PVRIG-PVRL2 interaction in vivo in preclinical tumor models have not been reported. Here, the authors elucidated the role of PVRIG in cancer by characterizing the expression profile of PVRIG and PVRL2 in cancer and the impact of PVRIG antagonism in tumor cell co-culture assays and in preclinical tumor models. The authors demonstrated that PVRIG had a differential expression profile on T cell subsets compared with TIGIT or CD96, and that PVRIG and PVRL2 expression were induced in cancer compared with normal adjacent tissues. In a variety of human in vitro assay systems, a high-affinity antagonistic monoclonal antibody to PVRIG (CHA.7.518.1.H4 (S241P)) enhances T cell function, particularly when combined with anti-TIGIT or αntu-PD1 antibody. In addition, the authors reported a novel characterization of PVRIG in mice using antagonistic antibodies or in PVRIG-deficient mice, and demonstrated that inhibition of the PVRIG-PVRL2 interaction reduces tumor growth rate, with the most potent effects when combined with PD-1 inhibition or genetic TIGIT deficiency . Taken together, these data demonstrate that PVRIG is a critical inhibitory receptor in the regulation of antitumor T cell responses and provide a rationale for studying CHA.7.518.1.H4 (S241P) in clinical trials in patients with cancer.

Materials and methods.

Studies of peripheral blood and tumor expression in humans.

Healthy human donor PBMC were obtained from Stanford University in accordance with the Declaration of Helsinki. Human tissues were provided by the United Human Tissue Network, a resource supported by the National Cancer Institute. Human cancer tissue and corresponding normal adjacent tissues were dissected into individual cells according to the protocol for

- 97 044486 manufacturer (Miltenyi Biotec). The resulting dissection cells were analyzed by flow cytometry for the expression of various targets on different cell subsets. For each target expression in a separate subset of cells, the fold expression value was calculated by dividing the MFI value of the target by the MFI value of the isotype control. Other researchers may have obtained samples from the same tissue material. Tumor type was determined based on review of the histopathological feature report for each specimen. For IHC studies, anti-PVRL2 (HPA-012759, Sigma) and PD-L1 (Sp142, SpringBio) antibodies were used to stain tumor microarrays (Biochain Institute) under conditions described in Supplementary Methods. Evaluation was performed by two independent examiners on duplicate nuclei from the same tumor.

Generation and characterization of antibodies to PVRIG

Anti-human PVRIG and anti-mouse PVRIG antibodies were generated as detailed in Supplementary Methods. Briefly, antibody binding specificity and affinity were assessed by selective binding to PVRIG-integrated cells, without detectable binding to cells that do not express the gene. The antagonistic activity of these anti-PVRIG antibodies was determined using ELISA and FACS-based assays in which the interaction of PVRIG with PVRL2 was disrupted. For characterization in cell-based assays, antibodies were tested in several T cell-target cell coculture assay systems consisting of target cells that express PVRL2 in culture with PBMCs or tumor-derived T cells. gp100-specific T cell lines were expanded from melanoma tumors as described previously (23). CMVpp65-reactive T cells were expanded from healthy donor PBMCs (CTL immunospot) with CMVpp65 (495-503), IL-2 and IL-7 for 10 days. For combination studies, antibodies to PD-1, TIGIT and PVRIG were used at a concentration of 10 μg/ml. Cytokine concentrations in conditioned media were determined cytometrically using a microsphere array (CBA), and FACS staining was performed as described in Supplementary Methods.

Characterization of mouse PVRIG expression and function.

Binding interactions of mouse PVRIG with mPVRL2 and mPVR were assessed by SPR and ELISA using recombinant PVRIG, PVRL2, and PVR proteins and by FACS using ectopically engineered cell lines overexpressing PVRIG and PVRL2 or cell lines transfected with PVR or PVRL2 siRNA. PVRIG- and TIGIT-deficient mice were generated as described in Supplementary Methods. Proximate assays were performed to examine PVRIG expression in spleen, lymph node, and tumor in different cell subsets. Functional cellular assays demonstrating T cell modulatory activity for mouse PVRIG were performed using WT and PVRIG ⁻ / ⁻ T cells and target cells ectopically expressed by PVRL2 Fc or PVRL2, as detailed in Supplementary Materials and Methods. CT26, MC38, and B16/Db-hmgp100 tumor models were performed as described in Supplementary Methods. All studies were approved by the Institutional Animal Care and Use Committee of Tel Aviv University (Tel Aviv, Israel) or Johns Hopkins University (Baltimore, USA).

Results.

PVRIG expression is highest on peripheral blood effector T cells and tumors.

The Ig superfamily (IgSF) consists of hundreds of proteins, but only a few are T cell inhibitory receptors. IgSF proteins tend to evolve rapidly (24) and, therefore, sequence similarity among these proteins is generally low and not optimal for identifying new immune receptors. To identify novel immune checkpoints, the authors developed bioinformatic algorithms based on common genomic and proteomic characteristics among known immune checkpoints, such as gene structure, protein domains, predicted cellular localization, and expression profile. Using these algorithms, the authors identified PVRIG as a novel immune receptor. A recent report also demonstrated that human PVRIG (CD112R) binds to PVRL2 and inhibits T cell function ( 15 ). However, the significance of this pathway in regulating tumor immunosurveillance has not been reported. Here, the authors assessed the expression and function of PVRIG and PVRL2 in human cancers and preclinical tumor models. In peripheral blood from healthy donors, PVRIG was expressed exclusively on lymphocytes, with the highest levels of expression on CD8 T cells and NK cells (Fig. 27A). Further analysis of T cell subsets demonstrated the highest expression of PVRIG in the CD8 or CD4 memory/effector T cell subsets compared to the Treg subset (Figure 27B, Figure 34A). The expression profile of memory T cells preferentially differentiates PVRIG from other family receptors (TIGIT, CD96), which tend to have equal or higher expression on Tregs compared to memory/effector T cells. The authors also compared the expression kinetics of PVRIG and TIGIT following T cell activation in two assay systems (CMV secondary response, Fig. 27C, DC-MLR, Fig. 27D, Fig. 34B) and demonstrated that PVRIG has slower induction kinetics and longer duration expression at a late time point compared to TIGIT. Preimu

- 98 044486 The significant expression of PVRIG on memory/effector cells compared to TIGIT suggests a unique role for PVRIG in the regulation of T cell responses.

The slow and prolonged induction of PVRIG expression on T cells following activation suggests that PVRIG may be expressed in the tumor microenvironment. We then analyzed PVRIG expression on leukocytes from dissected human tumors directly ex vivo using FACS. PVRIG expression was detected on CD8 T cells, CD4 T cells and NK cells from several tumor types (Figure 27E-G, Supplementary Figure 27C). PVRIG was coexpressed with PD-1 and TIGIT on CD4 and CD8 T cells (Figure 27F). On average, higher expression was found on CD4+ and CD8 ⁺ TILs from breast, endometrial, head and neck, lung, kidney, and ovarian tumor tissues compared with bladder, colorectal, and prostate tumor tissues. In tumor samples in which PVRIG expression was low/absent ex vivo, activation with anti-CD3 and anti-CD28 enhanced PVRIG expression, suggesting that PVRIG expression in TILs may be further induced following re-activation (Figure 34D ). For colon, lung, kidney, endometrial, and ovarian tumors, the authors were able to obtain normal adjacent tissue from the same patient and compare PVRIG expression on lymphocytes isolated from the tumor versus normal tissue. TILs showed significant induction of PVRIG on CD4 and CD8 T cells compared to cells isolated from matched normal adjacent tissues (NST) (FIG. 34E). As with PBMCs, the authors further compared the expression of PVRIG, TIGIT, and PD1 on Treg cells and CD8 T cells from lung, endometrial, and kidney tumors. On TILs, TIGIT expression was higher on Tregs compared to CD8 T cells, while for PVRIG and PD1, similar or higher expression was observed on CD8 T cells compared to Tregs (Fig. 27E). We then analyzed the co-regulation of PVRIG, TIGIT and PD-1 in T cell populations by correlating either the level of expression on ex vivo TILs or the magnitude of the fold change in expression between tumor and NCT. In both assays, CD4 and CD8 T cells showed a positive and significant correlation between PVRIG and PD1 or TIGIT (Figure 34F). Taken together, these data demonstrate that PVRIG is expressed on T cells and NK cells from a variety of human cancers, making PVRIG a novel inhibitory receptor target that may be critical for regulating T cell function in tumors.

PVRL2 expression is increased in tumor tissue compared to normal adjacent tissue.

Because PD-L1 expression has been demonstrated to help predict response to treatment with PD-1 inhibitors, we examined whether PVRL2 expression was concomitant with the expression of its cognate receptor, PVRIG, in human cancer tissues. Using an anti-PVRL2 antibody that we have validated for use in IHC (FIG. 35A), we stained tumor microarrays (TMAs) consisting of lung, colon, skin, breast, ovarian/endometrial, and kidney cancer tissues. With the exception of the kidney, PVRL2 expression was absent or minimal in most normal tissues of these organs. In tumor tissues, PVRL2 was induced in a significant number of lung, colon, skin, breast and ovarian/endometrial cancer samples (Fig. 28A). PVRL2 expression was detected on tumor cells and on immune cells of the invasive frontal surface of the tumor (Fig. 28B). To identify subsets of specific immune cells expressing PVRL2, we performed flow cytometry on freshly dissected tumors. Consistent with the IHC expression profile, PVRL2 expression was detected on CD45+ immune cells, particularly myeloid cells (eg, CD14+ tumor-associated macrophages (TAMs) and myeloid DCs) and on CD45' non-immune cells from a variety of tumor types (Fig. 28C, D). No PVRL2 expression was detected on lymphocytes (data not shown). Comparison of PVRL2 expression on ^CD45 cells and TAMs isolated from colon, lung, kidney, endometrial, and ovarian tumors demonstrated significant induction of PVRL2 on tumor-derived cells compared with cells isolated from matched normal adjacent tissues (NCTs). same donor (Fig. 36D). To assess which tumors expressed both PVRIG and PVRL2, we examined PVRIG expression on lymphocytes compared to PVRL2 on myeloid cells and on ^CD45 cells from a variety of tumor types. Of the cancer types analyzed, endometrial, lung, and kidney cancers had the highest frequency of PVRIG ^b “ ^c lymphocytes and PVRL2 ^b “ ^c TAM or CD45 ^- non-immune cells (Fig. 28E, Fig. 37). These data demonstrate that the PVRIG-PVRL2 pathway may be particularly important for modulating antitumor responses by regulating T cell–TAM interactions and T cell–tumor cell interactions in endometrial, lung, and kidney cancers.

Compared with PD-L1, PVRL2 expression is differentially regulated and present in PD-L1 tumors.

Because PVRIG and PD-1 can be co-expressed on tumor-infiltrating lymphocytes (TILs), we also examined the co-expression of PVRL2 and PD-L1 in the same tumor by staining serial sections of the same TMA. All PD-L1 positive tumors also expressed PVRL2, indicating some overlap in the regulation of these 2 pathways and

- 99 044486 provides the rationale for combining a PVRIG inhibitor with PD-1/PD-L1 inhibitors (Fig. 29A). In PDL1-negative tumors, PVRL2 was detected in the majority of these tumors across a variety of cancer types (FIG. 29A). This suggests that PVRL2 expression was more common than PD-L1 in some tumors and that targeting this pathway may be particularly effective in PD-L1-negative tumors. Because PD-L1 is reported to be induced in tumors by IFN-γ as part of an adaptive model of resistance (25), we further assessed the regulation of PVR, PVRL2, and PD-L1 expression by various inflammatory stimuli on bone marrow-derived dendritic cells and on tumor epithelial cell lines (Fig. 29D). Exposure of immature BM-DCs to anti-inflammatory signals generally results in increased expression levels of PVR, PVRL2, and PD-L1, demonstrating that the expression of PVR, PVRL2, and PD-L1 increases with DC maturation. In contrast, exposure of epithelial cells to IFN-γ increased PD-L1 expression but did not affect the high baseline expression of PVRL2 (Fig. 29E). For PVRL2, it has been reported that genomic stress, DNA damage, and tumor suppressor genes (26) (27) further support differential regulation of PVRL2 expression compared to PD-L1. Thus, these data indicate that PD-L1 and PVRL2 may be co-regulated on antigen-presenting cells such as DCs and may also be differentially regulated by IFN-γ on epithelial cells. The presence of PVRL2 in PD-L1-negative tumors suggests that targeting this pathway may have potential benefit in patients who do not respond to PD-1 inhibitor therapy.

CHA.7.518.1.H4 (S241P) is a high-affinity humanized monoclonal antibody against PVRIG that disrupts the interaction of PVRIG with PVRL2.

To study the functional consequences of antagonism of human PVRIG-PVRL2 interactions, we generated a high-affinity antagonistic antibody anti-PVRIG CHA.7.518.1.H4 (S241P), which blocks the interaction of PVRIG with PVRL2. This antibody selectively bound HEK293 cells ectopically expressing human PVRIG or cynomolgus PVRIG and also bound Jurkat cells that endogenously express PVRIG with subnanomolar affinity (Figure 30A). In biochemical assays, CHA.7.518.1.H4 (S241P) blocked the interaction of PVRIG Fc with PVRL2+ HEK293 cells (Fig. 30B) and also blocked the binding of PVRL2 Fc with PVRIG+ HEK293 cells (Fig. 30C). Using this antibody, the authors observed a functional effect of the antagonistic anti-PVRIG antibody in several T-cell assays. Artificial antigen presenting cells (aAPCs) ectopically expressing anti-CD3 cell surface antibody and human PVRL2 were generated and co-cultured with primary human CD4 T cells, in the presence of either anti-PVRIG (CHA.7.518.1.H4 (S241P)) , or isotype control. PVRIG expression was induced on proliferating CD4 T cells when co-cultured with CHO anti-CD3 aAPC (FIG. 38A). Antagonism of PVRIG with CHA.7.518.1.H4 (S241P) enhanced the proliferation of CD4 T cells from multiple donors (Fig. 30D). The authors also analyzed the effect of anti-PVRIG on two human gp100-reactive CD8 T cell lines that were derived from melanoma tumors. These T cell lines were individually cocultured with AAPs expressing HLA-A2 and PVRL2 (FIG. 38B) in the presence of isotype control IgG or anti-PVRIG antibodies. As noted in both lines, anti-PVRIG antibody increased IFN-γ and TNF-α production by ~20-50%. When assessing dose response, antibody CHA.7.518.1.H4 (S241P) exhibited single-digit nanomolar EC ₅₀ values in multiple assays (FIG. 38C, D). These data collectively demonstrate that antagonism of PVRIG-PVRL2 interactions with CHA.7.518.1.H4 (S241P) resulted in increased T cell activation.

CHA.7.518.1.H4 (S241P) in combination with TIGIT or PD-1 inhibitors results in a synergistic enhancement of T cell function.

Combined blockade of PVRIG and TIGIT synergistically increased CD4 T cell function in a T cell–dendritic cell coculture assay (15), suggesting a role for this pathway in regulating T cell–APC interactions. The effects of PVRIG and TIGIT blockade on CD8 T cells in tumor cell coculture settings have not been reported. Since expression profiling of tumor cells obtained by the authors demonstrated the expression of PVRL2 on CD45 immune cells, the authors further examined the effect of targeting this pathway in T cell-tumor cell co-cultures using two T cell assay systems. We first cocultured 2 tumor antigen-specific gp100 CD8 T cell lines with a melanoma cell line, MEL624, in the presence of anti-PVRIG, anti-TIGIT, or isotype control antibodies, alone or in combination. MEL624 cells expressed both PVR and PVLR2, and TIL-209 and TIL-463 expressed PVRIG, TIGIT and PD-1 (Figure 30F). On TIL-209, we observed that anti-PVRIG or anti-TIGIT alone did not increase IFN-γ production, while the combination of anti-PVRIG and anti-TIGIT synergistically increased IFN-γ production (Figure 30G). On TIL463, we observed that anti-PVRIG or anti-TIGIT did not significantly increase IFN-γ production, while the combination of anti-PVRIG and anti-TIGIT additively increased IFN-γ production (Figure 30G). In an additional assay system, the authors used CMVpp65-reactive CD8 T cells as moieties.

- 100 044486 efficient system for studying human T-cell responses. HLA-A2+ CMVpp65 CD8 T cells were expanded in the presence of CMVpp65 (495-503), and expression of PVRIG, TIGIT and PD-1 was observed at Day 10 (Fig. 30F). PVRIG was expressed on CMVpp65-specific CD8 T cells at levels similar to those observed in human cancer samples (Fig. 27). As target cells, we identified the PD- ^L1b " ^c (Panc05.04) and PD-L1 ^HU3K (Colo205) HLA-A2+ cancer cell lines, both of which expressed similar amounts of PVR and PVRL2 (Figure 30F). We then co-cultured CMVpp65-reactive T cells with HLA-A2+ tumor cell lines by introducing the pp65 peptide (495-503) in the presence of blocking antibodies to PVRIG, TIGIT and/or PD-1. We observed that anti-PVRIG Ab increased IFN-γ production by ~50% in co-culture with Panc05.04 cells and minimally increased IFN-γ production in co-culture with Colo205 (Figure 30H). Combining anti-TIGIT with anti-PVRIG Ab synergistically increased IFN-γ production in both target cell lines, resulting in a greater increase in IFN-γ levels compared to anti-PD-1 alone (Figure 30H). The combination of anti-PVRIG and anti-PD-1 also resulted in a synergistic increase in IFN-γ production compared to the antibody alone (Figure 30I). Taken together, these data suggest a potent synergy when combined blockade of PVRIG and TIGIT or PVRIG and PD1 is used to enhance activation of human CD8 T cells when interacting with tumor cells.

PVRIG deficiency resulted in increased T cell proliferation and decreased tumor growth rate.

Although the sequence for mouse PVRIG and its interaction with mouse PVRL2 have been reported, the expression profile and immunomodulatory activity of mouse PVRIG is not well understood. We first analyzed the expression and transcript of mPVRIG RNA in NK, NKT and T cells (Fig. 31A). Activated mouse CD8 T cells had increased levels of PVRIG transcripts with delayed induction kinetics compared to TIGIT (Fig. 31B). We confirmed that this recombinant mPVRIG bound to the mPVRL2 protein by surface plasmon resonance (SPR) and ELISA was performed in multiple assay orientations (Figure 39A-D). The authors also observed an interaction between mPVRIG and mPVR, although the affinity was approximately 10-fold less than the interaction with mPVRL2 (Fig. 39E). To determine whether PVR or PVRL2 is the dominant ligand for mPVRIG, we tested the binding of mouse PVRIG Fc to B16F10 cells that express PVR and PVRL2 (data not shown). PVRIG Fc exhibited dose-dependent binding to B16F10 cells, which was completely abrogated following siRNA knockdown of PVRL2 in B16F10 cells (Figure 39F). In comparison, binding of the PVRIG Fc fusion protein was negligible but consistently decreased after PVR knockdown (Figure 39E), indicating a very weak interaction between mPVRIG and mPVR. Taken together, these results demonstrate that in mice PVRL2 is the major ligand for PVRIG, as is the case in humans.

To determine the role of PVRIG in immune responses, we generated PVRIG C/'-deficient mice (FIG. 40). PVRIG mice^-/^- were born with the expected Mendelian ratio, did not show an obvious phenotype until 10 months of age, and at 8 weeks of age had a similar abundance of leukocyte cells (peripheral and lymphoid tissue) compared to wild-type mice (Fig. 41). Wild-type (WT) CD8 T cells and NK cells express PVRIG, and on cells PVRIG^/- PVRIG expression was not detected (Fig. 31C). To investigate the role of PVRIG in regulating mouse T cell responses, we examined the proliferation of WT and PVRIG^-/^- T cells in two analytical systems. WT or PVRIG^-/^- T cells were activated with immobilized anti-CD3 in the presence of soluble PVRL2 Fc or control Fc protein. Soluble PVRL2 Fc significantly inhibited WT CD4+ T cell proliferation in contrast to PVRIG proliferation^-/^- CD4+ T cells (Fig. 31D), suggesting that PVRIG^-/^- cells lack an inhibitory signal. To evaluate the role of mouse PVRIG in CD8 interaction⁺ T-cell tumor cells, from PVRIG mice^-/ generated pmel TCR transgenic mice that express a transgenic TCR specific for gp100₂₅_₃₃ (28). Activated PVRIGs^-/^- - or Pmel CD8+ T cells were cocultured with B16-Db/gp100 melanoma tumor cells that endogenously express PVRL2 (data not shown) and assessed for activation and effector function. PVRIG^-/^- pmel CD8⁺ T cells showed increased degranulation and production of effector cytokines (IFN-γ and TNF-α) compared to WT cells (Fig. 31E). These data indicate that mouse PVRIG inhibits the activation and effector function of tumor-specific T cells when co-cultured with PVRL2+ target tumor cells.

The authors then examined the effect of PVRIG deficiency on tumor growth in a syngeneic model of MS38. PVRIG ^-/- mice showed a significantly lower tumor growth rate compared to WT mice (Fig. 32A-B). Additionally, PVRIG ^−/− mice exhibited additional antitumor responses following PD-L1 blockade starting at day 14, as reflected by significant inhibition (p = 0.052) of tumor growth compared to WT or PVRIG ⁻ / ⁻ mice treated with anti-PD-L1, which was administered isotype control (Fig. 32C, D). Comparable to the reduction in tumor growth rate, PVRIG ^- / ^- mice treated with anti-PD-L1 showed a significant increase in the amount of IFN-γ ⁺ TNF-α ⁺ effect

- 101 044486 positive CD8 T cells when stimulated ex vivo compared with wild-type mice treated with antiPD-L1 and PVRIG'/' mice treated with isotype control (Fig. 32E). In addition, anti-PD-Ll-treated PVRIG' ^/ '-mice also had increased numbers of effector cytokine-producing CD8 ⁺ tumor-infiltrating lymphocytes (TILs) compared to anti-PD-Ll-treated wild-type mice, as well as PVRIG' ^/ '-mice receiving isotype control (Fig. 32F). Transcriptomic profiling of CD45+ immune cells from tumors collected midway through the experiment (day 18; mice received 2 doses of anti-PD-Ll or isotype control) demonstrated that gene signatures for TIL counts and cytotoxic TILs were significantly enhanced in PVRIG-deficient mice treated with anti-PD-Ll, relative to their wild-type equivalents (Fig. 32G-H). Significant changes in T cell mediated genes (GRZB, IFN-γ) were observed in the PVRIG'/' + anti-PD-Ll group compared to the other groups (Supplementary Fig. 42). Taken together, these data demonstrate that PVRIG deficiency, particularly in combination with PD-L1 blockade, results in increased T cell activation and decreased tumor growth rates in vivo.

Anti-mPVRIG antibody inhibited tumor growth when combined with PD-1 antibody or TIGIT deficiency.

After demonstrating that genetic deficiency of PVRIG resulted in reduced tumor growth rates, we subsequently attempted to demonstrate that antibody-mediated inhibition of the PVRIG-PVRL2 interaction could enhance antitumor immunity, particularly when combined with PD1 or TIGIT inhibitors, as demonstrated by from humans in vitro. To evaluate this, we generated a high-affinity anti-mPVRIG antagonist antibody. Anti-mPVRIG mAb affinity determined by FACS demonstrated a subnanomolar Kd value (0.33 nM on HEK293 mPVRIG, 0.39 nM on D10.G4.1 cells), similar to antibody CHA.7.518.1.H4 (S241P) (Fig. 39F-G). The specificity of this antibody was further confirmed as binding to D10.G4.1 cells was predominantly observed after mPVRIG knockdown (FIG. 39H). Anti-mPVRIG antibody was tested to disrupt the mPVRIG-mPVRL2 interaction by inhibiting mPVRIG Fc binding to B16F10 and mPVRIG Fc binding to mPVRIG-overexpressing HEK293 cells (FIG. 33A). Complete blocking of the PVRIG-PVRL2 interaction by the anti-mPVRIG antibody was observed in both assay formats (Figure 33A, Figure 39I), demonstrating an antagonistic anti-mPVRIG antibody. Next, the authors tested the effectiveness of mPVRIG blockade in vivo in a subcutaneous CT26 syngeneic colon tumor model. PVRIG expression was increased on NK and T cells in the tumor microenvironment compared with corresponding subsets of splenic or draining lymph nodes (Fig. 33B). Treatment of tumor-bearing mice with mPVRIG-blocking mAb monotherapy did not reduce tumor growth rate (data not shown). However, the combination of anti-PVRIG and anti-PD-L1 mAb effectively delayed CT26 tumor growth (Figure 33C) and significantly increased survival of treated mice with a complete response rate of 40% (Figure 33D). Consistent with the authors' analysis of human T cells, these data demonstrate that the combination of PD-1 inhibitors and PVRIG can reduce tumor growth rate.

The authors also analyzed the effect of ablation of PVRIG and TIGIT signals in the regulation of antitumor responses. For these studies, the authors analyzed the effectiveness of the antimPVRIG antibody in WT or TIGIT'/' mice inoculated with B16F10/Db-hmgp100 melanoma cells. Treatment of tumor-bearing WT mice with an mPVRIG-blocking mAb had little effect compared with isotype control (17% TGI on day 11 and 8% TGI at baseline on day 18). The effect of TIGIT deletion on tumor growth was also small compared to the WT control group (17% TGI at day 11 and 13% TGI at the time of study baseline assessment). However, when TIGIT deletion was combined with anti-PVRIG mAb, significant tumor growth inhibition was observed (63% at day 11 and 49% TGI at the time of study baseline assessment (FIG. 33E, F). Consistent with tumor growth inhibition , TIGIT'/' mice treated with anti-PVRIG mAb 407 exhibited increased survival compared with WT controls, but statistical significance was not achieved in this aggressive, rapidly growing tumor model (data not shown).Taken together, these data demonstrate synergistic activity of PVRIG inhibitors with PD1 or TIGIT inhibitors and are consistent with the authors' human functional data, providing a rationale for clinical trials of CHA.7.518.1.H4 (S241P) with PD1 or TIGIT inhibitors.

Discussion.

Although antibodies targeting immune T cell checkpoints such as CTLA4 and PD-1 improve survival in cancer patients, most cancer patients still do not benefit clinically from such drugs. One possible reason for this is the presence of additional T cell regulators that inhibit T cell antitumor immunity. In this example, the authors elucidated the role of PVRIG in regulating T cell effector functions and demonstrated that PVRIG antagonism enhances T cell antitumor responses and reduces growth rates

- 102 044486 tumors.

PVRIG is a new member of the family of nectins and nectin-like agents, placing it among a number of known immunoregulatory receptors in this family. Understanding the interactions of receptors in this family is critical to understanding the value and mechanism of action of PVRIG. Of these receptors, DNAM, TIGIT, and CD96 are most closely related to PVRIG in terms of cooperative interactions with the same ligands, PVR and PVRL2. DNAM binds to both PVR and PVRL2 and delivers a costimulatory signal to lymphocytes. TIGIT is reported to bind to PVR and weakly bind to PVRL2. The authors were unable to detect an interaction between TIGIT and PVRL2 by ELISA or SPR (data not shown), suggesting that PVR is the dominant ligand for TIGIT. Using similar methods, by the authors themselves and in a recent report, a high affinity interaction was found between PVRL2 and PVRIG, suggesting that PVRIG is the dominant inhibitory receptor for PVRL2. These data suggest that TIGIT and PVRIG contain dual signaling nodes on this axis and that blocking both proteins is necessary to maximize T cell activation in this family. In addition to interacting with various ligands, the authors observed that PVRIG has the highest expression level on effector or memory T cells, similar to PD-1, whereas TIGIT has the highest expression on regulatory T cells. In addition, the authors observed that PVRIG exhibited late induction after T cell activation compared to TIGIT. These data suggest that PVRIG plays a unique role in this family, interacting with high affinity with PVRL2 and having differential expression in memory cells and a late induction profile for TIGIT.

In this example, the authors also report a novel role for PVRIG in regulating antitumor T cell responses using PVRIG-deficient mice and anti-PVRIG antagonist antibodies. The authors demonstrate that mouse PVRIG was expressed on T cells and NK cells induced by lymphocyte activation and had the highest level of expression in the TME compared to peripheral tissues. In addition, the authors demonstrate that PVRIG deficiency results in increased T cell function in vitro and decreased tumor growth in vivo. A PVRIG antagonist antibody reduced tumor growth rates when combined with αntu-PD-L1 or genetic TIGIT deficiency, demonstrating an integral role for PVRIG in regulating T cell responses. These new data provide proof of concept using preclinical in vivo tumor models in which targeting PVRIG in combination with PD1 or TIGIT antagonism is a potential new cancer treatment option.

In this example, we report a high-affinity anti-human PVRIG antibody that disrupts the interaction of PVRIG and PVRL2, which we have developed for testing in clinical studies. To identify potential cancer signatures that could influence patient selection in clinical trials, we analyzed the expression profile of this axis in human cancers using FACS and IHC. For PVRIG, the authors observed that the mean expression level of PVRIG on CD4 and CD8 T cells, determined by FACS, was highest in endometrial, lung, kidney, and ovarian cancers, although this difference did not reach a statistical difference with other cancer types, indicating that determined by ANOVA using Tukey's multiple comparison test with the number of samples considered. Because PVRIG is induced upon T cell activation, and given that most tumor infiltrating T cells are antigen committed, it is apparent that, as would be expected, median PVRIG expression was similar across tumor samples and cancer types. The authors observed that PVRIG expression correlated with PD-1 and TIGIT expression, suggesting that the interaction of these 3 inhibitory receptors would be important in regulating the antitumor response. In this report, the authors observed a higher synergistic increase in T cell function when combining anti-PVRIG antibodies with anti-TIGIT antibodies in a tumor CD8 T cell co-culture, compared with combining PD1 with PVRIG or TIGIT inhibitors. These data, along with previous research demonstrating the role of PVRIG and TIGIT in regulating DC–T cell interactions, indicate that this pathway may be involved in the regulation of T cell–APC and T cell–tumor cell interactions and provide multiple mechanisms. by which targeting PVRIG can enhance the antitumor immune response.

Because PD-L1 expression correlated with clinical response to PD-1 inhibitors, the authors also analyzed PVRL2 expression in tumors using FACS and IHC to assess whether certain cancer types had higher levels of expression. By assessing cells from dissected tumors, the authors found that the average level of PVRL2 expression on macrophages from endometrial, head and neck, kidney, lung, and ovarian tumor samples was higher compared to other tumor types. The mean expression level of PVRL2 on CD45 non ^- immune cells was higher in breast, colorectal, endometrial, lung, ovarian and prostate cancers compared with other cancers. Based on the expression of PVRIG and PVRL2, the authors determined that cancers of the endometrium, head and neck, lung, kidney and ovary are more often characterized by high

- 103 044486 expression of PVRIG and PVRL2, and that these are potential types of cancers that may respond to therapy with inhibitors of this pathway.

The authors observed that PVRL2 expression could be modulated on antigen-producing cells in vitro by inflammatory mediators, whereas PVRL2 expression on cancer cells was not altered. These data suggest that PVRL2 expression on antigen-presenting cells may be regulated by inflammation and may be an indicator of an inflamed tumor. Indeed, the authors observed that all PD-L1+ tumors also express PVRL2, both on tumor cells and in the immune compartment. PVRL2 expression on myeloid cells may help predict responses to therapy with PVRIG inhibitors in combination with PD-1 or TIGIT to further enhance the antitumor effect. It is of interest that a subset of PDL1-negative tumors also express PVRL2, mainly on tumor cells rather than on immune cells. The expression of PVR and PVRL2 on epithelial cells is reported to be induced in tumorigenesis through xyz and in response to stress and DNA damage. These data are consistent with the authors' in vitro findings that the regulation of PVRL2 expression on tumor cells is independent of IFN-γ. Because PD-L1 is induced under conditions of adaptive resistance in response to IFN-γ and is associated with the inflammatory response, the presence of PVRL2 expression in the absence of PD-L1 suggests that PVRL2 expression is more widespread than PD-L1 and that PVRL2 is expressed in non-inflammatory tumors. Based on the above, it is possible that the presence of PVR and PVRL2 contribute to the suppression of immune responses independently of PD-L1, and that PVRIG and TIGIT inhibitors may be of particular importance for patients who are PD-L1 negative or non-responders/progressors to PD-1 inhibitor therapy .

In summary, this report provides several new insights into PVRIG biology, including characterization of expression of this axis in human cancers, demonstrating a significant role for PVRIG/TIGIT in regulating CD8-tumor cell interaction, and showing that PVRIG antagonism in combination with PD inhibition -1 or TIGIT deficiency lead to a synergistic reduction in tumor growth rates. These data expand the current understanding of PVRIG biology and provide a rationale for clinical trials of CHA.7.518.1.H4 (S241P), a high-affinity anti-PVRIG antibody, in patients with cancer.

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18. Stanietsky N, Simic H, Arapovic J, Toporik A, Levy O, Novik A, et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity. Proc Natl Acad Sci U S A 2009;106(42): 17858-63 doi 10.1073/pnas.0903474106.

19. Johnston RJ, Comps-Agrar L, Hackney J, Yu X, Huseni M, Yang Y, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8(+) T cell effector function. Cancer Cell 2014;26(6):923-37 doi 10.1016/j.ccell.2014.10.018.

20. Zhang B, Zhao W, Li H, Chen Y, Tian H, Li L, et al. Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155. Cancer Immunol Immunother 2016;65(3):305-14 doi 10.1007/s00262-016-1799-4.

21. Chan CJ, Martinet L, Gilfillan S, Souza-Fonseca-Guimaraes F, Chow MT, Town

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L, et al. The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions. Nat Immunol 2014;15(5):431-8 doi 10.1038/ni.2850.

22. Fuchs A, Celia M, Giurisato E, Shaw AS, Colonna M. Cutting edge: CD96 (tactile) promotes NK cell-target cell adhesion by interacting with the poliovirus receptor (CD155). J Immunol 2004; 172(7):3994-8.

23. Machienkin A, Uzana R, Frankenburg S, Eisenberg G, Eisenbach L, Pitcovski J, et al. Capture of tumor cell membranes by trogocytosis facilitates detection and isolation of tumor-specific functional CTLs. Cancer Res 2008;68(6):2006-13 doi 10.1158/0008-5472.CAN07-3119.

24. Ohtani H, Nakajima T, Akari H, Ishida T, Kimura A. Molecular evolution of immunoglobulin superfamily genes in primates. Immunogenetics 2011; 63 (7): 417-28 doi 10.1007/s00251-011-0519-7.

25. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, et al. Colocalization of inflammatory response with B7-hl expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med 2012;4(127):127ra37 doi 10.1126/scitranslmed.3003689.

26. Cerboni C, Fionda C, Soriani A, Zingoni A, Doria M, Cippitelli M, et al. The DNA Damage Response: A Common Pathway in the Regulation of NKG2D and DNAM-1 Ligand Expression in Normal, Infected, and Cancer Cells. Front Immunol 2014;4:508 doi 10.3389/fimmu. 2013.00508.

27. de Andrade LF, Smyth MJ, Martinet L. DNAM-1 control of natural killer cells functions through nectin and nectin-like proteins. Immunol Cell Biol 2014;92(3):237-44 doi 10.1038/icb.2013.95.

28. Overwijk WW, Tsung A, Irvine KR, Parkhurst MR, Goletz TJ, Tsung K, et al. gplOO/pmel 17 is a murine tumor rejection antigen: induction of self ¹ -reactive, tumoricidal T cells using high-affinity, altered peptide ligand. J Exp Med 1998; 188(2):277-86.

Example 6: Tumor cell killing assay.

The effect of anti-human TIGIT antibody and CHA.7.518.1.H4 (S241P) on tumor cell death, either alone or in combination, was assessed using an in vitro coculture assay with CMV-specific human CD8+ T cells. The target HLA-A2+ cell lines used in the assay were a melanoma cell line, Mel624, which stably expresses human PVR and PVRL2, and a pancreatic adenocarcinoma cell line, Panc05.04, which expresses endogenous levels of human PVR and PVRL2. Both tumor cell lines were stably transduced with the luciferase reporter gene via lentiviral transduction (System Biosciences). CMV pp65 peptide was introduced into Me1624 and Panc05.04 cells at a concentration of 0.0033 μg/ml or 0.01 μg/ml at 37°C for 1 hour, respectively. The cells were then washed and plated at a concentration of 20,000 cells/well. The reference antibody against human TIGIT and CHA.7.518.1.H4 (S241P) was added to the culture in combination or with the isotype control antibody hIgG4 at a concentration of 10 μg/ml. CMV-specific human CD8+ T cells from three different donors, designated Donor 4, Donor 72, and Donor 234, were added at a concentration of 100,000 cells/well. Co-cultures were incubated at 37°C for 16 h. After incubation, the plates were removed from the incubator and allowed to equilibrate to room temperature for 30 min. Bio-Glo luciferase substrate (Promega) was added to each well, and the mixture was equilibrated for 10 min at room temperature, protected from light. Luminescent or relative light units (RLU) were quantified on an En Vision multimarker reader (Perkin Elmer) with an ultrasensitive luminescence detector. The percentage of specific killing was calculated as [(RLU for therapeutic antibody RLU for media only)/RLU for media only] x 100.

Results.

In fig. 43A and B demonstrate the effect of anti-TIGIT and CHA.7.518.1.H4 (S241P) antibodies on killing Me1624 and Panc05.04 cells, respectively. When added to coculture alone, both anti-TIGIT antibody and CHA.7.518.1.H4 (S241P) induced significant T cell killing of tumor cell lines compared to isotype control antibody. For the anti-TIGIT antibody, the percentage of specific killing ranged from 19 to 41% for Me1624 cells, and

- 106 044486 3 to 44% for Panc05.04 cells from 3 different CMV-reactive donors tested. For CHA.7.518.1.H4 (S241P), the percentage of specific killing ranged from 16 to 20% for Me1624 cells, and from 0.21 to 29% for Panc05.04 cells. In some cases, an additive effect on the destruction of tumor cells was observed with the combined effect of anti-TIGIT antibody and CHA.7.518.1.H4 (S241P).

To determine whether the effect of anti-TIGIT and CHA.7.518.1.H4 (S241P) antibodies on tumor cell killing was dose-dependent, an assay was performed with a series of 10-point, 2-fold dilutions for each antibody, starting at 0. 5 μg/ml for anti-TIGIT antibodies and 10 μg/ml for CHA.7.518.1.H4 (S241P) (Fig. 44). Killing of Me1624 was reduced in a dose-dependent manner when either the anti-TIGIT antibody, BM26, or CPA.9.086, was combined with CHA.7.518.1.H4 (S241P). Stronger killing was observed with the combination of CPA.9.086 and CHA.7.518.1.H4 (S241P), with an EC50 value of 0.40 ± 0.49 nM compared to the combination of BM26 and CHA.7.518.1.H4 ( S241P), with an EC ₅₀ value of 2.6 ± 1.7 nM.

Example 7: Biophysical measurement of KD.

KinExA equilibrium experiments were performed using a KinExA 3200 instrument (Sapidyne Instruments, Boise, ID, USA) at 22°C. Recombinant His-tagged human TIGIT was obtained from Sino Biologicals (Beijing, China) and reconstituted in 1X PBS. All antigen and antibody samples for KinExA assays were prepared in degassed PBST buffer (PBS with 0.05% Tween 20) with 100 μg/ml filtered BSA and 0.02% sodium azide. The secondary detection antibody was a goat anti-human IgG antibody labeled with Alexa Flour 647 (H+L) (Jackson ImmunoResearch Laboratories), diluted 400-700 times in PBST buffer (with BSA and azide) as described above, from 0. 5 mg/ml stock solution in 1X PBS, pH 7.4. For each KinExA experiment, ~20 μg human TIGIT was diluted in 1 ml 50 mM sodium carbonate, pH 9.2, which was added directly to 50 mg azlactone beads (Ultralink Support, Thermo Scientific, Rockford, IL, USA) and vortexed for nights at 4°C. After shaking, the beads were washed once with 1 M Tris buffer, pH 8.5, containing 10 mg/ml BSA, and shaken for one hour at room temperature in the same buffer. Bound beads were added to the bead reservoir in the KinExA instrument and diluted to ~30 mL with 1 X HBS-N (0.01 M Hepes, 0.15 M NaCl, GE Healthcare) containing 0.02% sodium azide, which also worked buffer for the KinExA device. All antigen-associated garnoules were used immediately after preparation.

For two replicate K _D measurements of CPA.9.086 (Table A), 14 concentrations of TIGIT ranging from 957 aM to 212 pM were equilibrated at room temperature for ~72 h with 2.5 pM CPA.9.086 and 1.8 binding sites pM of CPA binding sites.9.086. For CPA.9.083, 14 concentrations of TIGIT ranging from 478 aM to 196 pM were equilibrated for ∼72 h with 1.8 pM CPA.9.083 binding sites. For duplicate measurements of the reference antibody, BM26 hIgG4, 14 concentrations of TIGIT ranging from 9.6 fM to 3.53 nM were equilibrated over ∼72 h with 20 pM BM26 binding sites and 8.0 pM BM26 binding sites. For CHA.9.547.13, 14 concentrations of TIGIT ranging from 10.5 fM to 2.2 nM were equilibrated over ∼72 h with 8 pM mAb CHA.9.547.13 binding sites. The volume flowing through the bead pack for each equilibrated sample for all experiments ranged from 4 mL to 11 mL at a flow rate of 0.25 mL/min. The data were fitted to a standard 1:1 equilibrium binding model using KinExA Pro software (version 4.2.10; Sapidyne Instruments) to estimate the KD and obtain the 95% confidence interval (CI) of the curve fit.

Results.

Both CPA.9.083 and CPA.9.086 bound to human TIGIT with femtomolar binding affinity, whereas CHA.9.547.13 and BM26 bound with picomolar binding affinity. In summary, CPA.9.083 and CPA.9.086 bind to human TIGIT with the highest affinity of the four different antibodies tested.

Table A

K _D measurements for hIgG4 anti-human TIGIT antibodies determined using KinExA

Antibody Kd ± 95% CI (n = 1) Kd ± 95% CI (n = 2) SNA.9.547.13 18.8 ±5.8 pM Undefined SPA.9.083 694 ± 277 04 Undefined SPA.9.086 553 ± 230 fM 665 ± 378 fM VM26 8.2 ± 2.8 pM 11.2±3.6pM

Example 8: Investigation and functional characterization of CPA.9.086, a novel therapeutic antibody targeting the TIGIT immune checkpoint.

Background: TIGIT is a coinhibitory receptor that is highly expressed on lymphocytes, including effector and regulatory CD4+ T cells (Tregs), effector CD8+ T cells, and NK cells, which infiltrate various tumor types. Interact

- 107 044486 vie TIGIT with its known ligands, poliovirus receptor (PVR) and PVR-like proteins (PVRL2 and PVRL3), directly inhibits lymphocyte activation. PVR is also widely expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be the dominant mechanism of immune evasion in cancer. Here, the authors report the biophysical and functional characterization of CPA.9.086, a therapeutic antibody targeting TIGIT. The authors also demonstrate that co-blockade of TIGIT and a novel checkpoint inhibitor, PVRIG, enhances T cell responses.

Materials and methods. Human phage display and mouse hybridoma antibody approaches have been pursued to generate therapeutic anti-TIGIT antibodies. The resulting antibodies were assessed for their ability to bind to recombinant and cell surface expressed human TIGIT with high affinity. Cross-reactivity of cynomolgus monkey and mouse anti-TIGIT antibodies was also examined. A subset of antibodies that bind with high affinity to human TIGIT and cross-react with cynomolgus TIGIT were further characterized for their ability to block the interaction between TIGIT and PVR. Blocking antibodies were screened for their ability to enhance antigen-specific T cell activation in vitro either alone or in combination with the anti-PVRIG antibody, CHA.7.518.1.H4 (S241P).

Results. The authors identified a lead antibody, CPA.9.086, that binds to human TIGIT with high femtomolar affinity. This TIGIT-related antibody was expressed endogenously on human CD8+ T cells with higher affinity than the reference antibodies tested and also cross-reacted with both cynomolgus and mouse TIGIT. When tested for in vitro activity, CPA.9.086 enhanced cytokine secretion and tumor cell killing by CMV-specific CD8+ T cells with superior or equivalent potency to the reference antibodies tested. The combination of CPA.9.086 with anti-PD1 antibody or CHA.7.518.1.H4 (S241P) resulted in increased activity of CMV-specific CD8+ T cells. In addition, the authors demonstrated that TIGIT is preferentially expressed on Tregs and effector CD8+ T cells from solid tumors compared to peripheral blood, suggesting that these populations are likely to be preferentially targeted by CPA.9.086.

Conclusion. The authors describe research into a very high affinity TIGIT antagonist antibody, CPA.9.086, which is currently in preclinical development. The authors theorize that the femtomolar affinity of CPA.9.086 may allow for lower and less frequent dosing in patients. CPA.9.086 may enhance human T cell activation alone or in combination with other checkpoint antibodies. In summary, the authors' findings demonstrate the benefit of targeting TIGIT, PD1, and PVRIG for cancer treatment.

Example 9: Analysis of the TIGIT/PVRIG axis in human cancer to support selection of indications and biomarkers for combination therapy.

Background: PVRIG and TIGIT were identified by Compugen's Predictive Discovery platform as immunostimulatory receptors that have been reported to inhibit antitumor activity. The authors are conducting a clinical study of antagonistic antibodies to PVRIG (eg, CHA7.518.1.H4 (S241P)) and to TIGIT (eg, CPA.9.083.H4 (S241P)). In this example, we analyzed primary human cancer tissues and immune cells to characterize expression along the TIGIT/PVRIG axis to support indication selection and combination strategy for these antibodies.

Methods: CHA7.518.1.H4 (S241P) and CPA.9.083.H4 (S241P) were identified based on their ability to block the interaction of PVRIG and TIGIT with their cognate ligands (PVRL2 and PVR, respectively) and screened for their ability to enhance antigen-specific activation of CD8 T -cells in co-culture with tumor cell lines. Immunohistochemistry and flow cytometry were performed to evaluate receptor/ligand expression in dissected bladder, breast, colorectal, head and neck, lung, kidney, ovarian, prostate, and gastric tumors.

Results. Among the cancer types examined, PVRIG and PVRL2 expression was highest in endometrial, lung, kidney, ovarian, and head and neck cancers compared with normal adjacent tissue. From dissected tumors, PVRIG expression was detected on T and NK TILs, whereas PVRL2 expression was detected on CD45 cells and myeloid cells. Coexpression analysis of PVRIG, TIGIT, and PD1 demonstrated that PVRIG was coexpressed with both TIGIT and PD1, and that PVRIG+TIGIT+PD1+ cells constituted the major proportion of CD8 TILs. Compared with PD-L1, PVRL2 expression was more common among several cancer types, b PVRL2 expression was detected in PD-L1-negative samples. In vitro, the combination of CHA7.518.1.H4 (S241P) with PD1 inhibitors or CPA.9.083.H4 (S241P) enhanced the production of CD8 cytokines and cytotoxic activity with the three-component antibody combination CHA7.518.1.H4 (S241P), CPA.9.083.H4 (S241P) and PD-1, which causes the greatest increase in functional activity. Several immune receptors were induced in response to blockade of PVRIG by the antibody CHA7.518.1.H4 (S241P) on CD8 T cells. Taken together, these data support the selection of indications and combination strategy for CHA7.518.1.H4 (S241P)

- 108 044486 and CPA.9.083.H4 (S241P) and potential biomarkers that may be indicators of response to therapy.

Conclusions. In summary, the authors demonstrated that PVRIG and PVRL2 are induced in the tumor microenvironment in human cancer, and the potential of CHA7.518.1.H4 (S241P) as an antitumor therapeutic agent, either as monotherapy or as two- or three-component antibody combination therapy , targeting TIGIT and PD-1. These data highlight the potential of this combination approach to expand the population of cancer patients who will respond to checkpoint inhibitor therapy, including patients who do not respond to PD-1 inhibitor therapy.

Example 10: PVRIG expression is associated with T cell exhaustion and synergizes with TIGIT to inhibit antitumor responses.

Summary.

Using a unique discovery computational platform, the authors identified a new family of checkpoint receptors consisting of 2 inhibitory receptors in the nectin family, TIGIT and PVRIG. Both PVRIG and TIGIT are expressed upon T cell activation but show differences in relative expression among T cell subsets and expression kinetics. PVRIG binds to PVRL2, whereas TIGIT binds to several ligands, among which the authors observed that PVR is the dominant functional ligand for TIGIT. The distinctive expression profile of PVRIG and the unique high-affinity PVRIG-PVRL2 interaction suggest that PVRIG plays a unique role in immune regulation. Using novel PVRIG'/' mice, the authors observed that genetic deficiency of PVRIG resulted in increased levels of T cell responses and decreased tumor growth rates in preclinical models, demonstrating the potential of targeting this pathway in cancer. To further define the clinical niche of the PVRIG antagonist, we examined the expression of the TIGIT/PVRIG and PD-1 axes in human tumor samples. Among the human cancers examined, expression of PVRIG and TIGIT on tumor-derived T cells was highest in endometrial, lung, kidney, and ovarian cancers. Co-expression analysis of PVRIG, TIGIT and PD1 demonstrated that PVRIG was correlated and co-expressed with both TIGIT and PD1, and that PVRIG+TIGIT+PD1+ cells constituted the major proportion of CD8 tumor-infiltrating lymphocytes (TILs). Of interest, expression of PVRIG, but not TIGIT, on CD8 ⁺ TILs was associated with the Eomes ^high T-bet ^low exhausted phenotype. PVR, PVRL2, and PD-L1 also showed tissue-specific differences in relative expression levels, with endometrial and ovarian tumors having a higher expression ratio of PVRL2 relative to PVR or PD-L1. Culture of primary human TILs with antagonistic antibodies anti-PVRIG (CHA7.518.1.H4 (S241P)) and anti-TIGIT (CPA.9.083.H4 (S241P)) enhanced T cell function to the same or greater extent compared to PD blockade -1. See fig. from 54 to 60.

CHA7.518.1.H4 (S241P) and CPA.9.083.H4 (S241P) target PVRIG and TIGIT in the family of nectins and nectin-like agents: Conclusions PVRIG and TIGIT are statically undefined checkpoint receptors and promising targets for cancer treatment.

In tumors with higher levels of PVRL2 than PVR, the PVRIG/PVRL2 interaction may be more dominant and require direct targeting of PVRIG.

Example 11: New preclinical data demonstrating the distinctive features of the PVRIG pathway in immuno-oncology and the potential of CHA7.518.1.H4 (S241P) in the treatment of multiple solid tumors.

The data further supports the rationale for the Clinical Study Design and biomarker strategy for CHA7.518.1.H4 (S241P).

CHA7.518.1.H4 (S241P) was safe when used at high doses in the GLP toxicity study.

New preclinical data are presented demonstrating the distinctive features of the PVRIG pathway in immunological oncology and the potential of CHA7.518.1.H4 (S241P), a first-in-class therapeutic antibody candidate targeting PVRIG in the treatment of multiple solid tumors. Data presented at the Keystone Symposia Conference, A3: T Cell Dysfunction, Cancer and Infection, January 16-20, 2018 (shown in Example 9 and related figures) demonstrate the possible dominance of the PVRIG/TIGIT axis in immuno-oncology and support the rationale Clinical trial design and biomarker strategy for CHA7.518.1.H4 (S241P) as monotherapy and in combination with CPA.9.083.H4 (S241P).

The poster entitled PVRIG Expression is Associated with T Cell Exhaustion and Synergizes with TIGIT to Inhibit Anti-Tumor Responses (Poster No. 2028) includes data demonstrating that the expression of PVRL2, a PVRIG ligand, is more dominant in some tumor types, including lung, breast, endometrial and ovarian tumors, compared with the expression of PVR, a ligand for TIGIT. These results suggest that PVRIG may be a dominant control point in patient populations with tumors expressing elevated levels of PVRL2, many of which do not respond to PD-1 inhibitor therapy. Therefore these

- 109 044486 patients may have an increased likelihood of response to monotherapy with the antibody CHA7.518.1.H4 (S241P).

In addition, expression studies demonstrate that PVRIG and TIGIT and their respective ligands are commonly expressed in the tumor types listed above, as well as in renal and head and neck cancer cells, indicating that in patient populations in which they are active two pathways, blockade of both TIGIT and PVRIG may be necessary to sufficiently stimulate an antitumor immune response. In addition, the findings also indicate that exhausted TILs found in multiple tumor types significantly coexpress three triple checkpoints: TIGIT, PD-1, and PVRIG, further supporting the significance of the triple combination in such patient populations.

A more complete understanding of the role of the PVRIG-TIGIT axis and the interactions between the various components of the axis also sheds light on the evolution of the PVRIG/PVRL2 pathway during the mouse-to-human transition, and is reflected in the reduced activity of the pathway in mice. The authors' data clearly demonstrate that studying mouse biology in relation to this pathway may underestimate the impact that this pathway may have on human antitumor immunity, suggesting that CHA7.518.1.H4 (S241P) may be even more therapeutic. effects than those observed in preclinical studies.

The potential dominance of the PVRIG pathway and its interaction with the TIGIT and PD-1 pathways demonstrated in our preclinical studies, coupled with expression profiles, provides the biological rationale to validate our clinical approach to testing the antibody CHA7.518.1.H4 (S241P) when used in as monotherapy and in two- and three-drug combinations as the authors prepare to begin their Phase 1b clinical trial. By following a study design for all patients, our biomarker strategy will be guided by these expression profiles to increase the number of patients most likely to respond to therapy with the CHA7.518.1.H4 (S241P) antibody,” said Anat Cohen-Dayag, PhD, President and CEO director of Compugen. We are also encouraged by the results of a GLP toxicity study on the antibody CHA7.518.1.H4 (S241P), which demonstrated that it is safe when used at high doses. Our data suggest that the PVRIG pathway and CHA7.518.1.H4 (S241P), our first-in-class therapeutic antibody, may have important clinical utility as the basis for novel cancer immunotherapies to address the needs of patient populations unresponsive or refractory to existing immune checkpoint inhibitor therapy.

About SNA7.518.1.N4 (S241P) and SPA.9.083.N4 (S241P)

CHA7.518.1.H4 (S241P) is a humanized hybridoma antibody that binds with high affinity to PVRIG, a novel B7/CD28-like immune checkpoint target candidate discovered by Compugen, indicating blocking of the target's interaction with PVRL2. Blockade of PVRIG with CHA7.518.1.H4 (S241P) demonstrated a potent, reproducible enhancement of T cell activation, consistent with the desired mechanism of action of T cell activation in the tumor microenvironment to generate antitumor immune responses. In addition, the antibody CHA7.518.1.H4 (S241P) in combination with antagonistic anti-PD-1 antibodies demonstrated a synergistic effect on stimulating human T cells, indicating the potential of these combinations to further enhance the immune response against tumors.

CPA.9.083.H4 (S241P), an antibody from Compugen targeting TIGIT, was developed for combination use with CHA7.518.1.H4 (S241P). Preclinical data strongly support dual blockade of the two negative co-stimulatory arms of the axis, TIGIT and PVRIG, which results in a more robust T cell response to antigenic stimulation and may therefore mediate an enhanced antitumor immune response.

Example 12: In vivo efficacy and survival of a three-component combination treatment.

Rationale and objectives.

This example demonstrates whether the combination of TIGIT, PVRIG, and mouse PD-L1 blockade can significantly enhance tumor growth inhibition (TGI) and survival in a syngeneic mouse tumor model.

Protocols.

Animals.

5-week-old female mice were purchased from Charles River Laboratories. Mice were housed in the Compugen USA animal facility with food and water provided ad libitum and acclimatized for a minimum of 6 days before the start of the study. All studies were approved by the Institutional Animal Care and Use Committee at Compugen USA (South San Francisco, CA).

Mouse syngeneic tumor model.

5x105 CT26 colon carcinoma cells (ATCC) were inoculated subcutaneously (s.c.) into the right flank of female Balb/c mice and grown for up to 8 days. Mice with tumors measuring 30-60 mm ³

- 110 044486 were randomized (the day of randomization is designated as day 0) into 3 groups of 10 mice per group. Animals received an intraperitoneal (i.p.) injection of 200 μl of either mouse isotype control IgG1 (mIgG1) antibody (designated Synagis), or a two-component combination of anti-TIGIT mIgG1 and anti-PVRIG mIgG1 antibodies, or a three-component combination of anti-TIGIT mIgG1, anti-PVRIG mIgG1 antibodies and anti-PD-L1 mIgG1. The anti-TIGIT mIgG1 antibody is a chimeric version of CPA.9.086 that contains the CPA.9.086 variable heavy and light chains and the human mIgG1 constant region. Antibodies were administered at fixed doses of 10 mg/kg anti-TIGIT, 10 mg/kg anti-PVRIG, and a suboptimal dose of 3 mg/kg anti-PD-L1 starting on day 8, three times a week for 2 weeks. Tumor growth was determined by measuring length (L) and width (W) with a caliper; the tumor size was calculated using the formula (L x W2)/2. Tumor size was required to be no larger than 2000 mm ³ , which was designated as the primary outcome parameter in the study, and mice were subsequently euthanized.

Statistical analysis.

Two-way repeated measures ANOVA followed by two-way repeated measures ANOVA for selected pairs of groups was performed using prism software. Analyzes of tumor growth measurements were performed by comparing tumor volumes measured on the last day on which all study animals were alive. Statistical differences in the percentage of tumor-free mice were determined using the Mantel-Cox log-rank test.

Results and summary.

This study examined the effects of blocking three different immune checkpoint pathways, TIGIT, PVRIG, and PD-L1, on TGI and survival in mice. Using a mouse model of CT26 colon carcinoma, it was demonstrated that the combination of anti-TIGIT mIgGl with anti-PVRIG mIgGl caused a small but significant TGI (20.7% TGI at day 25) compared to mice treated with isotype control antibody. When combining the three antibodies, the TGI increased to 58.3% on day 25, which was statistically and significantly more effective than the two-component combination (p < 0.001 by two-way ANOVA on day 25). Although none of the mice were tumor free (CR) by the end of the study (day 28), increased efficacy with the three-drug combination was associated with increased survival compared with the two-drug combination. Triple blockade demonstrated a significant improvement in overall survival with 90% survival at the end of the study (day 28). In addition to the antitumor efficacy reported herein, no significant changes in body weight were observed in all groups observed (data not shown). Taken together, these data demonstrate that the three-way combination was well tolerated and had superior antitumor effects in colon carcinoma in vivo compared with the two-way combination of TIGIT and PVRIG blockers. See fig. 61.

Claims (12)

CLAIM 1. Use of a three-component combination including anti-TIGIT antibody, anti-PVRIG antibody and anti-PD-1 antibody for the treatment of cancer. 2. The use of claim 1, wherein said anti-TIGIT antibody is an antibody selected from at least one of the following antibodies: CPA.9.083.H4 (S241P) (full-length heavy chain of the sequence SEQ ID NO: 70; full length light chain SEQ ID NO: 75), CPA.9.086.H4 (S241P) (full length heavy chain SEQ ID NO: 80; full length light chain SEQ ID NO: 85), CHA.9.547.7.H4 (S241P ) (full-length heavy chain of SEQ ID NO: 350; full-length light chain of SEQ ID NO: 355) and CHA.9.547.13.H4 (S241P) (full-length heavy chain of SEQ ID NO: 480; full-length light chain of SEQ ID NO: 485). 3. Use according to claim 1 or 2, characterized in that said anti-PVRIG antibody is an antibody selected from at least one of the following antibodies: CHA.7.518.1.H4 (S241P) (full-length heavy chain of the sequence SEQ ID NO: 1023; full-length light chain of SEQ ID NO: 1028) and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1033; full-length light chain of SEQ ID NO: 1038). 4. Use according to any one of claims 1 to 3, characterized in that said anti-PD-1 antibody is an antibody selected from at least one of pembrolizumab and nivolumab. 5. Application according to any one of claims 1-4, characterized in that said three-component combination is selected from CPA.9.083.H4 (S241P) (full-length heavy chain of SEQ ID NO: 70; full-length light chain of SEQ ID NO: 75), pembrolizumab and CHA.7.518.1.H4 (S241P) (full-length heavy chain of SEQ ID NO: : 1023; full length light chain - 111 044486 sequence SEQ ID NO: 1028); CPA.9.086.H4 (S241P) (full-length heavy chain of SEQ ID NO: 80; full-length light chain of SEQ ID NO: 85), pembrolizumab and CHA.7.518.1.H4 (S241P) (full-length heavy chain of SEQ ID NO: : 1023; full length light chain sequence SEQ ID NO: 1028); CHA.9.547.7.H4 (S241P) (full-length heavy chain of SEQ ID NO: 350; full-length light chain of SEQ ID NO: 355), pembrolizumab and CHA.7.518.1.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1023; full length light chain sequence SEQ ID NO: 1028); CHA.9.547.13.H4 (S241P) (full-length heavy chain of SEQ ID NO: 480; full-length light chain of SEQ ID NO: 485), pembrolizumab and CHA.7.518.1.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1023; full length light chain sequence SEQ ID NO: 1028); CPA.9.083.H4 (S241P) (full-length heavy chain of SEQ ID NO: 70; full-length light chain of SEQ ID NO: 75), pembrolizumab and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: : 1033; full length light chain sequence SEQ ID NO: 1038); CPA.9.086.H4 (S241P) (full-length heavy chain of SEQ ID NO: 80; full-length light chain of SEQ ID NO: 85), pembrolizumab and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: : 1033; full length light chain sequence SEQ ID NO: 1038); CHA.9.547.7.H4 (S241P) (full-length heavy chain of SEQ ID NO: 350; full-length light chain of SEQ ID NO: 355), pembrolizumab and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1033; full length light chain sequence SEQ ID NO: 1038); CHA.9.547.13.H4 (S241P) (full-length heavy chain of SEQ ID NO: 480; full-length light chain of SEQ ID NO: 485), pembrolizumab and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1033; full length light chain sequence SEQ ID NO: 1038); CPA.9.083.H4 (S241P) (full-length heavy chain of SEQ ID NO: 70; full-length light chain of SEQ ID NO: 75), nivolumab and CHA.7.518.1.H4 (S241P) (full-length heavy chain of SEQ ID NO: : 1023; full length light chain sequence SEQ ID NO: 1028); CPA.9.086.H4 (S241P) (full-length heavy chain of SEQ ID NO: 80; full-length light chain of SEQ ID NO: 85), nivolumab and CHA.7.518.1.H4 (S241P) (full-length heavy chain of SEQ ID NO: : 1023; full length light chain sequence SEQ ID NO: 1028); CHA.9.547.7.H4 (S241P) (full-length heavy chain of SEQ ID NO: 350; full-length light chain of SEQ ID NO: 355), nivolumab and CHA.7.518.1.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1023; full length light chain sequence SEQ ID NO: 1028); CHA.9.547.13.H4 (S241P) (full-length heavy chain of SEQ ID NO: 480; full-length light chain of SEQ ID NO: 485), nivolumab and CHA.7.518.1.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1023; full length light chain sequence SEQ ID NO: 1028); CPA.9.083.H4 (S241P) (full-length heavy chain of SEQ ID NO: 70; full-length light chain of SEQ ID NO: 75), nivolumab and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: : 1033; full length light chain sequence SEQ ID NO: 1038); CPA.9.086.H4 (S241P) (full-length heavy chain of SEQ ID NO: 80; full-length light chain of SEQ ID NO: 85), nivolumab and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: : 1033; full length light chain sequence SEQ ID NO: 1038); CHA.9.547.7.H4 (S241P) (full-length heavy chain of SEQ ID NO: 350; full-length light chain of SEQ ID NO: 355), nivolumab and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1033; full length light chain sequence SEQ ID NO: 1038); CHA.9.547.13.H4 (S241P) (full-length heavy chain of SEQ ID NO: 480; full-length light chain of SEQ ID NO: 485), nivolumab and CHA.7.538.1.2.H4 (S241P) (full-length heavy chain of SEQ ID NO: 1033; full-length light chain sequence SEQ ID NO: 1038). 6. Use according to any one of claims 1-5, characterized in that said antibodies are administered simultaneously - 112 044486 exactly. 7. Use according to claim 6, characterized in that the antibodies are administered as separate infusions or as a single infusion of a mixture of antibodies. 8. Use according to any one of claims 1 to 5, characterized in that said antibodies are administered sequentially. 9. Use according to claim 8, wherein the antibodies are administered sequentially over several hours or days. 10. Use according to any one of claims 1 to 9, characterized in that said cancer is selected from the group consisting of prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative cancer breast, pancreatic cancer, stomach cancer (stomach cancer), cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, kidney cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, germ cell testicular tumors, mesothelioma, esophageal cancer, Merkel cell cancer, high-MSI cancer, KRAS-mutant tumors, adult T-cell leukemia/lymphoma and myelodysplastic syndromes (MDS). 11. Use according to any one of claims 1 to 10, characterized in that said cancer is selected from the group consisting of triple negative breast cancer, stomach cancer (gastric cancer), lung cancer (small cell lung cancer, non-small cell lung cancer), cancer from Merkel cells, microsatellite-rich (MSI) cancers, KRAS-mutant tumors, adult T-cell leukemia/lymphoma, and myelodysplastic syndromes (MDS). 12. Use according to any one of claims 1 to 11, wherein the antibodies are provided in an administration kit with dosage units of each antibody packaged either separately in different dosage units or together as a mixture of antibodies in one dosage unit.