EA049282B1 - ANTIBODIES TO TIGIT - Google Patents

Abstract

The present invention provides antibodies that specifically bind to the T cell immunoreceptor with domains 1g and ITIM (TIGIT). The antibodies exhibit the ability to significantly activate T cells and natural killer cells by inhibiting TIGIT binding to CD 155. The antibodies can be used, among other applications, to treat cancer and infectious diseases.

Description

The present document, inter alia, provides antibodies that specifically bind to TIGIT and their use, among other applications, for the treatment of cancer and infectious disease.

Cross reference to related application

This application claims priority to U.S. Provisional Patent Application No. 63/033,609, filed June 2, 2020, the disclosure of which is incorporated herein by reference in its entirety, including all drawings.

Enabling a sequence list

This application contains a sequence listing, which is hereby incorporated by reference in its entirety. The attached text file containing the sequence listing, named 050658_531001WO_Sequence_Listing_ST25, was created on May 31, 2021 and is 143 KB in size.

Prerequisites for the creation of an invention

Tumors contain highly suppressive microenvironments in which T cell and NK cell function is regulated by cell surface checkpoint receptors, allowing cancer cells to evade immune detection. Functional blockade of inhibitory checkpoint receptors such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has yielded positive results in patients, raising considerable interest in identifying additional co-inhibitory molecules as potential targets.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT), a member of the immunoglobulin superfamily with an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail, is a coinhibitory receptor expressed by regulatory T cells (Treg), activated T cells, and natural killer (NK) cells. Several research groups reported that TIGIT expression was increased on tumor-infiltrating lymphocytes (TILs) and Treg cells in a variety of tumors. It was also reported that CD8+ effector T cells in blood during HIV infection and in lymphoid tissue during SIV infection exhibit higher levels of TIGIT. Moreover, TIGIT blockade resulted in increased activating activity in cultured human T cells and provided therapeutic effects in animal models of various tumors. Therefore, TIGIT plays an important role in antitumor immunity and may serve as a promising therapeutic target in the fight against cancer and various other diseases and conditions. Thus, there is a need for molecules that can interfere with TIGIT binding to achieve therapeutic goals.

Statement of the essence of the invention

The present invention relates, inter alia, to an antibody to TIGIT.

The present document provides an anti-TIGIT antibody or antigen-binding fragment thereof, comprising (a) a heavy chain variable region comprising a heavy chain complementarity determining region (CDR) 1 that is at least 80% identical to the sequence of SEQ ID NO: 36, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 37, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 38; and a light chain variable region (LC) comprising a light chain CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 39, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 40, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 41; (b) a heavy chain variable region comprising an HCCDR1 that is at least 80% identical to the sequence of SEQ ID NO: 42, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 43, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 44; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 45, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 46, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 47; (c) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 48, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 49, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 50; and a light chain variable region comprising an LCCDR1 that is at least 80% identical to the sequence of SEQ ID NO: 51, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 52, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 53; (d) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 54, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 55, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 56; and a light chain variable region comprising LC-CDR1, which is at least

- 1 049282

80% identical to the sequence of SEQ ID NO: 57, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 58, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 59; (e) a heavy chain variable region comprising an HCCDR1 that is at least 80% identical to the sequence of SEQ ID NO: 60, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 61, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 62; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 63, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 64, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65; (I) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 60, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 66, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 67; and a light chain variable region comprising an LCCDR1 that is at least 80% identical to the sequence of SEQ ID NO: 63, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 68, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65; (g) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 69, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 55, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 70; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 71, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 68, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65; (h) a heavy chain variable region comprising an HCCDR1 that is at least 80% identical to the sequence of SEQ ID NO: 72, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 73, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 63, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 68, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65; or (i) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 74, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 75, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 67; and a light chain variable region comprising an LCCDR1 that is at least 80% identical to the sequence of SEQ ID NO: 63, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 68, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65.

In some embodiments, an anti-TIGIT antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 1 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 2; (b) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 3 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 4; (c) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 5 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 6; (d) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 7 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 8; (e) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 9 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 10; (f) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 11 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 12; (g) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 13 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 14; (h) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 15 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 16; (i) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 17 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 12; (j) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 76 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 77; (k) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 78 and a

- 2 049282 a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 77; (l) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 76 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 79; or (m) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 78 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 79.

Provided herein are anti-TIGIT antibodies or antigen-binding fragments thereof that bind to an epitope that comprises at least one of the following TIGIT amino acid residues: T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope that comprises at least the following TIGIT residues: D72 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope that comprises at least the following TIGIT residues: E60 and D72 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80. In some embodiments, an anti-TIGIT antibody binds to an epitope that includes at least the following TIGIT residues: D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80. In some embodiments, an anti-TIGIT antibody binds to an epitope that includes at least the following TIGIT residues: E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80. In some embodiments, the antibodies have structural features of the CDR and variable region sequences as described herein.

The anti-TIGIT antibodies of the present invention may be an isolated antibody. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is a chimeric, humanized, or veneered antibody. In some embodiments, the chimeric antibody comprises a human IgG1/kappa Fab constant domain. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof is a human antibody. In some embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof inhibits TIGIT binding to CD155.

Provided herein is a pharmaceutical composition comprising an anti-TIGIT antibody as described herein and a pharmaceutically acceptable carrier. Provided herein is a method of treating or preventing cancer comprising administering to a subject having or at risk of developing cancer an effective regimen or administering to them a therapeutically effective amount of any anti-TIGIT antibody described herein. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is acute myeloid leukemia or adult T-cell leukemia. In some embodiments, the cancer is a solid tumor, non-small cell lung carcinoma, melanoma, cervical cancer, multiple myeloma, lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, gastric cancer, gastroesophageal junction adenocarcinoma, or esophageal cancer. In some embodiments, the subject is also administered tumor-infiltrating T cells. In some embodiments, the subject is further administered a vaccine that induces an anti-cancer immune response. In some embodiments, the vaccine comprises an antigen or fragment thereof expressed on the surface of cancer cells. In some embodiments, the subject is further administered natural killer cells whose anti-cancer cytotoxicity is enhanced by the antibody. In some embodiments, the subject is further administered a second antibody that specifically binds to an antigen expressed on the surface of cancer cells, such that the effector-mediated anti-cancer cytotoxicity of the second antibody is enhanced by the anti-TIGIT antibody of the invention. In some embodiments, the subject is further administered a second antibody that specifically binds to an antigen expressed on the surface of an immune cell. In some embodiments, the immune cell is a T cell or a natural killer cell. In some embodiments, the antigen is CTLA-4, PD-1, or PD-L1. In some embodiments, the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation therapy, cell therapy, and surgery. In some embodiments, the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands. In some embodiments, the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A _2a R, A _2b R, A _2a /A _2b R, arginase, CD39, CD73, IDO, and TDO. In some embodiments, the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, A _2a R, A _2b R, A _2a /A _2b R, arginase, CD39, and CD73. In some embodiments, the inhibitor is selected from the group consisting of ipilimumab, tremelimumab,

- 3 049282 nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tizlelizumab, zimberelimab, durvalumab and atezolizumab.

Provided herein is a method of assisting in the treatment of cancer comprising administering to a subject having a cancer a therapeutically effective amount of any of the anti-TIGIT antibodies described herein. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is acute myeloid leukemia or adult T-cell leukemia. In some embodiments, the cancer is a solid tumor, non-small cell lung carcinoma, melanoma, cervical cancer, multiple myeloma, lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, gastric cancer, gastroesophageal junction adenocarcinoma, or esophageal cancer. In some embodiments, the subject is further administered tumor-infiltrating T cells that are activated by the antibody. In some embodiments, the subject is further administered a vaccine that induces an anti-cancer immune response that is enhanced by the antibody. In some embodiments, the vaccine includes an antigen expressed on the surface of cancer cells, or a fragment thereof. In some embodiments, the subject is further administered natural killer cells whose anti-cancer cytotoxicity is enhanced by an anti-TIGIT antibody of the present invention. In some embodiments, the subject is further administered a second antibody that specifically binds to an antigen expressed on the surface of cancer cells, whereby the effector-mediated anti-cancer cytotoxicity of the second antibody is enhanced by the anti-TIGIT antibody of the present invention. In some embodiments, the subject is further administered a second antibody that specifically binds to an antigen expressed on the surface of an immune cell. In some embodiments, the immune cell is a T cell or a natural killer cell. In some embodiments, the antigen is CTLA-4, PD-1, or PD-L1. In some embodiments, the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation therapy, cell therapy, and surgery. In some embodiments, the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands. In some embodiments, the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A _2a R, A _2b R, A _2a /A _2b R, arginase, CD39, CD73, IDO, and TDO. In some embodiments, the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, A2aR, A2bR, A2a/A2bR, arginase, CD39, and CD73. In some embodiments, the inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tizlelizumab, zimberelimab, durvalumab, and atezolizumab.

Each of the aspects and embodiments described herein may be used together, except where expressly stated or clearly excluded from the context of the embodiment or aspect.

Brief description of graphic materials

Fig. 1A shows the amino acid sequence of mature 21F8 VH (SEQ ID NO: 1) and mature 21F8 VL (SEQ ID NO: 2). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 36), HC-CDR2 (SEQ ID NO: 37), and HC-CDR3 (SEQ ID NO: 38), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 39), LC-CDR2 (SEQ ID NO: 40), and LC-CDR3 (SEQ ID NO: 41), respectively.

Fig. 1B shows the amino acid sequence of mature 30M18 VH (SEQ ID NO: 3) and mature 30M18 VL (SEQ ID NO: 4). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 42), HC-CDR2 (SEQ ID NO: 43), and HC-CDR3 (SEQ ID NO: 44), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 45), LC-CDR2 (SEQ ID NO: 46), and LC-CDR3 (SEQ ID NO: 47), respectively.

Fig. 1C shows the amino acid sequence of mature 24F8 VH (SEQ ID NO: 5) and mature 24F8 VL (SEQ ID NO: 6). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively.

Fig. 1D shows the amino acid sequence of mature VH of 5J24 (SEQ ID NO: 7) and mature VL of 5J24 (SEQ ID NO: 8). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 54), HC-CDR2 (SEQ ID NO: 55), and HC-CDR3 (SEQ ID NO: 56), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 57), LC-CDR2 (SEQ ID NO: 58), and LC-CDR3 (SEQ ID NO: 59), respectively.

- 4 049282 of course.

Fig. 1E shows the amino acid sequence of mature 21B9 VH (SEQ ID NO: 9) and mature 21B9 VL (SEQ ID NO: 10). The amino acid sequences of CDR1, CdR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 60), HC-CDR2 (SEQ ID NO: 61), and HC-CDR3 (SEQ ID NO: 62), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 64), and LC-CDR3 (SEQ ID NO: 65), respectively.

Fig. 1F shows the amino acid sequence of mature 22B22 VH (SEQ ID NO: 11) and mature 22B22 VL (SEQ ID NO: 12). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 60), HC-CDR2 (SEQ ID NO: 66), and HC-CDR3 (SEQ ID NO: 67), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68), and LC-CDR3 (SEQ ID NO: 65), respectively.

Fig. 1G shows the amino acid sequence of mature 28P24 VH (SEQ ID NO: 13) and mature 28P24 VL (SEQ ID NO: 14). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 69), HC-CDR2 (SEQ ID NO: 55), and HC-CDR3 (SEQ ID NO: 70), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 71), LC-CDR2 (SEQ ID NO: 68), and LC-CDR3 (SEQ ID NO: 65), respectively.

Fig. 1H shows the amino acid sequence of mature 21B16 VH (SEQ ID NO: 15) and mature 21B16 VL (SEQ ID NO: 16). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 72), HC-CDR2 (SEQ ID NO: 73), and HC-CDR3 (SEQ ID NO: 67), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68), and LC-CDR3 (SEQ ID NO: 65), respectively.

Fig. 1I shows the amino acid sequence of mature VH 28012 (SEQ ID NO: 17) and mature VL 28012 (SEQ ID NO: 12). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 74), HC-CDR2 (SEQ ID NO: 75), and HC-CDR3 (SEQ ID NO: 67), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68), and LC-CDR3 (SEQ ID NO: 65), respectively.

Fig. 1J shows the amino acid sequence of mature Hu24F8.1 VH (SEQ ID NO: 76) and mature Hu24F8.1 VL (SEQ ID NO: 77). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively.

Fig. 1K shows the amino acid sequence of mature Hu24F8.2 VH (SEQ ID NO: 78) and mature Hu24F8.2 VL (SEQ ID NO: 77). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively.

Fig. 1L shows the amino acid sequence of mature Hu24F8.3 VH (SEQ ID NO: 78) and mature Hu24F8.3 VL (SEQ ID NO: 79). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively.

Fig. 1M shows the amino acid sequence of mature Hu24F8.4 VH (SEQ ID NO: 76) and mature Hu24F8.4 VL (SEQ ID NO: 79). The amino acid sequences of CDR1, CDR2, and CDR3 of VH are underlined and are designated HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of VL are underlined and are designated LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53), respectively.

Figure 2 shows that Ch24F8, Ch28O12, and Ch22B22 were able to bind to cynomolgus TIGIT expressed on cynomolgus CD4+ and CD8+ cells. Geometric mean fluorescence intensity (gMFI) was obtained and these data are presented as a fold comparison of gMFI to isotype control.

Fig. 3A shows a graph demonstrating the binding of humanized anti-TIGIT antibodies to Cno-Kι cells overexpressing human TIGIT.

Fig. 3B shows a graph demonstrating the binding of humanized anti-TIGIT antibodies to

- 5,049,282 CHO-K1 cells overexpressing cynomolgus macaque TIGIT.

Fig. 4A shows a graph demonstrating the binding of humanized anti-TIGIT antibodies to Cno-Kι cells overexpressing mouse TIGIT.

Fig. 4B shows a graph demonstrating the binding of humanized anti-TIGIT antibodies to CHO-K1 cells overexpressing rat TIGIT.

Fig. 5A shows a graph demonstrating binding of humanized anti-TIGIT antibodies to unactivated human CD8+ T cells.

Fig. 5B shows a graph demonstrating binding of humanized anti-TIGIT antibodies to activated human CD8+ T cells.

Fig. 6 is a graph showing the inhibition of human CD155 binding to human TIGIT-overexpressing CHO-K1 cells by humanized TIGIT antibodies.

Fig. 7 shows a graph demonstrating the inhibition of human CD155 binding to human TIGIT by humanized TIGIT antibodies in the Jurkat Dual Reporter cell line blockade assay.

Fig. 8 shows the binding of Fab24F8 to TIGIT.

Figure 9 shows TIGIT residues linked to Fab24F8 by hydrogen bonding, salt bridge, and van der Waals interaction forces.

Fig. 10A and Fig. 10B show that Fab24F8 blocks CD155 binding to TIGIT.

Fig. 10A shows a schematic representation of the complex structure of human CD155 (as a ribbon) associated with human TIGIT (represented as a molecular surface).

Figure 10B shows a superposition of CD155 in the same orientation as in Figure 10A, on a schematic representation of the Fab24F8 crystal structure complex bound to TIGIT (each represented by a molecular surface).

Figure 11 shows the response of IL-2 from one subject to SEA in the presence of Hu24F8.2-IgG1, AB122, AB122, and with Hu24F8.2-IgG1 or isotype control. Bars and error bars represent mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA with Šidák's multiple comparisons test (Hu24F8.2-IgG1 versus IgG1 at each concentration and AB122 + Hu24F8.2-IgG1 versus AB122 or with Hu24F8.2-IgG1).

Fig. 12A shows the response of IL-2 isolated from peripheral blood mononuclear cells (PBMCs) of a healthy or cancer patient to SEA in the presence of Hu24F8.2-IgG1 or isotype control. Each symbol represents an individual subject. *p<0.05; paired Student's t-test.

Fig. 12B shows the response of IL-2 isolated from healthy subject PBMC to SEA in the presence of AB122 compared with AB122 and Hu24F8.2-IgG1. Each symbol represents an individual subject. *p<0.05; paired Student's t-test.

Detailed description of the essence of the invention

The present invention provides, inter alia, antibodies that specifically bind to the extracellular domain of TIGIT. The antibodies of the present invention, also referred to herein as anti-TIGIT antibodies, inhibit the binding of TIGIT to CD155 and can thus activate T cells and/or NK cells. Among other uses, the antibodies can also be used to treat cancer and infectious disease. Additional structural and functional features of the anti-TIGIT antibody of the present invention are described in more detail below.

I. Definitions.

Unless otherwise defined, all terms of the art, designations and other scientific terms or terminology used herein have the commonly understood meanings as understood by those skilled in the art to which the present invention pertains. In some instances, terms with commonly understood meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a material difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well known and commonly used using standard methodology by those skilled in the art. The singular forms may imply the plural unless the context clearly dictates otherwise. For example, the term cell means one or more cells, including mixtures thereof. A and/or B is used herein to include all of the following alternatives: A, B, A or B, and A and B.

The term antibody refers to intact antibodies and binding fragments thereof that specifically bind to a single antigen or that specifically bind to multiple antigens (e.g., multispecific antibodies such as a bispecific antibody, trispecific antibody, etc.). Thus, any reference to an antibody is understood to refer to the antibody in intact form or a binding fragment unless the context otherwise requires. Additional functionalities (e.g., antigen binding) contemplated in the context of the present invention include fragments of anti-PD-1, anti-PD-LI, anti-TIM-3, anti-LAG-3,

- 6 049282 anti-PVRIG, anti-VISTA, aHTu-CTLA-4, anti-4-1BB, anti-BTLA, aHTu-CD39, aHTu-CD73, aHTu-OX40L and anti-OX40.

The term "binding fragment", which may be used interchangeably with the term "antigen-binding fragment", as used herein refers to a fragment of an antibody formed from a region of an antibody containing one or more CDRs, or to any other fragment of an antibody that specifically binds to an antigen but does not contain an intact native antibody structure. Examples of an antigen-binding fragment include, but are not limited to, a diabody, Fab, Fab', F(ab') ₂ , F(ab) _c , Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds-diabody), triabody, tetrabody, single chain antibody molecule (scFv), scFv dimer, multispecific antibody, camelized single domain antibody, nanobody, minibody, domain antibody, bivalent domain antibody, IgNAR, V-NAR, and hcIgG. Typically, the binding fragments compete for specific binding with the intact antibody from which they were derived. Binding fragments can be obtained by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins.

In relation to an antibody, Fab refers to that portion of an antibody that consists of a single light chain (with both variable and constant regions) disulfide-linked to the variable region and the first constant region of a single heavy chain.

Fab' stands for Fab fragment, which includes a portion of the hinge region.

F(ab')2 denotes the Fab' dimer.

Fc in relation to an antibody refers to that region of an antibody that consists of the second and third constant regions of the first heavy chain linked via a disulfide bond to the second and third constant regions of the second heavy chain. The Fc region of an antibody is responsible for various effector functions such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but lacks antigen-binding function.

Fv in relation to antibody refers to the smallest fragment of an antibody that contains a complete antigen-binding site. An Fv fragment consists of the variable region of a single light chain linked to the variable region of a single heavy chain.

The term single chain Fv antibody or scFv refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to each other directly or through a linker peptide sequence (Huston J. S. et al., Proc Natl Acad Sci USA, 85:5879(1988)).

The term single chain Fv-Fc antibody or scFv-Fc refers to an engineered antibody consisting of an scFv fused to the Fc region of an antibody.

The term camelized single-domain antibody, heavy chain antibody, or HCAb refers to an antibody that contains two V _H domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. December 10; 231(1-2); 25-38 (1999); Muyldermans S., J Biotechnol. June; 74(4):277-302 (2001); WO94/04678; WO94/25591; US Pat. No. 6,005,079). Heavy chain antibodies were originally derived from animals of the Camelidae family (camels, dromedaries, and llamas). Despite the absence of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428):446-8 (1993); Nguyen VK et al. Heavy-chain antibodies in Camelidae; a case of evolutionary innovation, Immunogenetics. April; 54(1):39-47 (2002); Nguyen VK et al. Immunology. May; 109(1):93-101 (2003)).

The variable domain of antibody heavy chains (VHH domain) is the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F et al., FASEB J. November; 21(13):3490-8. Epub 2007 Jun. 15 (2007)).

The term nanobody refers to an antibody fragment that consists of the VHH domain of the heavy chain antibody and two constant domains: CH2 and CH3.

The term diabodies includes small antibody fragments with two antigen-binding sites, the fragments comprising a V _H domain linked to a V _L domain in a single polypeptide chain (VH-VL or VL-VH) (see, e.g., Holliger P et al., Proc Natl Acad Sci USA. July 15; 90(14):6444-8 (1993); EP404097; WO93/11161). Using a linker that is too short to allow association between the two domains on one chain, the domains are forced together with the complementary domains of another chain, thus creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes).

The term domain antibody refers to an antibody fragment containing only the variable region of the heavy chain or the variable region of the light chain. In certain cases, two or more VH domains are covalently joined to a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

In certain embodiments, (dsFv) ₂ comprises three peptide chains: two V _H fragments linked by a peptide linker and connected by disulfide bridges to two V _L fragments.

- 7 049282

In certain embodiments, the term bispecific ds-diabody comprises V _H1 -V L ₂ (linked by a peptide linker) connected to V _L1 -V _H2 (also linked by a peptide linker) via a disulfide bridge between V H1 and V _L1 .

In certain embodiments, a bispecific dsFv or dsFv-dsFv comprises three peptide chains: a VH1-VH2 fragment, in which the heavy chains are linked by a peptide linker (e.g., a long flexible linker) and are connected to the V _L1 and V _L2 fragments, respectively, via disulfide bridges, wherein each disulfide-linked heavy and light chain has a different antigen specificity.

In certain embodiments, the ScFv dimer is a bivalent diabody or bivalent ScFv (BsFv) comprising V _H -V _L (linked by a peptide linker) dimerized with another fragment VH-VL such that the VH of one fragment is coordinated with the VL of the other fragment and forms two binding sites that can target the same antigens (or epitopes) or different antigens (or epitopes). In other embodiments, the scFv dimer is a bispecific diabody comprising V _H1 -V _L2 (linked by a peptide linker) linked to V _L1 -V _H2 (also linked by a peptide linker) such that V _H1 and V _L1 are coordinated and V _H2 and V _L2 are coordinated, and each coordinated pair has a different antigen specificity. An isolated antibody is an antibody that has been isolated from a component of its natural environment. In some embodiments, the isolated antibody is purified to a purity of greater than 95% or 99%, as determined by methods known in the art.

As used herein, the term monoclonal antibody refers to an antibody obtained from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone. The meaning of the term monoclonal antibody is not limited to antibodies obtained by any particular method. For example, monoclonal antibodies can be obtained using hybridoma techniques, as well as recombinant technologies, phage display technologies, synthetic technologies, or combinations of such technologies and other technologies well known in the art.

As used herein, the term humanized antibody refers to antibodies that contain sequences from both human and non-human (e.g., mouse or rat) antibodies.

As used herein, the term "human antibody" refers to an antibody that has or consists of an amino acid sequence(s), particularly antigen-binding residues, corresponding to a residue of an antibody produced by a human organism or a human immune cell, or obtained from a non-human source, such as a non-human transgenic animal, that utilizes human antibody repertoires or other sequences encoding human antibodies. In certain embodiments, a fully human antibody does not contain amino acid residues (particularly antigen-binding residues) derived from a non-human antibody.

The basic structural unit of an antibody, such as in a native intact antibody, is a tetramer of subunits. Each tetramer comprises two identical pairs of polypeptide chains, each pair having one light (about 25 kDa) and one heavy (about 50-70 kDa) chain. The amino-terminal region of each chain comprises a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes called the mature variable region. Thus, for example, the term mature light chain variable region refers to the light chain variable region without the light chain signal peptide. The carboxy-terminal region of each chain defines a constant region primarily responsible for effector function.

Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and these in turn determine the antibody isotypes: IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a J region of about 12 or more amino acids, with the heavy chain also including a D region of about 10 or more amino acids. (See Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7) (incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form the binding site of the antibody. Thus, an intact native antibody has two identical binding sites; a bispecific antibody has two non-identical binding sites; a trispecific antibody has three non-identical binding sites, etc. The mature variable regions of the heavy and light chains all have the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions, or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, allowing binding to a specific epitope. Both the light and heavy chains include the FR1, cDR1,

- 8 049282 fR2, cDR2, fR3, cDR3, and FR4 in the N-terminal to C-terminal direction. The amino acid assignments to each domain are according to the definitions given in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia & Lesk, J. Mol. Biol. 196:901–917 (1987); Chothia et al., Nature 342:878–883 (1989). Kabat also proposes a widely used numbering (Kabat numbering), in which corresponding residues from different heavy chains or different light chains are assigned the same numbers.

The term epitope refers to a site on an antigen to which an antibody binds. An epitope may be formed from contiguous amino acids or non-contiguous amino acids arranged near each other by the tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are generally preserved by exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally disrupted by treatment with denaturing solvents. Typically, an epitope includes at least 3, and more often at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance spectroscopy. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can be detected by a simple immunoassay demonstrating the ability of one antibody to compete with another antibody for binding to a target antigen. To identify contact residues, an antibody epitope can also be determined by X-ray crystallography of the antibody bound to its antigen. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or block binding of one antibody reduce or block binding of the other. Two antibodies have overlapping epitopes if some, but not all, amino acid mutations that reduce or block binding of one antibody reduce or block binding of the other.

Competition between antibodies is determined by an assay in which an antibody, under test conditions, inhibits the specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x, 90x, 100x or more, including fold values in between) inhibits binding of the reference antibody by at least about 50%, such as at least about 75%, 90% or 99%. In other embodiments, a test antibody competes with a reference antibody if an excess of the test antibody inhibits binding of the reference antibody by any of at least about 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as determined in a competitive binding assay. Antibodies detected in a competition assay (competing antibodies) include antibodies that bind to the same epitope as a reference antibody and antibodies that bind to an adjacent epitope that is close enough to the epitope bound by the reference antibody to create steric hindrance. The reference antibody may be a commercially available monoclonal antibody with functions similar to the candidate therapeutic antibody, a polyclonal antibody that functionally interacts with the target protein of interest, or an antibody reconstituted from sequences obtainable in the public domain. For example, but not limited to, a reference antibody that binds to TIGIT comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO: 93.

As used herein, the terms specific binding and specifically binds to refer to a measurable and reproducible interaction, such as binding of an antigen (e.g., TIGIT) to an antibody. For example, an antibody that specifically binds to an antigen is an antibody that binds to that target with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other antigens. The affinity and equilibrium dissociation constant (KD) of a molecule for an antigen are inversely proportional. High affinity for an antigen is measured by determining a low KD value. As used herein, an antibody that specifically binds to an antigen has a KD for the antigen of 10 ^-6 M or lower, alternatively 10 ^-7 M or lower, alternatively 10 ^-8 M or lower, alternatively 10 ^-9 M or lower, alternatively 10 ^-10 M or lower, alternatively 10 ^-11 M or lower; or a KD in the range of ^10-6 M to ^10-13 M, or ^10-9 M to 10-13 M, or 10-9 M to ^10-12 M, or ^10-10 M to ^10-13 M, or ^10-10 M to ^10-12 M, or ^10-11 M to ^10-13 M, or ^10-10 M to ^10-11 M, or ^10-11 M to ^10-12 M, as measured by surface plasmon resonance. In one embodiment ^, the term specific binding refers to binding in which the molecule binds to a particular polypeptide or an epitope on a particular polypeptide ^without substantially binding to any other polypeptide or polypeptide

- 9 049282 tide epitope.

As used herein, the term individual or subject includes animals such as humans (e.g., human individuals) and non-human animals. In some embodiments, the individual or subject is a patient under the care of a physician. Thus, the subject may be a human patient or an individual who has, is at risk for, or is suspected of having a disease of interest (e.g., cancer) and/or one or more symptoms of the disease. The subject may also be an individual who has been diagnosed at risk for having a condition of interest at the time of diagnosis or later. The term non-human animals includes all vertebrates, such as mammals, such as rodents, such as mice, non-human primates, and other mammals, such as, for example, sheep, dogs, chickens, as well as non-mammals, such as amphibians, reptiles, etc. As used herein, a reference to an amino acid residue of an amino acid sequence of interest corresponding to or in accordance with an amino acid residue of a reference amino acid sequence indicates that the amino acid residue of the sequence of interest is at a location that is homologous to or equivalent to a numbered residue in the reference amino acid sequence. One skilled in the art can determine whether the position of a particular amino acid residue in a polypeptide, such as a TIGIT polypeptide, corresponds to the position of that amino acid residue in a homologous reference sequence. For example, the TIGIT polypeptide sequence can be aligned with the reference sequence polypeptide using known techniques (e.g., basic local alignment search engine (BLAST), clustalW2, structural sequence alignment program (STRAP), or the like). In addition, the crystal structure coordinates of a reference sequence can be used to assist in determining the three-dimensional structure of a homologous polypeptide residue (Stengel et al., Proc. Natl. Acad. Sci. USA, 109:5399-5404, 2012). In another aspect, equivalent residues can be identified by determining homology at the tertiary structure level. Using such methods, the amino acid residues of a TIGIT polypeptide variant can be numbered according to the corresponding amino acid residue position numbering of the reference sequence. For example, the amino acid sequence of SEQ ID NO: 80 can be used to determine the position numbering of each amino acid residue of a human TIGIT variant or epitope of interest. In some embodiments, one amino acid sequence corresponds to another amino acid sequence if they have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. For classification purposes, amino acid substitutions with conservative or non-conservative amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions of amino acids within the same class. Non-conservative substitutions involve substitutions of a member of one of these classes for a member of another class. Percentage sequence identity values are determined with antibody sequences aligned as closely as possible using the Kabat numbering scheme. After alignment, if a region of interest of an antibody (e.g., the entire mature variable region of the heavy or light chain) maps to the same region of a reference antibody, the percent sequence identity between the regions of the subject and reference antibody is the number of positions occupied by the same amino acids in the region of the subject and reference antibody divided by the total number of aligned positions of the two regions, excluding gaps, multiplied by 100 to convert to a percentage.

Compositions or methods comprising or including (or any other grammatical variant thereof) one or more of the recited elements may include other elements not specifically recited. For example, a composition that includes an antibody may comprise the antibody alone or in combination with other ingredients. Specific ranges are provided herein with numerical values preceded by the term about. The term about is used herein in its original meaning of approximately and is intended to indicate a value that is substantially equal to the exact number that the term is preceded by, as well as a number that is close to or approximately the number that the term is preceded by. In determining whether a number is close to or approximately a specifically recited number, a close to or approximately unrecited number may be a number that, in the context in which it is presented, provides a substantial equivalent to the specifically recited number. For example, unless the degree of approximation is otherwise made clear by the context, approximately denotes either a range of plus or minus 10% of the stated value or rounds the value to the nearest significant digit in all cases, including the stated value. Where specified

- 10 049282 ranges, they include boundary values.

The term "substantially" and any grammatical variant thereof as used herein is a broad term and is used in its ordinary sense, including, without limitation, the meaning of almost completely or to a significant extent, but not completely. For example, the term may refer to a numerical value that may not be 100% of the complete numerical value, wherein the missing numerical value may be less than 0.1%, less than 0.5%, less than about 1%, less than about 2%, less than about 3%, less than about 4%, less than about 5%, less than about 6%, less than about 7%, less than about 8%, less than about 9%, less than about 10%, less than about 11%, less than about 12%, less than about 13%, less than about 14%, less than about 15%, less than about 16%, less than about 17%, less than about 18%, less than about 19%, or less than about 20% of the complete numerical value. For example, a subject antibody or antigen-binding fragment thereof may be substantially derived from a corresponding reference antibody or antigen-binding fragment thereof when the subject antibody or antigen-binding fragment thereof is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to the sequence of the corresponding reference antibody or antigen-binding fragment thereof. In another example, a CDR in a subject antibody can be derived substantially from a corresponding CDR in a reference antibody when the CDR in the subject antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical in sequence to the corresponding CDR in the reference antibody. In yet another example, without limitation, a CDR in a subject antibody can be derived substantially from a corresponding CDR in a reference antibody when no more than two amino acids are substituted, deleted, or added to the CDR in the subject antibody compared to the corresponding CDR in the reference antibody.

It should be understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be presented in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be presented separately or in any suitable subcombination. All combinations of embodiments related to the invention are specifically included in the present invention and are described herein as if each combination were expressly and distinctly individually disclosed. In addition, all subcombinations of various embodiments and elements thereof are also specifically included in the present invention and are described herein as if each such subcombination were expressly individually disclosed.

II. Target molecules.

Unless otherwise noted, TIGIT refers to human TIGIT (hTIGIT). Cyno TIGIT or cTIGIT refers to cynomolgus macaque TIGIT.

An exemplary hTIGIT sequence has been assigned Swiss-Prot accession number Q495A1. The complete hTIGIT sequence comprises 244 amino acids (SEQ ID NO: 80), with amino acids 1-21 constituting the signal peptide and amino acids 22-244 constituting the mature protein (SEQ ID NO: 81). Approximately residues 22-141 constitute the extracellular domain of hTIGIT (SEQ ID NO: 82). Approximately residues 142-162 constitute the transmembrane domain of hTIGIT and approximately residues 163-244 constitute the cytoplasmic domain of hTIGIT. In some embodiments, the extracellular domain of hTIGIT is HIS-tagged (SEQ ID NO: 83). An exemplary cynomolgus monkey TIGIT sequence has been assigned Swiss-Prot accession number A0A2K5UW92. The complete sequence of cynomolgus TIGIT comprises 312 amino acids (SEQ ID NO: 84). In some embodiments, the extracellular domain of cynomolgus TIGIT is HIS-tagged (SEQ ID NO: 85). Unless otherwise noted, CD155 refers to the human form of this protein. An exemplary human CD155 sequence has been assigned SwissProt accession number P15151 and is a 417 amino acid protein, of which approximately residues 120 are the signal peptide, 21-343 constitute the extracellular domain (SEQ ID NO: 86), 344-367 constitute the transmembrane domain, and 368-417 constitute the cytoplasmic domain.

Unless otherwise obvious from the context, reference to one of the above proteins means at least the extracellular domain of the protein and usually the complete protein other than the cleavable signal peptide.

III. Antibodies according to the invention.

A. Binding specificity and functional properties.

The present invention provides antibodies that specifically bind to TIGIT, and more particularly to epitopes within the extracellular domain of the TIGIT protein. In certain embodiments, the anti-TIGIT antibodies of the present invention have a KD for TIGIT of 10 ^-8 M or less (e.g., 10 ^-8 , 10 ^-9 , 10 ^-10 , etc.), as measured by surface plasmon resonance (SPR). In various embodiments, the anti-TIGIT antibodies of the present invention have a KD for TIGIT in the range of from about 1 x 10' ⁹ M to about 1 x 10' ¹³ M, or from about 1 x 10' ⁹ M to about 1 x 10 ^-12 M, or from about 1 x 10 ^-10 M to about 1 x 10 ^-13 M, or from about 1 x 10 ^-10 M to

- 11 049282 about 1 x 10 ^-12 M, or from about 1 x 10 ^-11 M to about 1 x 10 ^-13 M, or from about 1 x 10 ^-10 M to about 1 x 10 ^-11 M, or from about 1 x 10 ^-11 M to about 1 x 10 ^-12 M. The antibodies designated 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, and 28012 are nine such examples of mouse antibodies. The antibodies designated Ch22B22, Ch21B16, Ch28O12, Ch5J24, Ch21B9, Ch24F8, and Ch30M18 are seven such examples of chimeric antibodies.

The antibodies designated Hu24F8.1, Hu24F8.2, Hu24F8.3, and Hu24F8.4 are examples of humanized antibodies. The sequences of the mature heavy and light chain variable regions and CDRs of the mouse and humanized antibodies are shown in Table 1 and Table 2, respectively.

Table 1

Sequences of mature variable regions of heavy and light chains

Antibody Heavy chain variable region Light chain variable region 21F8 SEQIDNO: 1 SEQ GO NO: 2 30M18 SEQIDNO: 3 SEQ GO NO: 4 24F8 SEQIDNO: 5 SEQ ID NO: 6 5J24 SEQIDNO: 7 SEQIDNO: 8 21B9 SEQIDNO: 9 SEQIDNO: 10 22В22 SEQIDNO: 11 SEQIDNO: 12 28P24 SEQIDNO: 13 SEQIDNO: 14 21B16 SEQIDNO: 15 SEQIDNO: 16 28012 SEQIDNO: 17 SEQIDNO: 12 Hu24F8.1 SEQIDNO: 76 SEQ ID NO: 77 Hu24F8.2 SEQIDNO: 78 SEQ ID NO: 77 Hu24F8.3 SEQIDNO: 78 SEQIDNO: 79 Hu24F8.4 SEQIDNO: 76 SEQ ID NO: 79

Table 2

Heavy and light chain CDR sequences (as defined by Kabat)

Antibody HC-CDR1 (SEQ ID NO) HC-CDR2 (SEQ ID NO) HC-CDR3 (SEQ ID NO) LC-CDR1 (SEQ ID NO) LC-CDR2 (SEQ ID NO) LC-CDR3 (SEQ ID NO) 21F8 36 37 38 39 40 41 30M18 42 43 44 45 46 47 24F8 48 49 50 51 52 53 5J24 54 55 56 57 58 59 21B9 60 61 62 63 64 65 22B22 60 66 67 63 68 65 28P24 69 55 70 71 68 65 21B16 72 73 67 63 68 65 28012 74 75 67 63 68 65

Certain antibodies of the present invention bind to the same or overlapping epitopes as an antibody designated 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28012, or an antibody designated Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4. Other antibodies having such binding specificity can be obtained by immunizing mice with TIGIT or a portion thereof including the desired epitope and screening the resulting antibodies for binding to the extracellular domain of TIGIT, optionally in competition with 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, 28012, Hu24F8.1, Hu24F8.2, Hu24F8.3 or Hu24F8.4. Antibodies may also be screened against mutagenized forms of the TIGIT antigen to identify an antibody that exhibits the same or similar binding profile to mutational changes such as 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, 28012, Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4. The mutations may be systematic substitutions of alanine (or serine if alanine is already present) one residue at a time or broader intervals within the extracellular domain of the TIGIT antibody or within a region thereof known to contain an epitope. In some embodiments, some antibodies of the present invention bind to at least one of the following TIGIT epitope residues: T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80. In some embodiments,

- 12 049282 embodiments, some antibodies of the present invention bind to two, three, four, five or six of the following TIGIT epitope residues: T55, Q56, N58, E60, D72, S80 and K82 of SEQ ID NO: 80. In some embodiments, some antibodies of the present invention bind to the following TIGIT epitope residues: T55, Q56, N58, E60, D72, S80 and K82. In some embodiments, the anti-TIGIT antibody binds to an epitope that includes at least the following TIGIT residues: D72 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope that includes at least the following TIGIT residues: E60 and D72 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an epitope that includes at least the following TIGIT residues: D72 and K82 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80. In some embodiments, an anti-TIGIT antibody binds to an epitope that includes at least the following TIGIT residues: E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80.

Antibodies having the binding specificity of a selected murine antibody (e.g., 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28012) or a selected humanized antibody (e.g., Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4) can also be produced using a variant of the phage display method. See Winter, WO 92/20791. This method is particularly suitable for producing human antibodies. In this method, the variable region of the heavy or light chain of a selected murine antibody is used as the starting material. For example, if a light chain variable region is selected as the starting material, a phage library is constructed whose elements display the variable region of one light chain (i.e., the starting mouse material) and the variable region of different heavy chains. The heavy chain variable regions can be obtained, for example, from a library of rearranged human heavy chain variable regions. A phage that exhibits strong specific binding to TIGIT (e.g., at least 108 or at least 109 M ^-1 ) is selected. The heavy chain variable region from this phage then serves as the starting material for the construction of an additional phage library. In this library, each phage displays one heavy chain variable region (i.e., the region identified from the first phage display library) and a different light chain variable region. The light chain variable regions can be obtained, for example, from a library of rearranged human light chain variable regions. Again, a phage exhibiting strong specific binding to TIGIT is selected. As the starting material is murine, the resulting antibodies generally have the same or similar epitope specificity.

Some antibodies have a mature heavy chain variable region comprising HCCDR1, HC-CDR2 and HC-CDR3, and a mature light chain variable region comprising LCCDR1, LC-CDR2 and LC-CDR3, all or substantially from mAb 21F8. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3, and a mature light chain variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, all or substantially from mAb 30M18. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 and a mature light chain variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, all or substantially from mAb 24F8. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 and a mature light chain variable region comprising LC-CDR1, LCCDR2 and LC-CDR3, all or substantially from mAb 5J24. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 and a mature light chain variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, all or substantially from mAb 21B9. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 and a mature light chain variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, all or substantially from mAb 22B22. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 and a mature light chain variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, all or substantially from mAb 28P24. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 and a mature light chain variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3, all or substantially from mAb 21B16. Some antibodies have a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3, and a mature light chain variable region comprising LC-CDR1, LC-CDR2, and LC-CDR3, all or substantially from mAb 28012. The CDRs may be defined by any standard definition, including the Kabat, Chothia, Kabat and Chothia combined, AbM, or contact definitions, as shown in Table 3 below.

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Table 3

Loop Kabat AЬМ Chothia Contact definition L1 L-24-L34 L24-34 L24-L34 L30-L36 L2 L50-L56 L50-156 L50— L56 L46-L55 L3 L89-L97 L89-97 L89-L97 L89-L96 N1 N31-N35B (numbering according to Kabat) N26-N35Ь N26-N32..34 N30-N35V N1 NZ 1 -NZ 5 (Chothia numbering) N26-N35 N26-N32 N30-N35 N2 N50-N65 N50-N58 N52-N56 N47-N58 NZ N95-N102 N95-N102 N95-N102 N93-N10

Other antibodies can be obtained by mutagenesis of cDNA encoding the heavy and light chains of an exemplary antibody, such as 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28012. Also included in the present invention are antibodies that have an amino acid sequence identity of the mature heavy and/or light chain variable regions of at least any of the following: about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% relative to 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16 or 28012 and retain the functional properties of that sequence, and/or which differ from the corresponding antibody by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions or insertions from the reference. Amino acids in the framework residues of the variable region that are highly likely to play an important role in binding can be identified as described in the sections on humanization below. Also included are antibodies having at least one, and in some embodiments all six CDRs, as defined by Kabat, that are any of about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the corresponding CDRs 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28012.

In some embodiments, the antibodies have one or more of the following characteristics: (i) inhibition of binding of human TIGIT to human CD155, (ii) inhibition of binding of TIGIT to other ligands such as CD112 and CD113, (iii) enhancement of antigen-specific T cell responses, (iv) activation of natural killer cells, (v) stimulation of intrinsic T cell activation, and (vi) stimulation of production of one or more immunostimulatory cytokines and/or reduction of production of one or more immunosuppressive cytokines by T cells and other cells of the immune system.

In some embodiments, the antibodies described herein completely or partially inhibit the binding of TIGIT to CD155. Anti-TIGIT antibodies of the present invention can inhibit such interaction with a half-maximal inhibitory concentration (IC50) of from about 0.1 nM to about 10 nM, or from about 0.1 nM to about 8 nM, or from about 0.1 nM to about 5 nM, or from about 0.1 nM to about 4 nM, or from about 0.1 nM to about 3 nM, or from about 0.1 nM to about 2 nM, or from about 0.1 nM to about 1 nM, as measured in Example 1. In certain embodiments, some anti-TIGIT antibodies of the present invention can inhibit TIGIT binding to CD155 with an IC50 of from about 0.1 nM to about 2 nM or from about 0.2 nM to about 2 nM, as measured in Example 1. In certain embodiments, some anti-TIGIT antibodies of the present invention can inhibit TIGIT binding to CD155 with an IC50 of from about 0.2 nM to about 2 nM, from about 0.2 nM to about 0.8 nM, from about 0.4 nM to about 0.8 nM, or from about 0.6 nM to about 0.8 nM, as measured in Example 1. Some antibodies can inhibit such interaction with a half-maximal inhibitory concentration ( _IC50 ) of any of about 25-300 ng/mL, 25-75 ng/mL, 25-50 ng/mL, 40-75 ng/mL, 50-75 ng/mL, 50-90 ng/mL, 50-100 ng/mL, 75-100 ng/ml, 50-150 ng/ml, 75-175 ng/ml, 100-200 ng/ml, 125-225 ng/ml, 100-250 ng/ml, 150-300 ng/ml, 175-250 ng/ml, 200-300 ng/ml, 25-275 ng/ml, 250-300 ng/ml, 49+/-10% ng/ml, 65+/-10% ng/ml, or 76+/-10% ng/ml, as measured in Example 1. In other embodiments, the antibodies can completely or partially inhibit TIGIT binding to CD155 with an _IC50 of any of at least about 25 ng/ml, 50 ng/mL, 75 ng/mL, 100 ng/mL, 125 ng/mL, 150 ng/mL, 175 ng/mL, 200 ng/mL, 225 ng/mL, 250 ng/mL, 275 ng/mL, or 300 ng/mL or more, including concentrations in the range thereof. In addition, some antibodies can enhance antigen-specific T cell responses by 1.5-3-fold, such as by any of about 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, or 3-fold or more. Alternatively or additionally, some antibodies can increase production by 1.5-3 times

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1, 2, 3, or all of IL-2, IL-6, TNFa, and IFNy by NK cells and/or T cells, for example, the fold increase can be any of about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 times or more. Alternatively or additionally, some antibodies may increase intrinsic T cell activation by 1.5-3-fold, wherein the fold increase may be any of about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3-fold or more. Alternatively or additionally, some antibodies may inhibit cancer or an infectious disease, as demonstrated in an animal model or in a clinical study. Animal models of cancer are widely available in which human cancer cells are introduced into an immunodeficient laboratory animal, such as a mouse or rat. In one embodiment, the antibody specifically binds to TIGIT and comprises a mature heavy chain variable region comprising HC-CDR1, HC-CDR2, and HC-CDR3, and a mature light chain variable region comprising LC-CDR1, 1C-CDR2, and LCCDR3, all or substantially from antibody 24F8. In various embodiments, the antibody can (i) have an equilibrium binding constant (KD) of from about 0.01 x 10'11 M to about 100 x 10'11 M, from about 0.1 x 10'11 M to about 100 x 10'11 M, from about 0.1 x 10'11 M to about 10 x 10'11 M, from about 1 x 10'11 M to about 100 x 10'11 M, as measured by surface plasmon resonance, and/or (ii) block the binding of soluble human CD155 ligand to human TIGIT on the cell surface with a half-maximal inhibitory concentration (IC50) of from about 0.2 nM to about 2 nM, from about 0.2 nM to about 0.8 nM, from about 0.4 nM to about 0.8 nM or from about 0.6 nM to about 0.8 nM, as measured in Example 1. Alternatively and/or in addition to the above individual binding and blocking properties or combinations thereof, in some embodiments, the antibody binds to an epitope that includes at least the following TIGIT residues: (i) D72 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80, (ii) E60 and D72 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80, (iii) D72 and K82 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80, (iv) E60, d72 and K82 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58 and S80 of SEQ ID NO: 80 or (v) T55, Q56, N58, E60, D72, S80 and K82 of SEQ ID NO: 80.

Humanization or chimerization of antibodies increases the half-life in vivo compared to the parent murine antibodies. The resulting half-life can be 10-50 days, for example in humans. Half-life can be measured by pharmacokinetic studies such as those described by Kim et al, in Eur J of Immunol 24:542 (1994).

B. Non-human antibodies.

The production of other non-human antibodies against TIGIT, such as those from mouse, guinea pig, primate, rabbit, chicken, or rat, can be accomplished, for example, by immunizing the animal with TIGIT or a fragment thereof, or with cells bearing TIGIT. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused or otherwise complexed with a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvants can be used, as described below. For immunization of laboratory animals, complete Freund's adjuvant can be used followed by incomplete adjuvant. Rabbits or guinea pigs are typically used to produce polyclonal antibodies. Mice are typically used to produce monoclonal antibodies. The antibodies are screened for specific binding to TIGIT. Optionally, the antibodies are further screened for binding to a specific region of TIGIT. Such screening can be performed by determining binding of the antibody to a mutant set of TIGIT deletions and determining which mutant deletions bind to the antibody. Binding can be assessed, for example, by Western blotting, fluorescence-activated cell sorting (FACS), or enzyme-linked immunosorbent assay (ELISA).

C. Humanized antibodies.

Reduction or elimination of the HAMA (human anti-mouse antibody (also applicable to human anti-rat, human anti-rabbit, or human anti-hamster, etc.)) response is an important aspect of the clinical development of acceptable therapeutic agents. See, e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al, J. Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res. (1990), 50:1495. As described herein, the present invention provides antibodies humanized such that the HAMA response is reduced or eliminated. Variants of these antibodies can be further prepared using standard techniques known in the art, some of which are further described below.

A humanized antibody is a genetically engineered antibody in which the CDRs from an antibody derived from a non-human donor are grafted onto the acceptor sequences

- 15,049,282 human antibody activity (see, for example, Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539, Carter, US 6,407,213, Adair, US 5,859,205 6,881,557, Foote, US 6,881,557). The antibody acceptor sequences may be, for example, mature human antibody sequences, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody with some or all of the CDRs that are wholly or substantially taken from a donor antibody, and the variable region framework sequences and constant regions, if present, are wholly or substantially derived from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and typically all three CDRs that are taken in whole or in essence from a donor antibody heavy chain, and a heavy chain variable region framework sequence and a heavy chain constant region, if present, that are essentially taken from a human heavy chain variable region framework sequence and constant region sequences. Similarly, a humanized light chain has at least one, two, and typically all three CDRs that are taken in whole or in essence from a donor antibody light chain, and a light chain variable region framework sequence and a light chain constant region, if present, that are essentially taken from a human light chain variable region framework sequence and constant region sequences. As used herein, as in any embodiment, a CDR in a subject antibody is taken substantially from a corresponding CDR in a reference antibody when the corresponding residues (as defined by Kabat) of the corresponding CDRs are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; however, CDR H2 (as defined by Kabat) in a subject antibody is taken substantially from the corresponding CDR in a reference antibody when the corresponding residues (as defined by Kabat) of the corresponding CDRs are at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical. The antibody chain variable region framework or antibody chain constant region sequences are substantially taken from a human variable region framework or human constant region sequence, respectively, when the corresponding residues (as defined by Kabat) are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

Although humanized antibodies often include all six CDRs (e.g., as defined by Kabat) from a non-human (e.g., murine) antibody, they can also be produced with fewer CDRs (e.g., at least 3, 4, or 5) from a non-human antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000).

In some antibodies, only a subset of the CDRs, namely a subset of the CDR residues required for binding, called the SDRs, are needed to maintain binding in the humanized antibody.

CDR residues that do not contact either the antigen or the SDRs can be identified based on previous studies (e.g., residues H60-H65 in CDR H2 are often not required), starting from the Kabat CDR regions outside the Chothia hypervariable loops (Chothia, J. Mol. Biol. 196:901, 1987), by molecular modeling and/or empirically, or as described in Gonzales et al., Mol. Immunol. 41: 863, 2004. In such humanized antibodies, at positions that lack one or more residues of the donor CDR or that lack the entire donor CDR, the amino acid occupying the position may be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor sequence of the antibody. The number of such substitutions of acceptor amino acids for donor amino acids in the CDRs intended for inclusion in the antibody reflects the balance of competing views. Such substitutions potentially provide the advantage of reducing the number of mouse amino acids in the humanized antibody and thus reducing potential immunogenicity. However, the substitutions may also cause changes in affinity, but significant reductions in affinity can be avoided. The positions for substitution within the CDRs and amino acids can also be chosen empirically.

Although the acceptor sequence may be identical to the selected human framework sequence, whether derived from a human immunoglobulin or a human consensus framework, it is contemplated in the present invention that the acceptor sequence may include pre-existing amino acid substitutions relative to the human immunoglobulin sequence or the human consensus framework sequence. These pre-existing substitutions may be minimal; typically, the amino acid differences are four, three, two, or one relative to the human immunoglobulin sequence or the consensus framework sequence. The acceptor sequences of human antibodies may

- 16 049282 may optionally be selected from a plurality of known human antibody sequences to provide a high degree of sequence identity (e.g., 65-85% identity) between the human variable region acceptor framework sequences and the corresponding variable region framework sequences of the donor antibody chains. Specific amino acids from the human variable region framework residues may be selected for substitution based on their potential impact on CDR conformation and/or antigen binding. Investigation of such potential impacts is accomplished by modeling, studying the characteristics of amino acids at specific locations, or empirically observing the effects of substitution or mutagenesis of specific amino acids.

For example, when there is an amino acid difference between a non-human variable region framework residue and a selected human variable region framework residue, the human framework amino acid may be substituted with an equivalent non-human antibody framework amino acid if the amino acid is reasonably expected to be:

(1) directly non-covalently binds antigen, (2) is adjacent to the CDR region, (3) otherwise interacts with the CDR region (e.g., is within approximately 6 A of the CDR region).

Other candidates for substitution are amino acids from the acceptor human framework that are not typically found in human immunoglobulin at that position. These amino acids can be substituted by amino acids found at equivalent positions in the non-human donor antibody or by amino acids found at equivalent positions in more typical human immunoglobulins. Other candidates for substitution are amino acids from the acceptor human framework that are not typically found in human immunoglobulin at that position.

In some embodiments, the humanized anti-TIGIT antibody has a mature heavy chain variable region comprising a CDR1 of the amino acid sequence of SEQ ID NO: 48 with zero to two amino acid substitutions or deletions, and a CDR2 of the amino acid sequence of SEQ ID NO: 49 with zero to two amino acid substitutions or deletions, and a CDR3 of the amino acid sequence of SEQ ID NO: 50 with zero to two amino acid substitutions or deletions, and framework regions that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the framework regions of with GenBank accession number AAV40102.1 or framework regions with GenBank accession number ADX65334.1; and also has a mature light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 51 with zero to two amino acid substitutions or deletions, and a CDR2 having the amino acid sequence of SEQ ID NO: 52 with zero to two amino acid substitutions or deletions, and a CDR3 having the amino acid sequence of SEQ ID NO: 53 with zero to two amino acid substitutions or deletions, and framework regions that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the framework regions of accession number GenBank ACY78416.1 or framework regions with GenBank accession number ADU32611.1. Framework regions with accession numbers AAV40102.1, ADX65334.1, ACY78416.1 and ADU32611.1 are indicated as defined by Kabat, see Example 2 or see SEQ ID NO: 76-79, which contain the sequences of framework regions with accession numbers AAV40102.1, ADX65334.1, ACY78416.1 and ADU32611.1, as well as donor CDRs. In some embodiments, the mature heavy chain variable region is linked to at least a portion of the heavy chain constant region, and the mature light chain variable region is linked to at least a portion of the light chain constant region. In some embodiments, to express a full-length antibody, the mature variable region of the heavy chain is linked to a constant region of the heavy chain, and the mature variable region of the light chain is linked to a constant region of the light chain. Suitable constant regions are described in more detail in Section III (F). In certain embodiments of the above, the constant region of the heavy chain has the functional ability to bind to the FcyR receptor. In yet further embodiments, the constant region of the heavy chain comprises or consists of SEQ ID NO: 94, and the constant region of the light chain comprises or consists of SEQ ID NO: 95. In certain embodiments of the above, the constant region of the heavy chain has an attenuated functional ability to bind to FcyR. In additional embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 97 and the light chain constant region comprises or consists of SEQ ID NO: 95. In yet further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 101 and the light chain constant region comprises or consists of SEQ ID NO: 95. In certain embodiments of the above, the heavy chain constant region has enhanced functional ability to bind to FcyR. In additional embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 99 and the light chain constant region comprises or consists of SEQ ID NO: 95.

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In some embodiments, the humanized anti-TIGIT antibody has a mature heavy chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 76, and a mature light chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 77. In some embodiments, any change may occur in the framework residues of the variable region, except for those residues that have been determined to be likely important for binding. In some embodiments, any change is a conservative amino acid substitution. In some embodiments, the antibody comprises a mature heavy chain variable region of SEQ ID NO: 76 and a mature light chain variable region of SEQ ID NO: 77. The Hu24F8.1 antibodies of the present invention comprise a mature heavy chain variable region of SEQ ID NO: 76 and a mature light chain variable region of SEQ ID NO: 77. In some embodiments of the above, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region and the mature light chain variable region is linked to at least a portion of a light chain constant region. In some embodiments, for expression of a full-length antibody, the mature heavy chain variable region is linked to a heavy chain constant region and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in more detail in Section III (F). In certain embodiments of the above, the heavy chain constant region can induce Fcγ receptor (FcyR)-mediated signaling as measured by commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In yet further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 94, and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce FcyR-mediated signaling as measured by commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions.

In some embodiments, the humanized anti-TIGIT antibody has a mature heavy chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 78, and a mature light chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 77. In some embodiments, any change may occur in the framework residues of the variable region, except for those residues that have been determined to be likely important for binding. In some embodiments, any change is a conservative amino acid substitution. In some embodiments, the antibody comprises a mature heavy chain variable region of SEQ ID NO: 78 and a mature light chain variable region of SEQ ID NO: 77. The Hu24F8.2 antibodies of the present invention comprise a mature heavy chain variable region of SEQ ID NO: 78 and a mature light chain variable region of SEQ ID NO: 77. In some embodiments of the above, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region and the mature light chain variable region is linked to at least a portion of a light chain constant region. In some embodiments, for expression of a full-length antibody, the mature heavy chain variable region is linked to a heavy chain constant region and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in more detail in Section III (F). In certain embodiments of the above, the heavy chain constant region can induce Fcy receptor (FcyR)-mediated signaling, which is measured using commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In yet further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 94, and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcy receptor (FcyR)-mediated signaling, which is measured using commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 97 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 101 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments,

- 18 049282 embodiments, the heavy chain constant region induces enhanced Fcy receptor (FcyR)-mediated signaling as measured by commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 99, and the light chain constant region comprises or consists of SEQ ID NO: 95.

In some embodiments, the humanized anti-TIGIT antibody has a mature heavy chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 76, and a mature light chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 79. In some embodiments, any change may occur in the framework residues of the variable region, except for those residues that have been determined to be likely important for binding. In some embodiments, any change is a conservative amino acid substitution. In some embodiments, the antibody comprises a mature heavy chain variable region of SEQ ID NO: 76 and a mature light chain variable region of SEQ ID NO: 79. The Hu24F8.3 antibodies of the present invention comprise a mature heavy chain variable region of SEQ ID NO: 78 and a mature light chain variable region of SEQ ID NO: 79. In some embodiments of the above, the mature heavy chain variable region is linked to at least a portion of a heavy chain constant region and the mature light chain variable region is linked to at least a portion of a light chain constant region. In some embodiments, for expression of a full-length antibody, the mature heavy chain variable region is linked to a heavy chain constant region and the mature light chain variable region is linked to a light chain constant region. Suitable constant regions are described in more detail in Section III (F). In certain embodiments of the above, the heavy chain constant region can induce Fcy receptor (FcyR)-mediated signaling, which is measured using commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In yet further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 94, and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcy receptor (FcyR)-mediated signaling, which is measured using commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 97 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 101 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region induces enhanced Fcy receptor (FcyR)-mediated signaling as measured by commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 99 and the light chain constant region comprises or consists of SEQ ID NO: 95.

In some embodiments, the humanized anti-TIGIT antibody has a mature heavy chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 78, and a mature light chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 79. In some embodiments, any change may occur in the framework residues of the variable region, except for those residues that have been determined to be likely important for binding. In some embodiments, any change is a conservative amino acid substitution. In some embodiments, the antibody comprises a mature heavy chain variable region of SEQ ID NO: 78 and a mature light chain variable region of SEQ ID NO: 79. The Hu24F8.4 antibodies of the present invention comprise a mature heavy chain variable region of SEQ ID NO: 76 and a mature light chain variable region of SEQ ID NO: 79. In some embodiments of the above, the mature heavy chain variable region is linked to at least a portion of the heavy chain constant region and the mature light chain variable region is linked to at least a portion of the light chain constant region. In some embodiments, for expression of a full-length antibody, the mature heavy chain variable region is linked to the heavy chain constant region and the mature light chain variable region is linked to the light chain constant region. Suitable Constant Regions

- 19 049282 are described in more detail in Section III (F). In certain embodiments of the above, the heavy chain constant region can induce Fcγ receptor (FcyR)-mediated signaling as measured by commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In yet further embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 94 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce FcyR-mediated signaling as measured by commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 97 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 101 and the light chain constant region comprises or consists of SEQ ID NO: 95. In other embodiments, the heavy chain constant region induces enhanced Fcy receptor (FcyR)-mediated signaling as measured by commercially available antibody-dependent cell-mediated toxicity bioassay kits according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or consists of SEQ ID NO: 99 and the light chain constant region comprises or consists of SEQ ID NO: 95.

In further embodiments, for each of the above cases, the humanized anti-TIGIT antibodies may (i) have an equilibrium binding constant (KD) of from about 0.01x10'11 M to about 100x10'11 M, from about 0.1x10'11 M to about 100x10'11 M, from about 0.1x10'11 M to about 10x10'11 M, from about 1x10'11 M to about 100x10'11 M, or even from about 1x10'11 M to about 10x10'11 M, as measured by surface plasmon resonance techniques, and/or (ii) block the binding of soluble human CD155 ligand to human TIGIT on the cell surface with a half-maximal inhibitory concentration (IC50) of from about 0.2 nM to about 2 nM, from about 0.2 nM to about 0.8 nM, from about 0.4 nM to about 0.8 nM, or from about 0.6 nM to about 0.8 nM, as measured in Example 1. Alternatively and/or in addition to the individual binding and blocking properties noted above, or combinations thereof, in some embodiments, the humanized anti-TIGIT antibody binds to an epitope that includes at least the following TIGIT residues: (i) D72 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80, (ii) E60 and D72 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80, (iii) D72 and K82 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58, E60 and S80 of SEQ ID NO: 80, (iv) E60, d72 and K82 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58 and S80 of SEQ ID NO: 80 or (v) T55, Q56, N58, E60, D72, S80 and K82 of SEQ ID NO: 80.

D. Chimeric and veneered antibodies.

The present invention further provides chimeric and veneered forms of non-human antibodies, in particular, for example, antibodies 21F8, 30M18, 24f8, 5J24, 21B9, 22B22, 28P24, 21B16 and 28O12.

A chimeric antibody is an antibody in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., a mouse antibody) are combined with the constant regions of human light and heavy chains. Such antibodies retain substantially or completely the binding specificity of the non-human antibody and are approximately two-thirds human in sequence.

A veneered antibody is a type of humanized antibody in which some or generally all of the CDRs and some of the framework residues of the variable regions of a non-human antibody are retained, but other framework residues of the variable regions that may interact with B- or T-cell epitopes, such as cell surface residues (Padlan, Mol. Immunol. 28:489, 1991), are replaced with residues at corresponding positions in the human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially taken from the non-human antibody and the variable region frameworks of the non-human antibody are largely humanized by the substitutions. The present invention includes veneered forms of antibodies 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28012.

In some embodiments, the chimeric anti-TIGIT antibody is a chimeric mouse-human chimera having mouse variable domains and human IgG1/kappa constant domains. In one embodiment, the chimeric anti-TIGIT antibody is a chimeric Fab mVH+mVL constructed from the mouse 21F8VH (SEQ ID NO: 1) and 21F8VL (SEQ ID NO: 2) domains and a human IgG1/kappa Fab constant domain (Ch21F8). In one embodiment, the chimeric anti-TIGIT antibody is a chimeric mVH+mVL Fab constructed from the mouse 30M18VH (SEQ ID NO: 3) and 30M18VL (SEQ ID NO: 4) domains and the human IgG1/kappa Fab constant domain (Ch30M18). In one embodiment, the chimeric anti-TIGIT antibody is

- 20 049282 is a chimeric Fab mVH+mVL constructed from the mouse 24F8VH (SEQ ID NO: 5) and 24F8VL (SEQ ID NO: 6) domains and the constant domain of a human IgG1/kappa Fab (Ch24F8). In one embodiment, the chimeric anti-TIGIT antibody is a chimeric Fab mVH+mVL constructed from the mouse 5J24VH (SEQ ID NO: 7) and 5J24VL (SEQ ID NO: 8) domains and the constant domain of a human IgG1/kappa Fab (Ch5J24). In one embodiment, the chimeric anti-TIGIT antibody is a chimeric mVH+mVL Fab constructed from the mouse 21B9VH (SEQ ID NO: 9) and 21B9VL (SEQ ID NO: 10) domains and a human IgG1/kappa Fab constant domain (Ch21B9). In one embodiment, the chimeric anti-TIGIT antibody is a chimeric mVH+mVL Fab constructed from the mouse 22B22VH (SEQ ID NO: 11) and 22B22VL (SEQ ID NO: 12) domains and a human IgG1/kappa Fab constant domain (Ch22B22). In one embodiment, the chimeric anti-TIGIT antibody is a chimeric mVH+mVL Fab constructed from the mouse 28P24VH (SEQ ID NO: 13) and 28P24VL (SEQ ID NO: 14) domains and a human IgG1/kappa Fab constant domain (Ch28P24). In one embodiment, the chimeric anti-TIGIT antibody is a chimeric mVH+mVL Fab constructed from the mouse 21B16VH (SEQ ID NO: 15) and 21B16VL (SEQ ID NO: 16) domains and a human IgG1/kappa Fab constant domain (Ch21B16). In one embodiment, the chimeric anti-TIGIT antibody is a chimeric mVH+mVL Fab constructed from the mouse 28O12VH (SEQ ID NO: 17) and 28O12VL (SEQ ID NO: 12) domains and the constant domain of a human IgG1/kappa Fab (Ch28O12).

E. Human antibodies.

Human antibodies against TIGIT are provided using a variety of techniques described below. Certain human antibodies are selected by competition binding experiments using the Winter phage display method described above or otherwise to have the same epitope specificity as a particular mouse antibody, such as one of the monoclonal mouse antibodies described in the Examples. Human antibodies can also be screened for specificity for a particular epitope using only a fragment of TIGIT as the target antigen and/or antibodies specific for a set of TIGIT deletion mutants can be screened. Methods for producing human antibodies include the trioma method, see Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666, using transgenic mice, including the use of human immunoglobulin genes (see, e.g., Lonberg et al., WO93/12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991), as well as phage display methods (see, for example, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743 and US 5,565,332).

F. Selecting a constant region.

Each of the variable regions of the heavy and light chains of the chimeric, humanized (including veneered) or human antibodies can be linked to at least a portion of a human constant region. In some embodiments, the variable domain of the heavy chain described in the sections above is linked to a portion of a human heavy chain constant region, and the variable domain of the light chain described in the sections above is linked to a portion of a human light chain constant region. In some embodiments, the variable domain of the heavy chain described in the sections above is linked to a portion of a human heavy chain constant region, and the variable domain of the light chain described in the sections above is linked to a full-length human light chain constant region. The constant region of the heavy chain includes the Fc region (crystallizable fragment), which is the tail region of the antibody that interacts with cell surface receptors (Fc receptors) and certain proteins of the complement system. In some embodiments, the heavy chain variable domain described in the sections above is linked to a full-length human heavy chain constant region, and the light chain variable domain described in the sections above is linked to a full-length human light chain constant region.

The choice of the constant region (or truncated portion thereof) depends in part on whether effector functions are desired or even to be enhanced. The term effector functions refers to the biological activity attributable to the Fc region of an antibody and varies depending on the antibody isotype. Non-limiting examples of antibody effector functions include: C1q binding at the C1 complex and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., the B cell receptor) and B cell activation. Human antibodies are classified into five isotypes (IgM, IgD, IgG, IgA, and IgE) based on their heavy chain, each of which provides different functions. IgG consists of four human subclasses (IgG1, IgG2, IgG3, and IgG4), each containing a distinct heavy chain. They are highly homologous and differ primarily in the hinge region and the extent to which they can activate the host immune system.

- 21 049282 For example, human IgG1 and IgG3 isotopes can mediate complement-mediated cytotoxicity, while human IgG2 and IgG4 isotypes cannot do so or can do so to a very small extent. The constant regions of the light chain can be of the lambda or kappa subclasses. For anticancer immunotherapy or immunotherapy against a pathogen that does not express TIGIT, in addition to human IgG1 and IgG3, human IgG2 or IgG4 or an attenuated form of human IgG1 with reduced effector function can be used. For human IgG4, a genetically engineered mutation S228P (EU numbering) can be included in the heavy chain to prevent Fab-arm exchange. However, to eliminate cancer cells expressing TIGIT (e.g., T cell or NK cell tumors), human IgG1 or IgG3 can be used for immunosuppression. For example, antibodies with Fc effector function (e.g., human IgG1 or IgG3) can be used to directly kill cancer cells expressing TIGIT (e.g., in some hematological malignancies) or for immunosuppression. Suitable sequences for human IgG1 or IgG3 are known in the art and include, for example, SEQ ID NO:94, and human IgG3 described in U.S. Patent No. 5,624,821.

Human constant regions exhibit allotypic variation and isoallotypic variation among individuals, i.e., between individuals, the constant regions may differ at one or more polymorphic positions. Isoallotypes differ from allotypes in that a serum recognizing an isoallotype binds to a nonpolymorphic region of one or more other isotypes. A reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying polymorphic positions in natural allotypes.

One or more amino acids at the amino terminus or carboxy terminus of the light and/or heavy chain, such as lysine at the C-terminus of the heavy chain, may be absent or present in a derivative 10 form in some or all molecules. The N-terminal glutamine of the heavy or light chain may be substituted with a glutamate residue to prevent pyroglutamate formation. Substitutions can be made in constant regions to reduce or enhance effector function, such as complement-mediated cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent 15 No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to increase half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Examples of substitutions include Gln at position 250 and/or Leu at position 428 (EU numbering) to increase the half-life of the antibody. In some embodiments, the antibodies described herein comprise a wild-type heavy chain constant region as described above. In some embodiments, the wild-type heavy chain constant region is SEQ ID NO: 94. SEQ ID NO: 92 is an example of a heavy chain amino acid sequence comprising the wild-type constant region of SEQ ID NO: 94. In other embodiments, the antibodies described herein have a wild-type variant heavy chain constant region (or a truncated portion thereof) selected from a variant human IgG1, a variant human IgG2, a variant human IgG3, or a variant human IgG4. In some embodiments, the variant heavy chain region is SEQ ID NO: 97, SEQ ID NO: 99, or SEQ ID NO: 101. SEQ ID NOs: 96, 98, and 100 are example of heavy chain amino acid sequences comprising a variant heavy chain constant region.

Some antibodies of the invention are engineered by introducing constant region mutation(s) to reduce effector functions such as CDC and ADCC or antibody-dependent cellular phagocytosis (ADCP) compared to the same antibody without the mutation(s). In some embodiments, each or all of these effector functions are reduced by at least 50%, 75%, 90%, or 95% compared to antibodies without the mutation. Other assays 35 are described in Shields et al, 2001 J. Biol. Chem., Vol. 276, p 6591-6604; Chappel et al, 1993 J. Biol. Chem., Vol 268, p 25124-25131; Lazar et al, 2006 PNAS, 103; 4005-4010.

A substitution at any or all of positions 234, 235, 236 and/or 237 reduces the affinity of Fcγ receptors, in particular the FcγRI receptor (see, e.g., U.S. Patent 6,624,821). In some embodiments, an alanine residue is used for the substitution, such as in the double mutation L234A/L235A, 40 to reduce effector function. Other combinations of mutations with reduced effector 35 functions include L234A/L235A/G237A, E233P/L234V/L235A/AG236, A327G/A330S/P331S, K322A, L234A and L235A, L234F/L235E/P331S (EU numbering). Optionally, positions 234, 236, and/or 237 in human IgG2 are replaced by alanine and position 235 by glutamine (see, e.g., US 5,624,821). Two amino acid substitutions in the complement-fixing site C1q at EU positions 330 and 331 reduce complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitutions in human IgG1 with IgG2 residues at positions 233–236 and IgG4 residues at positions 327, 330, and 331 reduce ADCC and CDC (see, e.g., Armour KL et al., 1999

- 22 049282

Eur J Immunol. 29(8):2613–24; and Shields RL et al., 2001. J Biol Chem. 276(9):6591–604). Mutations N297A, n297Q, or N297G (EU numbering) reduce glycosylation and thus reduce effector functions.

The antibodies of the present invention can be engineered in some embodiments to enhance Fc effector function. For example, FcγR binding can be enhanced by amino acid engineering. In some embodiments, this can be accomplished by substituting one or more amino acids in the Fc region. Desired mutations can be identified, for example, either by alanine scanning or by rational library design and screening. Using these technologies, IgG variants with enhanced FcR binding and enhanced effector function can be identified. Alternatively, a number of Fc receptor region mutations are known in the art, for example as described in Smith P. et al (2012) PNAS 6181-6186.

In some embodiments, the antibodies described herein comprise a modified IgG1 constant domain that enhances the ability of the antibody to mediate ADCC compared to wild-type IgG1 without modification. The modified IgG1 domain may be characterized by amino acid substitutions at one or more of L235V, S239D, F243L, R292P, A330L, I332E, P396L (EU numbering). In other embodiments, the modified IgG1 domain is characterized by substitutions at S239D, A330L, and I332E (EU numbering). In some embodiments, a therapeutically effective amount of an antibody as described herein is capable of inducing cell death in at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, or at least 65% of TIGIT-expressing cells within 1, 2, or 3 hours, as determined by methods described in the art. Alternatively, the glycoform can be used to enhance the Fc-mediated therapeutic function of the antibody. N-linked Fc glycosylations on IgG1 antibodies are important for effector function. The binding and activity of IgG molecules can be affected by sialylation, galactosylation, sugar bisection, and fucosylation. Glycosylation patterns on therapeutic antibodies can be manipulated in a number of different ways. The cell type producing the recombinant antibody and the conditions under which it is cultured can influence the glycosylation and activity of therapeutic antibodies. In addition, bioreactor conditions and subsequent processing can also influence glycan microheterogeneity. Antibodies with low levels of fucose residues or afucosylated antibodies have been shown to enhance Fc-mediated properties. Numerous methods for achieving this reduction in fucose levels through glycoengineering are well known in the art. One method is to manipulate enzymes involved in the post-translational modification of antibodies. These may include overexpression of glucosidases such as β-1-4-N-apetylglucosaminyltransferase III, knockout of genes encoding fucosyltransferases, or the use of cell lines that are inherently fucose-deficient or have been mutated to express low levels of fucosylation. In addition, N-linked glucosidase inhibitors such as castanospermine can also be used to produce low-fucose IgG molecules. In some embodiments, engineered amino acid variants can have a broader enhanced affinity for multiple FcyRs, while a modified glycoform of the antibody can substantially have a more specific affinity with enhanced binding to FcyRIIIa. Glycoforms interact with proximal amino acids in the Fc region, and substitution of the amino acid that contacts the Ig oligosaccharides can result in the formation of different glycoform structures.

G. Expression of recombinant antibodies.

Chimeric, humanized (including veneered), and human antibodies are typically produced by recombinant expression. Accordingly, the present invention also provides polynucleotides encoding the anti-TIGIT antibodies described in this Section IIIA-G, vectors comprising the polynucleotides, and host cells comprising the vectors. The polynucleotides encoding the anti-TIGIT antibodies of the present invention can be inserted into a vector for amplification, expression, or further optimization. A variety of vectors are available. In some embodiments, the vector system includes mammalian systems, bacterial systems, yeast systems, etc. and includes plasmids such as, but not limited to, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., as well as other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1a), and a transcription termination sequence. To achieve expression, recombinant polynucleotide constructs typically include an expression control sequence, a functional

- 23 049282 naturally linked to coding sequences of the antibody chains, including naturally associated or heterologous promoter regions. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. After introduction of the vector into an appropriate host cell, the cell is incubated under conditions suitable for high-level expression of the nucleotide sequences, as well as for harvesting and purifying the recombinant antibodies. Vectors containing a polynucleotide sequence encoding an anti-TIGIT antibody of the present invention can be introduced into a host cell for cloning or expression of the genes. Suitable host cells for cloning or expression of the polynucleotide sequences in the vectors described herein include prokaryotic and eukaryotic cells. Non-limiting examples of suitable prokaryotes include eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescans and Shigella, and Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces. In addition to prokaryotes, suitable cloning or expression hosts for vectors encoding anti-TIGIT antibodies include eukaryotic microorganisms, such as filamentous fungi or yeasts. Non-limiting examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe; Kluyveromyces host cells such as, for example, K. lactis, K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500), K. drosophilarum (ATCC 36.906), K. thermotolerans, and K. marxianus; yarrowia (EP 402.226); Pichia pastoris (EP 183.070); Candida; Trichoderma reesia (EP 244.234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium, as well as Aspergillus such as A. nidulans and A. niger. Acceptable host cells can also be obtained from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants and corresponding acceptable host cells have been identified from insects such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of transfection virus strains are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used herein as the virus of the invention, particularly for transfection of Spodoptera frugiperda cells. Cotton, corn, potato, soybean, petunia, tomato, and tobacco plant cell cultures can also be used as host cells. In some embodiments, mammalian cells are used as host cells for the expression of nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myeloma cells including Sp2/0 and NS0. In some embodiments, the cells are non-human. Expression vectors for these cells can include expression control sequences such as an origin of replication, a promoter, an enhancer (Queen et al, Immunol. Rev. 89:49 (1986)), as well as necessary information processing sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. In some embodiments, the expression control sequences are promoters derived from endogenous genes, cytomegalovirus, sV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

Host cells are transformed with the above-described expression or cloning vectors for the production of antibodies to TIGIT and cultured in standard nutrient medium modified as appropriate for induction of promoters, selection of transformants, or amplification of genes encoding the desired sequences. After expression, the antibodies can be purified according to standard procedures in the art, including HPLC, column chromatography, gel electrophoresis, etc. (see, in general, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

IV. Therapeutic applications.

The TIGIT antibodies of the present invention can be used to enhance immune responses in the treatment of cancer and infectious diseases. Diseases treatable with the antibodies of the present invention include, but are not limited to, cancers including hematological malignancies, solid tumors, Merkel cell carcinoma, urothelial tumor, squamous cell carcinoma of the head and neck, B-cell lymphomas, cancer of the uterus, cervix, testicles, gastrointestinal tract (e.g., esophagus, gastroesophageal junction, oropharynx, stomach, small or large intestine, colon or rectum), bladder, bone, bone marrow, skin, gallbladder, heart, lung, salivary glands, adrenal glands, thyroid gland, ganglion, central nervous system (CNS) and peripheral nervous system (PNS), and hematopoietic cancer

- 24 049282 system and the immune system (eg, the spleen or thymus).

The present invention also provides methods for treating or preventing other cancer-related diseases, disorders, or conditions, including, for example, immunogenic tumors, non-immunogenic tumors, latent tumors, virus-induced tumors (e.g., epithelial cell cancer, endothelial cell cancer, squamous cell carcinoma, and oncogenic papillomavirus), adenocarcinomas, teratocarcinomas, chemically induced cancers, metastasis, and angiogenesis. In particular embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, or leukemia. Use of the term(s) cancer-related diseases, disorders, and conditions is intended to refer broadly to conditions associated directly or indirectly with cancer, and includes, for example, angiogenesis and precancerous conditions such as dysplasia. In certain embodiments, the cancer may be metastatic or at risk of becoming metastatic or may occur in diffuse tissue, including cancer of the blood or bone marrow (e.g., leukemia).

Such cancers may or may not express TIGIT or CD155. Anti-TIGIT antibodies are effective against cancers that do not express TIGIT because inhibition of the interaction of TIGIT with CD155 stimulates an immune response against such cancers. Examples of hematologic malignancies include leukemias, lymphomas, and myelomas, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocytic leukemia, acute lymphoblastic leukemia, T-cell lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma. Examples of solid tumors include, but are not limited to, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell and non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcomas, and brain tumors (e.g., gliomas such as glioblastomas). The methods of the present invention can be practiced in an adjuvant therapy setting. The term adjuvant therapy setting refers to a clinical setting in which the subject has a history of a proliferative disease, particularly cancer, and has responded substantially (but not necessarily) favorably to therapy, which includes, but is not limited to, surgery, radiation therapy, and/or chemotherapy. However, due to a history of proliferative disease, these subjects are considered to be at risk of developing this disease. Treatment or administration in an adjuvant setting refers to a further method of treatment. In some embodiments, provided herein is a method of treating or preventing cancer, comprising administering to a subject having cancer or at risk of developing cancer, a therapeutically effective amount of any of the antibodies disclosed herein, in an adjuvant setting.

The methods provided herein may also be practiced in a neoadjuvant setting, i.e., the method may be performed prior to primary/definitive therapy. In some embodiments, the subject has previously received treatment. In some embodiments, the subject has not previously received treatment. In some embodiments, the treatment is first-line therapy. In some embodiments, provided herein is a method of treating or preventing cancer, comprising administering to a subject having or at risk of developing cancer, a therapeutically effective amount of any of the antibodies disclosed herein, in a neoadjuvant setting. Other disorders treatable with the antibodies of the present invention include infectious diseases caused by viruses, bacteria, fungi, protozoa and other pathogens (e.g., hepatitis (A, B or C)), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTLV), dengue fever virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, HIV, simian immunodeficiency virus (SIV), and arbovirus (tick-borne) encephalitis, Chlamydia, Rickettsia, Mycobacterium, Staphylococcus, Streptococcus, Pneumococcus, Conococcus, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria, Salmonellosis, Bacillus, Cholera, Tetanus, Botulism, Anthrax, Plague, Leptospirosis, and Lyme Disease.

A. Introduction of antibodies.

The antibodies described herein are administered in an effective regimen that includes a dosage, route of administration, and frequency of administration that delays the onset of the disorder, reduces the severity of the disorder, inhibits further deterioration, and/or alleviates at least one sign or symptom of the disorder. If the subject already suffers from the disorder, the regimen may be referred to as a therapeutically effective regimen. If the subject is at increased risk of developing the disorder from

- 25 049282 relative to the general population but is not yet symptomatic, the regimen may be called a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy may be observed in an individual subject relative to historical controls or prior experience in the same subject. In other cases, therapeutic or prophylactic efficacy may be demonstrated in a preclinical or clinical study in a population of treated subjects compared to a control population of untreated subjects.

In some cases, the subject is defined as PD-L1 positive, CD155 positive, TIGIT positive, microsatellite instability high (mSI high), infiltrating T cells, activated T cells, high levels of molecules associated with antigen processing and presentation, high tumor mutational burden (TMB high), or any combination of these features. In some embodiments, the patient is selected for treatment with the antibodies described herein based on high TIGIT expression, such as on CD8+ cells and/or CD4+ cells and/or NK cells, compared to a control population. In some cases, the subject is defined as having an oncogene-driven cancer and has a mutation in at least one gene selected from the group consisting of TP53, VHL, KRAS, BRAF, MET, FUBP1, RAC1, EGFR, CDK4, CTCF, PGR, RET, RASA1, JAK1, PHF6, NF1, CIC, ARID1A, ZFHX3, ZCCHC12, GNA11, SMAD4, USP9X, CDKN2A, FAT1, PIK3R1, SCAF4, PMS2, RNF43, SMC1A, BCOR, FGFR2, COL5A1, ATM, KMT2B, CTNNB1, MYC, RAD21, PTEN, AXL, HIF1A, EPAS1, PAK4, RHOB, TBL1XR1, KEAP1, ZFP36L2, FGFR3, FOXA1, FLT3, TRAF3, RNF111, PPP2R1A, TXNIP, STAG2, RIT1, TGIF1, FOXQ1, ATR, CYSLTR2, PCBP1, PIK3R2, ASXL1, HIST1H1C, KLF5, PIK3CB, SPOP, MECOM, CACNA1A, CTNND1, DACH1, XPO1, ZNF750, FBXW7, MUC6, KDM6A, GATA3, ZBTB20, PIK3CA, RBI, SOX17, SMARCA4, KIT, CHD8, CHD4 and AROV.

In some aspects, any of the methods described herein include administering to subjects in need thereof a therapeutically effective amount of one or more anti-TIGIT antibodies described herein. As used herein, the terms therapeutically effective amount or therapeutically effective dosage for an anti-cancer therapy (such as administering any of the anti-TIGIT antibodies described herein) mean an amount sufficient to achieve beneficial or desired results. For therapeutic use, the terms beneficial or desired results include, but are not limited to, clinical results such as a reduction in one or more symptoms resulting from cancer, an increase in the quality of life of subjects suffering from cancer, a reduction in the dose of other drugs required to treat cancer, an enhancement of the effect of another drug, such as by targeting, a delay in disease progression, and/or an extension of life. An effective dosage can be administered in one or more administration sessions. For purposes of the present invention, an effective dosage of an anti-cancer therapy is an amount sufficient to achieve the goals of therapeutic or prophylactic treatment, directly or indirectly. As is clear from the clinical context, a therapeutically effective dosage of an anticancer therapy may or may not be achieved in combination with another anticancer therapy.

Exemplary dosages for any of the antibodies described herein are about 0.1-20 mg/kg or 0.5-5 mg/kg body weight (e.g., about 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg), or 10-1600 mg (e.g., any dose of the less than 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg or 1600 mg or more, including values in the range between these values) as a fixed dosage. In one embodiment, the antibody described herein is given in an amount of from about 300 to 1500 mg every three weeks. In another embodiment, the antibody described herein is given in an amount of about 300 to 1800 mg every four weeks. The dosage depends on the condition of the subject and the response to previous treatment, if any, regardless of whether the treatment is prophylactic or therapeutic and whether the disorder is acute or chronic, among other factors.

The administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intratumoral, local, intranasal or intramuscular. In some embodiments, administration into the systemic circulation is carried out by intravenous or subcutaneous administration. Intravenous administration may be carried out, for example, by infusion over a period of, for example, 30-90 minutes.

The frequency of administration depends on, among other factors, the half-life of the antibody in the bloodstream, the condition of the subject, and the route of administration. The frequency may be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the condition of the subject or the progression of the disorder for which the subject is being treated. In one embodiment, the frequency may be biweekly cycles. In another embodiment, the frequency may be

- 26 049282 set three-week cycles. In another embodiment, the frequency is four-week cycles. In another embodiment, the frequency is six-week cycles. An approximate frequency for intravenous administration is from once a week to once a quarter during a continuous course of treatment, although more or less frequent administration is also possible. For subcutaneous administration, an approximate frequency of administration is from once a day to once a month, although more or less frequent administration is also possible. The number of dosages administered depends on whether the disorder is acute or chronic, as well as the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, a number of doses from 1 to 10 are often sufficient. Sometimes a single bolus dose, optionally divided into several administrations, is sufficient for an acute disorder or an acute exacerbation of a chronic disorder. Treatment can be repeated when the acute disorder recurs or when there is an acute exacerbation. For chronic disorders, the antibody may be administered at regular intervals, such as weekly, biweekly, monthly, quarterly, every six months for at least 1, 5, or 10 years or for the life of the subject.

Treatment that includes an antibody to TIGIT can alleviate disease, as demonstrated by an increase in median progression-free survival or overall survival time of subjects with cancer by at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,

58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,

77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

96%, 97%, 98%, 99%, or even 100% compared to control subjects, or in an increase in any of these periods by 2 weeks, 1, 2, or 3 months, or by 4 or 6 months, or even 9 months or a year. Additionally or alternatively, treatment comprising an antibody to TIGIT can increase the complete response rate, partial response rate, or objective response rate (complete + partial) in subjects by at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,

40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,

59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,

78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,

97%, 98%, 99%, or even 100% compared to control subjects. Control subjects received the same treatment as subjects receiving the anti-TIGIT antibody, except for the anti-TIGIT antibody. Thus, control subjects may receive placebo or a combination of placebo and some chemotherapy drug other than the anti-TIGIT antibody, if the subjects receiving the anti-TIGIT antibody also receive them.

The anti-TIGIT antibodies disclosed herein can enhance NK cell-mediated cytotoxicity of CD155-expressing cells (such as, but not limited to, K562 cells) by any of about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35% or more, relative to the NK cell-mediated cytotoxicity of CD155-expressing cells in the absence of one of the anti-TIGIT antibodies disclosed herein.

Typically, in a clinical trial (e.g., a phase II, phase II/III, or phase III trial), the increase in median progression-free survival and/or response rate of subjects treated with an anti-TIGIT antibody relative to a control group of subjects is statistically significant, e.g., at a p level of 0.05 or 0.01 or even 0.001. Complete and partial response rates are determined using objective criteria commonly used in clinical trials of cancer, such as those listed or accepted by the National Cancer Institute and/or the Food and Drug Administration, which may include, for example, but not limited to, tumor volume, number of tumors, metastases, survival time, and quality of life.

Pharmaceutical compositions for parenteral administration may be sterile and substantially isotonic and prepared under conditions that meet Good Manufacturing Practice (GMP) standards. The pharmaceutical compositions may be provided in unit dosage form (i.e., single-dose dosage). The pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or additives. The composition depends on the selected route of administration. For injection, the antibodies may be formulated in aqueous solutions, such as physiologically compatible buffers such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the antibodies may be in lyophilized form for reconstitution with a suitable vehicle, such as sterile, pyrogen-free water, before use. The antibody concentration in liquid formulations can vary from, for example, approximately 10-150 mg/mL. In some formulations, the concentration is approximately 20-80 mg/L.

- 27 049282

B. Combination therapies.

The present invention provides for the use of an antibody to TIGIT alone or in combination with one or more active therapeutic agents. The additional active therapeutic agents may be small chemical molecules, macromolecules such as proteins, antibodies, peptibodies, peptides, DNA, RNA or fragments of such macromolecules, or components of cell or gene therapy. Combination therapy may target different but complementary mechanisms of action and thus have a synergistic therapeutic or prophylactic effect on a disease, disorder or condition associated with these mechanisms. Additionally or alternatively, combination therapy may provide for a reduction in the dose of one or more agents, thereby alleviating, reducing or eliminating adverse effects associated with one or more of the agents.

The active therapeutic agents in such combination therapy may be formulated as a single composition or as separate compositions. When administered separately, each therapeutic agent in the combination may be given at the same or about the same time or at different times. Moreover, during the course of therapy of a patient, the therapeutic agents are administered in combination, even if they have different forms of administration (e.g., capsules for oral administration and preparations for intravenous administration), they are administered at different dosing intervals, with one therapeutic agent given in a constant dosing regimen and the other administered with an increase in dose, a decrease in dose, or discontinued, or each therapeutic agent in the combination is independently administered with an increase in dose, a decrease in dose, discontinued and/or resumed. If the combination is formulated as separate compositions, in some embodiments, the separate compositions are presented together in a kit. In certain embodiments, any of the anti-TIGIT antibodies disclosed herein are administered or used sequentially with one or more additional active therapeutic agents, such as when the one or more additional active therapeutic agents are administered before or after administration of the anti-TIGIT antibody of the present invention. In other embodiments, the antibodies are administered concurrently with one or more additional active therapeutic agents, such as when the anti-TIGIT antibody is administered at or about the same time as one or more of the additional therapeutic agents; the anti-TIGIT antibody and one or more of the additional therapeutic agents may be present in two or more separate formulations or may be combined into a single formulation (i.e., presented as a combination dosage form). For purposes of the present invention, whether the additional agent(s) are administered sequentially or concurrently with the anti-TIGIT antibody, they are considered to be administered in combination.

The antibodies of the present invention may be used in combination with at least one other (active) agent in any manner acceptable in the circumstances. In one embodiment, treatment with at least one active agent and at least one anti-TIGIT antibody of the present invention is maintained over a period of time. In another embodiment, treatment with at least one active agent is tapered or discontinued (e.g., when the subject is stable) and treatment with the anti-TIGIT antibody of the present invention is maintained on a constant dosing schedule. In a further embodiment, treatment with at least one active agent is tapered or discontinued (e.g., when the subject is stable) and treatment with the anti-TIGIT antibody of the present invention is tapered (e.g., at a lower dose, less frequent administration, or a shorter treatment schedule). In another embodiment, treatment with at least one active agent is performed at a dose reduction or is discontinued (e.g., when the subject's condition is stable), and treatment with an anti-TIGIT antibody of the present invention is performed at a dose increase (e.g., at a higher dose, more frequent administration, or a longer treatment regimen). In another embodiment, treatment with at least one active agent is maintained unchanged, and treatment with an anti-TIGIT antibody of the present invention is performed at a dose reduction or is discontinued (e.g., at a lower dose, less frequent administration, or a shorter treatment regimen). In another embodiment, treatment with at least one active agent and treatment with the anti-TIGIT antibodies of the present invention are performed at a dose reduction or are discontinued (e.g., at a lower dose, less frequent administration, or a shorter treatment regimen).

Treatment with antibodies of the present invention can be combined with other treatments that are effective against the disorder being treated. If the treatment of a proliferative condition, cancer, tumor or precancerous disease, disorder or condition involves the use of antibodies of the present invention, which can be combined with chemotherapy, radiation therapy (e.g., localized radiation therapy or total body radiation therapy), stem cell therapy, surgery or other biological treatment.

The antibodies of the present invention can be administered with vaccines that induce an immune response against cancer. Such an immune response is enhanced by the antibody of the present invention. Vaccine

- 28 049282 may include an antigen expressed on the surface of a cancer cell and/or tumor fragment thereof, effective for inducing an immune response, optionally associated with a carrier molecule.

In some embodiments, the one or more additional therapeutic agents are immunomodulatory agents. Suitable immunomodulatory agents that may be used in the present invention include CD40L, B7, and B7RP1, activating monoclonal antibodies (mAbs) to stimulatory receptors such as anti-CD40, anti-CD38, anti-ICOS, and 4-IBB ligand; antigen loading of dendritic cells (in vitro or in vivo); cancer vaccines such as dendritic cell tumor vaccines; cytokines/chemokines such as IL1, IL2, IL12, IL18, ELC/CCL19, SLC/CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC, IFNa/β, M-CSF, IL-3, GM-CSF, IL-13 and anti-IL-10; bacterial lipopolysaccharides (LPS); indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors and immunostimulatory oligonucleotides. In certain embodiments, the present invention provides methods of suppressing tumor growth comprising administering an anti-TIGIT antibody described herein in combination with a signal transduction inhibitor (STI) to achieve additive or synergistic tumor suppressive effects. As used herein, the term "signal transduction inhibitor" refers to an agent that selectively inhibits one or more steps in a signaling pathway. Signal transduction inhibitors (STIs) provided by the present invention include: (i) bcr/abl kinase inhibitors (e.g., imatinib mesylate, GLIVEC®); (ii) epidermal growth factor (EGF) receptor inhibitors, including kinase inhibitors (e.g., gefitinib, erlotinib, afatinib, and osimertinib) and antibodies; (iii) her-2/neu receptor inhibitors (e.g., HERCEPTIN®); (iv) inhibitors of Akt family kinases or the Akt signaling pathway (e.g., rapamycin); (v) cell cycle kinase inhibitors (e.g., flavopiridol); and (vi) phosphatidylinositol kinase inhibitors. Agents involved in immunomodulation may also be used in combination with the anti-TIGIT antibody described herein to suppress tumor growth in patients with cancer.

In some embodiments, one or more of the additional therapeutic agents is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; Androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Adrenal suppressants such as aminoglutethimide, mitotane, trilostane; Folic acid supplement such as folinic acid; Aceglatone; Aldophosphamide glycoside; Aminolevulinic acid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elphormitin; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllinic acid; 2-ethylhydrazide; Procarbazine; Razoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosin; Arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids such as paclitaxel, nab-paclitaxel, and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes such as cisplatin, carboplatin, and oxaliplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; anthracyclines and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

Chemotherapeutic drugs also include antihormonal agents, which work by regulating or inhibiting the action of hormones on tumors, such as anti-

- 29 049282 estrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone, and toremifene; and antiandrogens such as abiraterone, enzalutamide, apalutamide, darolutamide, flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. In certain embodiments, the combination therapy includes a chemotherapy regimen including one or more chemotherapeutic agents. In certain embodiments, the combination therapy includes administering a hormonal or hormone-related agent.

Additional treatment options that may be used in combination with an anti-TIGIT antibody include radiation therapy, an antibody against a tumor antigen, an antibody-toxin complex, a T-cell adjuvant, bone marrow transplant, or antigen-presenting cell transplant (e.g., dendritic cell-based cell therapy), including TLR agonists that are used to stimulate such antigen-presenting cells.

In certain embodiments, the present invention provides for the use of an anti-TIGIT antibody described herein in combination with RNA interference therapy to silence gene expression. RNAi begins with the cleavage of longer double-stranded RNAs into small interfering RNAs (siRNAs). One strand of the siRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC), which is then used to identify mRNA molecules that are at least partially complementary to the incorporated strand of the siRNA. RISC can bind to or cleave the mRNA, both of which inhibit translation. In certain embodiments, the present invention provides for the use of an anti-TIGIT antibody described herein in combination with agents that modulate adenosine levels. Such therapeutic agents may act on ectonucleotides that catalyze the conversion of ATP to adenosine, including ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1, also known as CD39 or cluster of differentiation 39), which hydrolyzes ATP to ADP and ADP to AMP, and a 5'-nucleotidase known as ecto (NT5E or 5NT, also known as CD73 or cluster of differentiation 73), which converts AMP to adenosine. In one embodiment, the present invention provides a combination of CD73 inhibitors, such as those described in WO 2017/120508, WO 2018/094148, and WO 2018/067424. In one embodiment, the CD73 inhibitor is AB680. In another approach, the targets are adenosine A2a and A2K receptors. A combination of A2a and/or A2K receptor antagonists is also provided. In one embodiment, the present invention provides a combination with adenosine receptor antagonists described in WO/2018/136700 or WO 2018/204661. In one embodiment, the adenosine receptor antagonist is AB928 (etrumadenant).

In certain embodiments, the present invention provides for the use of an anti-TIGIT antibody described herein in combination with inhibitors of phosphatidylinositol 3-kinases (PI3K), particularly the PI3Ky isoform. PI3Ky inhibitors can stimulate an anti-cancer immune response by modulating myeloid cells, such as by inhibiting myeloid-derived suppressor cells, attenuating the effects of immunosuppressive tumor-infiltrating macrophages, or by stimulating macrophages and dendritic cells to produce cytokines that contribute to effective T cell responses, resulting in a reduction in the development and spread of cancer. Examples of PI3Ky inhibitors that can be combined with an anti-TIGIT antibody described herein include those described in WO 2020/0247496 A1. In one embodiment, the PI3Ky inhibitor is IPI-549.

In certain embodiments, the present invention provides for the use of an anti-TIGIT antibody described herein in combination with inhibitors of arginase, which has been shown to be either responsible for or involved in inflammation-induced immune dysfunction, in the process of cancer cell immune evasion, immunosuppression, and infectious disease immunopathology. Examples of arginase compounds can be found, for example, in PCT/US2019/020507 and WO/2020/102646. In certain embodiments, the present invention provides for the use of an anti-TIGIT antibody of the present invention with inhibitors of HIF-2a, which play an important role in the cellular response to low oxygen availability. Under hypoxic conditions, hypoxia-inducible factors (HIFs) can activate the expression of genes that regulate metabolism, angiogenesis, cell proliferation and survival, as well as immune evasion and inflammatory response. Overexpression of HIF2α is associated with poor clinical outcomes in patients with various cancers; hypoxia is also prevalent in many acute and chronic inflammatory disorders, such as inflammatory bowel disease and rheumatoid arthritis.

The present invention also provides for the use of an anti-TIGIT antibody described herein in combination with one or more inhibitors of the RAS signaling pathway. Oncogenic mutations in the RAS gene family, such as HRAS, KRAS, and NRAS, are associated with a variety of

- 30 049282 cancer. For example, mutations G12C, G12D, G12V, G12A, G13D, Q61H, G13C, and G12S in the KRAS gene family, among others, have been observed in many tumor types. Direct and indirect inhibitory strategies have been investigated to inhibit the mutant RAS signaling pathway. Indirect inhibitors target effectors other than RAS in the RAS signaling pathway and include, among others, inhibitors of RAF, MEK, ERK, PI3K, PTEN, SOS (e.g., SOS1), mTORC1, SHP2 (PTPN11), and AKT. Non-limiting examples of indirect inhibitors in development include RMC-4630, RMC-5845, RMC-6291, RMC-6236, JAB-3068, JAB-3312, TNO155, RLY-1971, BI1701963. Direct inhibitors of mutant RAS have also been studied and typically target the KRAS-GTP complex or the KRAS-GDP complex. Examples of direct RAS inhibitors in development include sotorazib (AMG510), MRTX849, mRNA-5671, and ARS1620, among others. In some embodiments, the one or more RAS signaling pathway inhibitors are selected from the group consisting of RAF inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, PTEN inhibitors, SOS1 inhibitors, mTORC1 inhibitors, SHP2 inhibitors, and AKT inhibitors. In other embodiments, the one or more RAS signaling pathway inhibitors directly inhibit mutant RAS.

In some embodiments, the present invention relates to a combination of an anti-TIGIT antibody of the present invention with one or more anexelekto (i.e., AXL) inhibitors. The AXL signaling pathway is associated with tumor growth and metastasis, and is believed to mediate cancer resistance to various therapies. Various AXL inhibitors are in development that also inhibit other TAM family kinases (i.e., TYRO3, MERTK), as well as other receptor tyrosine kinases, including, but not limited to, MET, FLT3, RON, and AURORA. Examples of multi-kinase inhibitors include gilteritinib, merestinib, cabozantinib, BMS777607, and foretinib. Specific AXL inhibitors have also been developed, such as SGI-7079, TP-0903 (i.e., dubermatinib), BGB324 (i.e., bemcentinib), and DP3975. In certain embodiments, the present invention provides for the use of an anti-TIGIT antibody described herein in combination with adoptive cell therapy, a new and promising form of personalized immunotherapy in which immune cells with anti-tumor activity are administered to patients with cancer. Adoptive cell therapy is being investigated using tumor infiltrating lymphocytes (TILs) and T cells engineered to express, for example, chimeric antigen receptors (CARs) or T cell receptors (TCRs). Adoptive cell therapy essentially involves collecting T cells from an individual, genetically modifying them to target a specific antigen or to enhance their antitumor efficacy, amplifying them to sufficient numbers, and infusing the genetically modified T cells into a patient with cancer. T cells may be collected from a patient who is later re-infused with expanded cells (e.g., autologous), or they may be collected from donor patients (e.g., allogeneic).

T cell-mediated immunity involves multiple sequential steps, each of which is regulated by balancing stimulatory and inhibitory signals to optimize the response. Although nearly all inhibitory signals in the immune response ultimately modulate intracellular signaling pathways, many are initiated through membrane receptors whose ligands are membrane-bound or soluble (cytokines). Although costimulatory and inhibitory receptors and ligands that regulate T cell activation are often underexpressed in cancer tissues relative to normal tissues, inhibitory ligands and receptors that regulate T cell effector functions in tissues are commonly overexpressed on tumor cells or on nontransformed cells associated with the tumor microenvironment. The functions of soluble and membrane-bound receptor (immune checkpoint ligands) can be modulated using agonist antibodies (for costimulatory pathways) or antagonist antibodies (for inhibitory pathways). Thus, unlike most antibodies currently approved for use in cancer therapy, immune checkpoint blocking or agonist antibodies do not target tumor cells directly, but rather target lymphocyte receptors or their ligands to enhance endogenous antitumor activity. [See Pardoll, (April 2012) Nature Rev. Cancer 12:252–64].

Examples of immune checkpoints (ligands and receptors) that are candidates for blockade, some of which are selectively upregulated in different tumor cell types, include PD-1 (programmed cell death protein 1); PD-L1 (programmed cell death receptor 1 ligand 1); BTLA (B and T lymphocyte attenuator); CTLA4 (cytotoxic T lymphocyte-associated antigen 4); TIM-3 (T-cell immunoglobulin and mucin-containing protein 3); LAG-3 (lymphocyte activation gene 3); TIGIT (T-cell immunoreceptor with Ig and ITIM domains); and inhibitory receptors on killer cells, which can be divided into two classes based on their structural features: 1) the killer cell immunoglobulin-like receptors (KIRs) and ii) the C-type lectin receptors (members of the type II transmembrane receptor family). Other less well-defined immune checkpoints have been described in the literature, including both receptors (e.g., the 2B4 receptor (also known as CD244)) and ligands (e.g., some ligands)

- 31 049282 B7 family genes such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, b7x, and VCTN1)). [See Pardoll, (April 2012) Nature Rev. Cancer 12:252-64].

The present invention provides for the use of an anti-TIGIT antibody described herein in combination with inhibitors of the aforementioned immune checkpoint receptors and ligands and of as-yet uncharacterized immune checkpoint receptors and ligands. Several immune checkpoint modulators are currently approved, and many more are in development. In 2011, after being approved for use in the treatment of melanoma, the fully humanized anti-CTLA4 monoclonal antibody ipilimumab (e.g., YERVOY®; Bristol Myers Squibb) became the first immune checkpoint inhibitor to receive regulatory approval in the United States. Fusion proteins including CTLA4 and an antibody (CTLA4-Ig; abatacept (eg, ORENSIA®; Bristol Myers Squibb)) have been used to treat patients with rheumatoid arthritis, and other fusion proteins have been shown to be effective in renal transplant patients who are sensitized to the Epstein-Barr virus. The next class of immune checkpoint inhibitors that have been approved by regulators are inhibitors against PD-1 and its ligands PD-L1 and PD-L2. Approved anti-PD-1 antibodies include nivolumab (eg, OPDIVO®; Bristol Myers Squibb) and pembrolizumab (eg, KEYTRUDA®; Merck) for the treatment of patients with a variety of cancers, including squamous cell carcinoma, classical Hodgkin lymphoma, and urothelial carcinoma. Approved anti-PD-L1 antibodies include avelumab (eg, BAVENCIO®; EMD Serono & Pfizer), atezolizumab (eg, TECENIQ®; Roche/Genentech), and durvalumab (eg, IMFINZI®; AstraZeneca) for the treatment of patients with certain cancers, including urothelial carcinoma. In some combinations provided herein, the immune checkpoint inhibitor is selected from MEDI-0680 nivolumab, pembrolizumab, avelumab, atezolizumab, budigalimab, BI-754091, camrelizumab, cosibelimab, durvalumab, dostarlimab, cemiplimab, sintitlimab, tiselizumab, toripalimab, retifanlimab, sasanlimab, and zimberelimab (AB122). In some embodiments, the immune checkpoint inhibitor is MEDI-0680 (AMP-514; WO2012/145493) or pidilizumab (CT-011). Another approach to targeting the PD-1 receptor uses a recombinant protein called AMP-224, which consists of the extracellular domain of PD-L2 (B7-DC) fused to the Fc region of IgG1. In one embodiment, the present invention provides for the use of an anti-TIGIT antibody according to the present invention with an anti-PD-1 antibody. In one specific embodiment, the anti-PD-1 antibody is zimberelimab. In some embodiments, the anti-TIGIT antibody is provided in an amount of about 200 to 1500 mg every three weeks, and the anti-PD-1 antibody is provided in an amount of about 100 to 1200 mg every three weeks. In another embodiment, the anti-TIGIT antibody described herein is given in an amount of about 300 to 1800 mg every four weeks, and the anti-PD-1 antibody is provided in an amount of about 200 to 1500 mg every four weeks. In another embodiment, the anti-PD-1 antibody is zimberelimab and is provided in an amount of about 360 mg or 480 mg every three or four weeks.

In another aspect, the present invention provides a combination with a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-B, VEGF and other immunosuppressive cytokines) or with a cytokine that stimulates T cell activation, to stimulate an immune response.

In another aspect, T cell responses can be stimulated by a combination of a disclosed anti-TIGIT antibody and one or more of (i) an antagonist of a protein that inhibits T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, PVRIG, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and/or (ii) an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD2. Other agents that can be combined with the anti-TIGIT antibody of the present invention for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, the anti-TIGIT antibody described herein can be combined with KIR antagonists such as lirilumab.

Other agents for combination therapies include agents that inhibit or deplete macrophages or monocytes, including, but not limited to, CSF-1R antagonists such as CSF-1R antagonist antibodies including RG7155 (WO 11/70024, WO 11/107553, WO 11/131407, WO 13/87699, WO 13/119716, WO 13/132044) or FPA-008 (WO 11/140249; WO 13169264; WO 14/036357). In another aspect, the disclosed anti-TIGIT antibody can be combined with one or more of: agonist agents that ligate positive costimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of antitumor T cells, agents that bypass certain immunosuppressive signaling pathways within

- 32 049282 tumor microenvironment (e.g., blocking the engagement of inhibitory receptors (e.g., PD-L1/PD-1 interactions), depleting or inhibiting Tregs (e.g., using a CD25 monoclonal antibody (e.g., daclizumab) or by ex vivo depletion of microbead-bound CD25 antibodies), or reversing/preventing T cell anergy or exhaustion), as well as agents that trigger innate immune activation and/or inflammation at tumor sites.

In one aspect, the immuno-oncology agent is a CTLA-4 antagonist, such as an anti-CTLA-4 antagonist antibody. Suitable anti-CTLA-4 antibodies include, for example, ipilimumab (e.g., YERVOY®; Bristol Myers Squibb) or tremelimumab. In another aspect, the immuno-oncology agent is a PD-L1 antagonist, such as an anti-PD-L1 antagonist antibody. Suitable PD-L1 antibodies include, for example, atezolizumab (MPDL3280A; WO 2010/077634) (e.g., TECentriQ®; Roche/Genentech), durvalumab (MEDI4736), BMS-936559 (WO 2007/005874), and MSB0010718C (WO2013/79174). In another aspect, the immuno-oncology agent is a LAG-3 antagonist, such as an anti-LAG-3 antagonist antibody. Suitable anti-LAG-3 antibodies include, for example, BMS-986016 (WO 10/19570, WO 14/08218), or IMP-731 or IMP-321 (WO08/132601, WO09/44273). In another aspect, the immuno-oncology agent is a CD137 (4-1BB) agonist, such as an anti-CD137 agonist antibody. Suitable anti-CD137 antibodies include, for example, urelumab and PF-05082566 (WO 12/32433). In another aspect, the immuno-oncology agent is a GITR agonist, such as an anti-GITR agonist antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO 06/105021, WO 09/009116), and MK-4166 (WO 11/028683). In another aspect, the immuno-oncology agent is an OX40 agonist, such as an OX40 agonist antibody. Suitable OX40 antibodies include, for example, MEDI-6383 or MEDI-6469. In another aspect, the immuno-oncology agent is an OX40L antagonist, such as an OX40L antagonist antibody. Suitable OX40L antagonists include, for example, RG-7888 (WO06/029879). In another aspect, the immuno-oncology agent is a CD40 agonist, such as an anti-CD40 agonist antibody. In another embodiment, the immuno-oncology agent is a CD40 antagonist, such as an anti-CD40 antagonist antibody. Suitable anti-CD40 antibodies include, for example, lucatumumab or dacetuzumab. In another aspect, the immuno-oncology agent is a CD27 agonist, such as an anti-CD27 agonist antibody. Suitable anti-CD27 antibodies include, for example, varlilumab. In another aspect, the immuno-oncology agent is MGA271 (anti-B7H3) (WO 11/109400). In another embodiment, a combination of the anti-TIGIT antibodies of the present invention with a Trop-2 targeting agent is provided, for example, the antibody drug conjugate, sacituzumab govitecan-hsi. In another embodiment, a combination of the TIGIT antibodies described herein is provided with an agent that inhibits the CD47-SIRPa signaling pathway. An example of an anti-CD47 antibody is magrolimab.

Examples of therapeutic agents used in combination therapy to treat patients with diseases, disorders, and conditions related to the cardiovascular system and/or metabolism include statins (eg, CRESTOR®, LESCOL®, LIPITOR®, MEVACOR®, PRAVACOL®, and ZOCOR®), which inhibit the enzymatic synthesis of cholesterol; bile acid sequestrants (eg, CHOLESTYDE®, LO-CHOLEST®, PREVALIT®, QUESTRAN®, and WELHOL®), which lower cholesterol and prevent its absorption; ezetimibe (ZETIA®), which blocks cholesterol absorption; fibric acid (eg, TRICOR®), which lowers triglyceride levels and may modestly increase high-density lipoprotein (HDL) levels; niacin (e.g., NIACOR®), which modestly lowers low-density lipoprotein (LDL) cholesterol and triglycerides; and/or a combination of the above (e.g., VYTORIN® (ezetimibe with simvastatin)). Alternative treatments for high cholesterol that may be candidates for use in combination with the anti-TIGIT antibody described herein include various supplements and herbs (e.g., garlic, policosanol, and guggul).

Examples of therapeutic agents used in combination therapy of diseases, disorders or conditions related to immunity and inflammation include, but are not limited to, the following: non-steroidal anti-inflammatory drug (NSAID) such as aspirin, ibuprofen and other propionic acid derivatives (alminoprofen, benoxaprofen, buclonic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidomethacin and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam,

- 33 049282 piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and pyrazolones (apazone, bezpiperilone, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone). Other combinations include cyclooxygenase-2 (COX-2) inhibitors. Other active agents for combination include steroids such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone or hydrocortisone. Such a combination may provide special advantages since one or more undesirable effects of the steroid may be reduced or even eliminated by reducing the required dose of steroid. Additional examples of active agents that may be used in combination, for example, for the treatment of patients with rheumatoid arthritis, include cytokine suppressor anti-inflammatory drug(s) (CSAD); antibodies to or antagonists of other human cytokines or growth factors, such as TNF, LT, IL-10, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF or PDGF.

Specific combinations of active agents can intervene at different points in the autoimmune and subsequent inflammatory cascade and include TNF antagonists such as chimeric, humanized, or human anti-TNF antibodies, REMICADE®, HUMIRA®, anti-TNF antibody fragments (eg, CDP870), and soluble p55 or p75 TNF receptors, their derivatives, p75TNFRIgG (ENBREL®) or p55TNFR1gG (lenercept), soluble IL-13 receptor (sIL-13), and TNFa-converting enzyme (TACE) inhibitors; IL-1 inhibitors (eg, interleukin-1-converting enzyme inhibitors) may similarly be effective. Other combinations include interleukin 11, anti-β7 antibodies, and p-selectin glycoprotein ligand (PSGL). Other examples of agents used in combination with the A2AR/A2BR inhibitors described herein include interferon-131a (AVONEX®); interferon-131b (BETASERON®); Copaxone; hyperbaric oxygenation; intravenous immunoglobulin; clabribine; and antibodies to or antagonists of other human cytokines or growth factors (e.g., anti-CD40 ligand and anti-CD80 antibodies).

In some embodiments, the combination is an antibody of the invention with a second antibody directed to a surface antigen preferentially expressed on cancer cells relative to a control normal tissue. Some examples of antibodies that may be administered as part of combination therapy with antibodies of the invention for the treatment of cancer include Herceptin® (trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) against VEGF, or EGF receptor antibodies such as Erbitux® (cetuximab) and Vectibix® (panitumumab). Other agents that may be administered include antibodies or other inhibitors of any of PD-1, PD-L1, CTLA-4, 4-1BB, BTLA, PVRIG, VISTA, TIM-3, and LAG-3; or other inhibitors that inhibit inward signaling, such as mTOR and GSK3e inhibitors; and cytokines such as interferon-γ, IL-2, and IL-15. Some specific examples of additional agents include: ipilimumab, pazopanib, sunitinib, dasatinib, pembrolizumab, INCR024360, dabrafenib, trametinib, atezolizumab (MPDL3280A), erlotinib (e.g., TARCEVA®), cobimetinib, nivolumab, and zimberelimab. The choice of a second antibody or other agent for combination therapy depends on the type of cancer being treated. Optionally, the cancer is tested for expression or preferential expression of an antigen to guide the selection of the appropriate antibody. In some embodiments, the isotype of the second antibody is human IgG1 to promote effector functions such as ADCC, CDC, and phagocytosis.

Similar types of combination therapy can be used to treat or prevent infectious disease such as viral, bacterial, fungal and parasitic diseases, disorders and conditions, and related disorders.

For example, an antibody of the present invention can be combined with an antibody directed against a pathogen or a vaccine against a pathogen, such as, for example, palivizumab against Rous sarcoma virus. The vaccine can be a protein of the pathogen or a fragment thereof effective to induce an immune response. The antibody of the present invention enhances the immune response of the antibody or vaccine directed against the pathogen. The antibody of the present invention can also be administered with T cells or natural killer cells expanded ex vivo. Such combination therapy includes antiviral agents that target different stages of the viral life cycle and have different mechanisms of action, including, but not limited to, the following: viral deenvelopment inhibitors (e.g., amantadine and rimantadine); reverse transcriptase inhibitors (e.g., acyclovir, zidovudine, and lamivudine); integrase targeting agents; agents that block the attachment of transcription factors to viral DNA; agents (e.g., antisense molecules) that affect translation (e.g., fomivirsen); agents that modulate translation/ribozyme function; protease inhibitors; modulators of viral assembly (e.g., rifampin); antiretroviral agents such as, for example, analog nucleoside reverse transcriptase inhibitors (e.g., azidothymidine (AZT), ddl, ddC, 3TC, d4T); non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz, nevirapine); analog nucleotide reverse transcriptase inhibitors; and agents that prevent the release of viral particles (e.g., zanamivir and oseltamivir). Treatment and/or prophylaxis of certain viral infections (e.g.,

- 34 049282

HIV) often involves the use of a group (cocktail) of antiviral agents.

Other antiviral agents contemplated for use in combination with any of the anti-TIGIT antibodies described herein include, but are not limited to, the following: abacavir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, ATRIPLA®, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, fosphonet, ganciclovir, ibacitabin, imunavir, idoxuridine, imiquimod, indinavir, inosine, various interferons (e.g., peginterferon alfa-2a), lopinavir, loviride, maraviroc, moroxidine, methisazone, nelfinavir, nexavir, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, ritonavir, pyramidine, saquinavir, stavudine, telaprevir, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, TRUVADA®, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zalcitabine.

The present invention contemplates the use of any of the TIGIT antibodies disclosed herein in combination with antiparasitic agents. Such agents include, but are not limited to, thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide, bithionol, oxamiquin, metrifonate, ivermectin, albendazole, eflornithine, melarsoprol, pentamidine, benznidazole, nifurtimox, and nitroimidazole. Other agents known to those skilled in the art may be useful in the treatment of parasitic disorders.

Embodiments of the present invention provide for the use of any of the anti-TIGIT antibodies disclosed herein in combination with agents useful for the treatment or prevention of bacterial disorders. Antibacterial agents can be classified in a variety of ways, including based on their mechanism of action, based on their chemical structure, and based on their spectrum of activity. Examples of antibacterial agents include those that target the bacterial cell wall (e.g., cephalosporins and penicillins) or the cell membrane (e.g., polymyxins), or interfere with essential bacterial enzymes (e.g., sulfonamides, rifamycins, and quinolines). Most antibacterial agents that target protein synthesis (e.g., tetracyclines and macrolides) are bacteriostatic, while agents such as aminoglycoside are bactericidal. Another way to categorize antibacterial agents is based on their target specificity; Narrow-spectrum agents target only specific types of bacteria (e.g., gram-positive bacteria such as Streptococcus), while broad-spectrum agents have activity against a wider range of bacterial types. Those skilled in the art know the types of antibacterial agents that are suitable for use against specific bacterial infections.

Embodiments of the present invention provide for the use of any of the anti-TIGIT antibodies described herein in combination with agents useful for treating or preventing fungal disorders. Antifungal agents include polyenes (e.g., amphotericin, nystatin, and pimaricin); azoles (e.g., fluconazole, itraconazole, and ketoconazole); allylamines (e.g., naftifine and terbinafine), and morpholines (e.g., amorolfine), and antimetabolites (e.g., 5-fluorocytosine).

The present invention encompasses pharmaceutically acceptable salts, acids or derivatives of any of the above.

V. Other applications.

The TIGIT antibodies of the present invention can be used to detect TIGIT in the context of clinical diagnosis or treatment or in research. For example, the antibodies can be used to detect the presence of TIGIT on T cells, natural killer cells and cancer cells as an indication that a subject has a treatable cancer or infectious disease. Expression of TIGIT on T cells, natural killer cells and/or cancer cells of a subject suffering from a cancer or infectious disease also provides an indication that the cancer or infectious disease may be treatable with the antibodies of the present invention. The antibodies can also be sold as research reagents for laboratory studies to detect T cells, natural killer cells and cancer cells and their response to various stimuli. In such cases, the antibodies may be labeled with one or more detectable signals, including, but not limited to, fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and may be provided in the form of a kit with all the necessary reagents to perform a TIGIT assay. The TIGIT antibodies of the present invention may also be used to purify TIGIT, such as by affinity chromatography.

VI. Sets.

As components of a kit, the anti-TIGIT antibodies may be combined with any of the second antibodies or agents described for use in combination therapy. The invention disclosed herein provides one or more kits comprising one or more of the antibodies disclosed herein and one or more pharmaceutically acceptable auxiliary excipients or carriers (such as, but not limited to, phosphate buffered saline solutions, water,

- 35 049282 sterile water, polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil, emulsions such as oil-in-water or water-in-oil emulsions, microemulsions, nanocarriers and various types of wetting agents). Additives such as alcohols, oils, glycols, preservatives, flavors, colorants, suspending agents and the like can also be included in the kits of the present invention along with the carrier, diluent or excipient. In one embodiment, a pharmaceutically acceptable carrier suitable for use in the antibody compositions disclosed herein is sterile, pathogen-free and/or is otherwise safe for administration to a subject without the risk of concomitant infection and other undue unwanted side effects. In a kit, the respective agents may be provided in separate vials with instructions for combination for subsequent administration or with instructions for separate administration. The kit may also include written instructions for the proper handling and storage of any of the TIGIT antibodies disclosed herein.

VII. Implementation options.

Embodiment 1. An anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, comprising (a) a heavy chain variable region comprising a heavy chain complementarity determining region (CDR) (HC) 1 that is at least 80% identical to the sequence of SEQ ID NO: 36, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 37, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 38; and a light chain variable region comprising a light chain CDR1 (LC) that is at least 80% identical to the sequence of SEQ ID NO: 39, an LCCDR2 that is at least 80% identical to the sequence of SEQ ID NO: 40, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 41; (b) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 42, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 43, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 44; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 45, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 46, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 47; (c) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 48, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 49, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 50; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 51, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 52, and an LCCDR3 that is at least 80% identical to the sequence of SEQ ID NO: 53; (d) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 54, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 55, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 56; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 57, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 58, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 59; (e) a heavy chain variable region comprising an HCCDR1 that is at least 80% identical to the sequence of SEQ ID NO: 60, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 61, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 62; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 63, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 64, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65; (f) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 60, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 66, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 63, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 68, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65; (g) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 69, an HC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 55, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 70; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 71, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 68, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65; (h) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 72;

- 36 049282 identical to the sequence of SEQ ID NO: 72, HC-CDR2, which is at least 80% identical to the sequence of SEQ ID NO: 73, and HC-CDR3, which is at least 80% identical to the sequence of SEQ ID NO: 67; and a light chain variable region comprising LC-CDR1, which is at least 80% identical to the sequence of SEQ ID NO: 63, LC-CDR2, which is at least 80% identical to the sequence of SEQ ID NO: 68, and LC-CDR3, which is at least 80% identical to the sequence of SEQ ID NO: 65; or (i) a heavy chain variable region comprising an HC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 74, an HCCDR2 that is at least 80% identical to the sequence of SEQ ID NO: 75, and an HC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 that is at least 80% identical to the sequence of SEQ ID NO: 63, an LC-CDR2 that is at least 80% identical to the sequence of SEQ ID NO: 68, and an LC-CDR3 that is at least 80% identical to the sequence of SEQ ID NO: 65.

Embodiment 2. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 1, comprising (a) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 36, an HC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 37, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 38; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that has identity to SEQ ID NO: 39, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 40, and an LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 41; (b) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 42, an HC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 43, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 44; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 45, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 46, and an LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 47; (c) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 48, an HCCDR2 having an amino acid sequence that comprises SEQ ID NO: 49, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 50; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 51, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 52, and an LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 53; (d) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 54, an HC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 55, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 56; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 57, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 58, and an LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 59; (e) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 60, an HC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 61, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 62; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 64, and an LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 65; (f) a heavy chain variable region comprising an HC-CDRI having an amino acid sequence that comprises SEQ ID NO: 60, an HC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 66, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 68, and an LCCDR3 having an amino acid sequence that comprises SEQ ID NO: 65; (g) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 69, an HC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 55, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 70; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 71, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 68, and an LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 65; (h) a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 72 ...3 having an amino acid sequence that comprises SEQ ID NO: 73, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 74.

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CDR2 having an amino acid sequence that comprises SEQ ID NO: 73, and HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 67; and a light chain variable region comprising LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 63, LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 68, and LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 65; or (i) a heavy chain variable region comprising HC-CDRI having an amino acid sequence that comprises SEQ ID NO: 74, HC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 75, and HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 67; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 63, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 68, and an LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 65.

Embodiment 3. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising (a) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 1 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 2; (b) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 3 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 4; (c) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 5 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 6; (d) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 7 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 8; (e) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 9 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 10; (f) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 11 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 12; (g) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 13 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 14; (h) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 15 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 16; (i) a variable region of a heavy chain that is at least 80% identical to the sequence of SEQ ID NO: 17 and a variable region of a light chain that is at least 80% identical to the sequence of SEQ ID NO: 12; (j) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 76 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 77; (k) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 78 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 77; (l) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 76 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 79; or (m) a heavy chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 78 and a light chain variable region that is at least 80% identical to the sequence of SEQ ID NO: 79.

Embodiment 4. The TIGIT antibody or antigen-binding fragment thereof according to any one of embodiments 1-3, wherein the TIGIT antibody or antigen-binding fragment thereof is a monoclonal antibody.

Embodiment 5. The anti-TIGIT antibody or antigen-binding fragment thereof of any one of embodiments 1-4, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a chimeric, humanized, or veneered antibody. Embodiment 6. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 5, wherein the chimeric antibody comprises a human IgG1/kappa Fab constant domain.

Embodiment 7. An anti-TIGIT antibody or antigen-binding fragment thereof according to any one of embodiments 1-3, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a human antibody.

Embodiment 8. An anti-TIGIT antibody or antigen-binding fragment thereof of any of the preceding embodiments that inhibits binding of TIGIT to CD155, wherein optionally the anti-TIGIT antibody or antigen-binding fragment thereof inhibits binding with an _IC50 of from about 0.1 nM to about 10 nM, from about 0.1 nM to about 5 nM, from about 0.2 nM to about 2 nM, from about 0.2 nM to about 0.8 nM, from about 0.4 nM to about 0.8 nM, or from about 0.6 nM to about 0.8 nM, as measured in Example 1.

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Embodiment 9. An anti-TIGIT antibody or antigen-binding fragment thereof according to any one of embodiments 1-5 or 7-8, wherein the antibody further comprises a variant heavy chain constant region selected from variant human IgG1, variant human IgG2, variant human IgG3, or variant human IgG4, and optionally a human light chain constant region.

Embodiment 10. The TIGIT antibody or antigen-binding fragment thereof of embodiment 9, wherein the variant heavy chain constant region has enhanced or diminished effector function compared to the wild-type heavy chain constant region.

Embodiment 11. The TIGIT antibody or antigen-binding fragment thereof of embodiment 10, wherein the variant region of the human IgG heavy chain comprises SEQ ID NO: 97, SEQ ID NO: 99, or SEQ ID NO: 101.

Embodiment 12. An anti-TIGIT antibody or antigen-binding fragment thereof according to any one of embodiments 1-5 or 7-8, wherein the antibody further comprises a wild-type constant region of a human IgG heavy chain and, optionally, a human light chain constant region.

Embodiment 13. The TIGIT antibody or antigen-binding fragment thereof of embodiment 12, wherein the wild-type human IgG heavy chain constant region comprises SEQ ID NO: 94.

Embodiment 14. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 12 or 13, comprising a human light chain kappa constant region, optionally wherein the human light chain constant region comprises SEQ ID NO: 95. Embodiment 15. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiments 1-5 or 7-8, wherein the antibody has a heavy chain and a light chain, wherein (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 92 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 96 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 98 and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 100 and the light chain has an amino acid sequence comprising SEQ ID NO: 93.

Embodiment 16. The anti-TIGIT antibody or antigen-binding fragment thereof of any one of the preceding embodiments, wherein the antibody or binding fragment thereof (a) has an equilibrium binding constant (KD) of from about 0.01x10'11 M to about 100x10'11 M, from about 0.1x10'11 M to about 100x10'11 M, from about 0.1x10'11 M to about 10x10'11 M, from about 1x10'11 M to about 100x10'11 M, or from about 1x10'11 M to about 10x10'11 M, as measured by surface plasmon resonance; (b) blocks the binding of soluble human CD155 ligand to the cell surface of human TIGIT with a half-maximal inhibitory concentration (IC50) of from about 0.2 nM to about 2 nM, from about 0.2 nM to about 0.8 nM, from about 0.6 nM to about 0.8 nM, or from about 0.6 nM to about 0.8 nM, as measured in Example 1; (c) binds to an epitope that includes at least the following residues of TIGIT: (i) D72 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80; (iii) D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80; (iv) E60, D72, and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80; or (v) T55, Q56, N58, E60, D72, S80, and K82 of SEQ ID NO: 80; or (d) any combination of (a), (b), and (c). Embodiment 17. An anti-TIGIT antibody or antigen-binding fragment thereof according to embodiment 16, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof according to any one of embodiments 1-17 for binding to TIGIT.

Embodiment 18. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 16 or 17, wherein the excess antibody or antigen-binding fragment thereof competes with a reference antibody for binding to TIGIT by at least about 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% in a competition binding assay, wherein the reference antibody comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92, and a light chain having an amino acid sequence comprising SEQ ID NO: 93.

Embodiment 19. An antibody to TIGIT or an antigen-binding fragment thereof that specifically binds to human TIGIT and comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO: 93.

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Embodiment 20. A method of inhibiting binding of TIGIT to CD155 comprising contacting TIGIT with an anti-TIGIT antibody or antigen-binding fragment thereof of any of the preceding embodiments.

Embodiment 21. A method of treating a subject infected with a pathogen, comprising administering to the subject an effective regimen or a therapeutically effective amount of an antibody of any of the preceding embodiments. Embodiment 22. The method of embodiment 21, wherein the pathogen is a virus, bacteria, fungi, or protozoa.

Embodiment 23. The method of embodiment 22, wherein the pathogen is HIV, simian immunodeficiency virus (SIV), hepatitis, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTLV), dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arbovirus encephalitis virus, chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, conococci, klebsiella, proteus, serratia bacteria, pseudomonas, Legionella, diphtheria bacteria, salmonella, bacilli, cholera bacteria, tetanus bacteria, botulism spores, anthrax bacteria, plague bacteria, leptospirosis bacteria and Lyme disease bacteria.

Embodiment 24. The method of any one of embodiments 21-23, wherein the subject is treated with a vaccine that induces an immune response against a pathogen that is enhanced by an antibody.

Embodiment 25. The method of embodiment 24, wherein the vaccine comprises a pathogen protein or fragment thereof.

Embodiment 26. The method of any one of embodiments 21-25, wherein the subject is further administered a second antibody against the pathogen, wherein the antibody enhances the effector-mediated cytotoxicity of the second antibody against the pathogen.

Embodiment 27. The method of any one of embodiments 21-26, wherein the subject is further administered one or more of an antiviral agent, an antiparasitic agent, an antibacterial agent, or an antifungal agent.

Embodiment 28. A method for treating or preventing cancer, comprising administering to a subject having cancer or at risk of developing cancer an effective regimen or administering to them a therapeutically effective amount of any of the TIGIT antibodies or antigen-binding fragments thereof according to any of the preceding embodiments.

Embodiment 29. The method of embodiment 28, wherein the cancer is a hematological malignancy, a solid tumor, Merkel cell carcinoma, urothelial cancer, squamous cell carcinoma of the head and neck, B-cell lymphoma, uterine cancer, cervical cancer, testicular cancer, gastrointestinal cancer, bladder cancer, bone cancer, bone marrow cancer, skin cancer, gallbladder cancer, heart cancer, lung cancer, salivary gland cancer, adrenal cancer, thyroid cancer, ganglia cancer, central nervous system (CNS) and peripheral nervous system (PNS) cancer, and hematopoietic system cancer, immune system cancer.

Embodiment 30. The method of embodiment 28 or 29, wherein the subject is administered tumor-infiltrating T cells that are activated by an antibody or antigen-binding fragment thereof.

Embodiment 31. The method of any one of embodiments 28-30, wherein the subject is administered a vaccine that induces an immune response against cancer that is enhanced by an antibody or antigen-binding fragment thereof.

Embodiment 32. The method of embodiment 31, wherein the vaccine comprises an antigen or fragment thereof expressed on the surface of cancer cells.

Embodiment 33. The method according to any one of embodiments 28-32, wherein the subject is administered natural killer cells whose anti-cancer cytotoxicity is enhanced by an antibody or antigen-binding fragment thereof.

Embodiment 34. The method of any one of embodiments 28-33, wherein the subject is further administered a second antibody against an antigen expressed on the surface of cancer cells, whereby the effector-mediated anti-cancer cytotoxicity of the second antibody is enhanced by the antibody or antigen-binding fragment thereof.

Embodiment 35. The method according to any one of embodiments 28-33, wherein the subject is further administered a second antibody against an antigen expressed on the surface of an immune cell.

Embodiment 36. The method of embodiment 35, wherein the immune cell is a T cell or a natural killer cell.

Embodiment 37. The method of embodiments 35 or 36, wherein the antigen is CTLA-4, PD-1, or PD-L1.

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Embodiment 38. The method of any one of embodiments 28-37, wherein the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation therapy, cell therapy, and surgery.

Embodiment 39. The method of any one of embodiments 28-38, wherein the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands.

Embodiment 40. The method of embodiment 39, wherein the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A _2a R, A _a R, A ^/A ^ R, arginase, CD39, CD73, IDO, and TDO.

Embodiment 41. The method of embodiment 39, wherein the inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tislelizumab, zimberelimab, durvalumab, and atezolizumab.

Embodiment 42. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1-19 and a pharmaceutically acceptable carrier.

Embodiment 43. An anti-TIGIT antibody or antigen-binding fragment thereof that binds to an epitope of human TIGIT comprising at least one of the following amino acid residues from SEQ ID NO 80: T55, Q56, N58, E60, D72, S80, and K82.

Embodiment 44. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 43, wherein the antibody or antigen-binding fragment thereof binds to an epitope that comprises at least the following residues of TIGIT: (i) D72 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) E60 and D72 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80; (iii) D72 and K82 of SEQ ID NO: 80, and optionally at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80; (iv) E60, D72 and K82 of SEQ ID NO: 80 and optionally at least one of T55, Q56, N58 and S80 of SEQ ID NO: 80; or (v) T55, Q56, N58, E60, D72, S80 and K82 of SEQ ID NO: 80.

Embodiment 45. An anti-TIGIT antibody or antigen-binding fragment thereof according to embodiment 43 or 44, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof according to any one of embodiments 1-19 for binding to TIGIT.

Embodiment 46. The anti-TIGIT antibody or antigen-binding fragment thereof of embodiment 43 or 44, wherein the excess antibody or antigen-binding fragment thereof competes with a reference antibody for binding to TIGIT by at least about 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% in a competition binding assay, wherein the reference antibody comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92, and a light chain having an amino acid sequence comprising SEQ ID NO: 93.

Embodiment 47. The TIGIT antibody or antigen-binding fragment thereof of any one of embodiments 1-19, wherein the antibody or antigen-binding fragment thereof binds to an epitope of human TIGIT comprising at least one of the following amino acid residues of SEQ ID NO 80: T55, Q56, N58, E60, D72, S80, and K82.

Each maximum numerical limitation set forth in this specification shall be intended to include every lesser numerical limitation, as if such lesser numerical limitation were expressly written herein. Each minimum numerical limitation set forth in this specification shall include every greater numerical limitation, as if such greater numerical limitation were expressly written herein. Each numerical range set forth in this specification shall include every narrower numerical range that is within such broader numerical range, as if all such narrower numerical ranges were expressly written herein.

All patent applications, websites, other publications, accession numbers, etc., referenced above or below are, for all purposes, herein incorporated by reference in their entirety to the same extent as if each individual item were individually and separately indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number on the effective filing date of this application shall be deemed to be the earlier of the actual filing date or the filing date of the priority application on which the accession number is referenced (if applicable). Likewise, if different versions of a publication, website, etc. are published at different times, the most recently published version on the effective filing date of the application shall be deemed to be the most recently issued version unless otherwise indicated. Unless otherwise specifically indicated, any feature, step,

- 41 049282 an element, embodiment or aspect of the invention may be used in combination with any other of the above.

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Examples

The following examples discuss the production, characterization, and humanization of anti-human TIGIT antibodies, and provide examples of methods by which the binding characteristics by which the antibodies described herein can be determined can be determined.

Example 1. Obtaining antibodies to TIGIT.

Anti-TIGIT antibodies were obtained from immunized mice. The extracellular domains of His-tagged human TIGIT protein SEQ ID NO: 83 (hTIGIT-His) and cynomolgus monkey TIGIT protein SEQ ID NO: 85 (cTIGIT-His) were transiently expressed in HEK293 cells and purified by anti-His affinity chromatography. Booster immunization at multiple sites (RIMMS) of BALB/c mice was performed using a mixture of recombinant hTIGIT-His and cTIGIT-His protein. Plasma titers were tested against the immunogens before the final challenge by ELISA to confirm a good titer. After the final challenge, terminal blood was collected along with splenic, inguinal, brachial, axillary and cervical lymph nodes. Prior to fusion, purified B cells were isolated from the collected material to generate hybridomas for primary screening.

Primary screening of hybridomas was performed using ELISA 10 days post-fusion. 384-well ELISA plates were coated with hTIGIT-His protein at 1 μg/ml and after blocking the plate, 20 μl of hybridoma supernatants were added and allowed to bind to TIGIT in the plate coating. After incubation at room temperature, the plate was washed and antibody bound to TIGIT in the plate coating was detected using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG.

Hybridoma cells were then expanded in 48-well plates and supernatants were collected for antibody specificity testing in ELISA assays. ELISA plates were coated with either hTIGIT-His or cTIGIT-His protein and CD47-His protein (Acro Biosystems, Cat# CD7-H5227) was used as a selectivity control to remove antibodies recognizing the His tag of the immunogenic protein. Antibodies showing positive binding to both human and cynomolgus TIGIT but negative binding to the His-tagged control protein were then tested for blocking functional TIGIT/CD155 binding. The extracellular domain of human TIGIT was fused to a mouse Fc sequence, and this protein, hTIGIT-mFc (SEQ ID NO: 87), was expressed in HEK293 cells and purified by protein A affinity chromatography. Recombinant human CD155 containing the extracellular domains fused to the human Fc sequence (hCD155-hFc) from R&D Systems (Cat# 9174-CD-01M) was used to perform the CD155/TIGIT blockade assay. To test the functional blockade activity of the antibodies, hTIGIT-mFc protein was used at a concentration of 0.5 μg/mL to coat the ELISA plate; after blocking, the hybridoma supernatant was added together with hCD155-hFc protein at a concentration of 0.5 μg/mL. Following incubation, the ELISA plate was washed and bound hCD155-hFc was detected using an HRP-conjugated anti-human IgG antibody. Clones were identified that were able to bind to both human and cynomolgus monkey TIGIT, and this antibody binding was able to block the CD155/TIGIT interaction.

These clones were further expanded and the antibodies were purified using a Protein G column. These purified antibodies were tested for binding to cell surface-expressed human and cynomolgus monkey TIGIT by flow cytometry. Stable Cno-Kι cell lines expressing clones 2A7 with full-length human TIGIT (Swiss-Prot Q495A1; SEQ ID NO: 80) or clone C10 with full-length cynomolgus monkey TIGIT (Swiss-Prot A0A2K5UW92; SEQ ID NO: 84) were developed. For flow assays, cells were harvested and incubated in 100 μl HBSS buffer in the presence or absence of various concentrations of antibodies for 1 h at 4°C. After washing with HBSS, antibody binding to the cells was detected using A1exa488-labeled goat anti-mouse IgG at 2 μg/ml (ThermoFisher Scientific, Cat# A-11001) for 30 min at 4°C. The cells were then washed and resuspended in phosphate-buffered saline (PBS) and subjected to flow cytometry using an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA, USA). The geometric mean fluorescence intensity of the entire single-cell population was obtained. Antibodies capable of binding to both human and cynomolgus monkey TIGIT expressed on the cell surface were further tested for their blocking activity against the binding of recombinant human CD155 to human TIGIT on the CHO cell surface. CHO-hTIGIT cells (105 cells) were incubated with hCD155-Fc protein at a concentration of 2.5 μg/ml in the presence of various concentrations of antibody at room temperature for 1 h. After washing with HBSS buffer, hCD155-Fc binding to hTIGIT-CHO cells was detected using conjugated

- 42 049282 PerCP-eFluor 710-coated anti-CD155 antibody (ThermoFisher, Cat. #1550-42). The cells were then washed and analyzed by flow cytometry. Table 4 shows the half-maximal effective concentration (EC ₅₀ ) (n = 2) of the antibodies for binding to human TIGIT and cynomolgus monkey TIGIT, and the IC ₅₀ for inhibition of CD155 binding to TIGIT.

Table 4

Purified mouse anti-TIGIT antibodies that bind to cell surface expressed TIGIT

a-TIGIT clone hTIGIT ECso (nM) cTIGIT ECso (nM) hTIGIT/hCD155 ICso (nM) 22В22 0,507 0,580 0.83 21B16 0,498 1,201 1,15 28012 0,638 0,710 1,16 5J24 0,704 1,634 1.46 21B9 0.506 0,631 1.66 21F8 0.952 1,789 1.67 28P24 0,466 1,260 1.70 24F8 1,306 1,846 1.79 30M18 0.825 1,166 2,19

The best hybridoma cell lines were selected based on their binding affinity to human TIGIT and cynomolgus monkey TIGIT and their ability to block CD155 binding to TIGIT. Hybridomas from these clones were expanded and the variable regions of the heavy and light chains (VH and VL, respectively) of mouse anti-TIGIT antibodies were sequenced using standard procedures. The amino acid sequences of mature VH and VL of antibodies 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, and 28O12 are shown in Fig. 1A–1I, where their CDRs are underlined. The designations of CDR sequences and the numbering of amino acid positions are as defined by Kabat.

The seven antibodies shown in Fig. 1A-1I were expressed recombinantly as mouse-human chimeras with mouse variable domains and human IgG1/kappa constant domains. The recombinant proteins were expressed in HEK293 cells and purified by protein A affinity chromatography.

The ability of these chimeric anti-TIGIT antibodies to bind to the cell surface overexpressing human TIGIT and cynomolgus TIGIT was confirmed using flow cytometric analysis as described previously. Following incubation of the chimeric anti-TIGIT antibody with hTIGIT-CHO or cTIGIT-CHO, bound antibody was detected using A1exa488-conjugated goat anti-human IgG (ThermoFisher Scientific, Cat.# A-11013). The functional activity of the antibodies in inhibiting the binding of recombinant hCD155-hFc to hTIGIT-CHO-K1 cells was also determined using flow cytometric analysis as described previously. The EC ₅₀ of the chimeric TIGIT antibodies binding to human and cynomolgus monkey TIGIT and the IC ₅₀ for inhibiting hCD155 binding to hTIGIT-expressing cells were determined and are presented in Table 5.

Table 5. Recombinant chimeric mouse-human antibodies to TIGIT that bind to TIGIT overexpressed on the surface of Cno-Kι cells, as well as their blocking activity, which inhibits the binding of human CD155 to human TIGIT.

Table 5

Antibody to α- TIGIT hTIGIT-CHO ECso (nM) cTIGIT-CHO ECso (nM) hTIGIT-CHO/hCD155 ICso (nM) Ch22B22 0.16 0.24 1,16 Ch21B16 0,12 0.42 0,20 C1128O12 0.159 0.358 0.48 Ch5J24 0,131 0,212 0,70 Ch21B9 0,102 0.18 0.80 C1124F8 0,15 0,199 0.62 Ch30M18 0.336 6,138 2,18

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The ability of the chimeric anti-TIGIT antibodies to bind to endogenously expressed TIGIT on isolated human CD4+ and CD8+ cells was tested by flow cytometric analysis. Human CD4+ or CD8+ T cells were isolated from human whole blood using RosetteSep™ Human CD4+ T cell Enrichment Cocktail (Stemcell, Cat.# 15022) or Human CD8 ⁺ T cell enrichment Cocktail (Stemcell, Cat.# 15022), respectively. As shown in Table 6, comparable EC _{50 s} were observed for the recombinant anti-TIGIT antibodies binding to human CD4 ⁺ or CD8 ⁺ cells, consistent with comparable binding affinities for full-length human TIGIT overexpressed on Cho-Kι cells. However, differences were found between the clones with respect to their maximal binding activity (MFImax).

Table 6 Recombinant chimeric mouse-human antibodies to TIGIT that bind to isolated human T cells

Antibody to a- TIGIT CD4 ⁺ ECso (hM) CD4 ⁺ MFImax CD8 ⁺ ECso (hM) CD8 ⁺ MFImax Ch24F8 0,130 210 0,177 5800 Ch5J24 0,073 125 0,117 4500 Ch21B9 0,029 150 0,062 4500 Ch22B22 0,097 140 0,175 5000 Ch21B16 0,119 180 0,160 5200 Ch28O12 0,141 190 0.249 7200 Ch30M18 0.197 125 0.293 3500

Recombinant chimeric anti-TIGIT antibodies binding to cynomolgus monkey whole blood were tested to confirm the ability of the antibody to bind to endogenous cynomolgus monkey TIGIT protein on CD4 ⁺ and CD8 ⁺ cells. Cynomolgus monkey whole blood was incubated with recombinant chimeric anti-TIGIT antibodies at concentrations of 20 μg/mL, 5 μg/mL, 1 μg/mL, and 0.2 μg/mL. After 30 min of incubation at 4°C, cells were lysed with RBCs for 15 min at room temperature. Cells were then washed and collected by centrifugation and blocked with a cocktail containing Fc-block (BD Biosciences, Cat. #564219) and Live-dead fixable Aqua (Invitrogen, Cat. #L34957). Cell-bound anti-TIGIT antibodies were detected using anti-human IgG Fc-Biotin (Southern Biotech, Cat. #9040-08) and incubation for 30 min at 4°C, followed by washing and centrifugation of the cells and a second incubation for 30 min at 4°C with peroxidase-conjugated streptavidin (Invitrogen, Cat. #12-4317-87). Wild-type human IgG1 was used as an isotype control, and directly peroxidase-conjugated anti-human TIGIT antibody (TIGIT-PE (eBiosciences, Cat. #12-9500-42)) was used as a positive control. In Fig. Figure 2 shows that Ch24F8, Ch28O12, and Ch22B22 were able to bind to cynomolgus TIGIT expressed on cynomolgus CD4 ⁺ and CD8 ⁺ cells. Geometric mean fluorescence intensity (gMFI) was obtained and these data are presented as a fold comparison of gMFI to isotype control.

The kinetic binding of these recombinant anti-TIGIT antibodies to human TIGIT was determined by surface plasmon resonance (SPR) using a Bio-Rad ProteOn XPR36 instrument. The recombinant antibodies were immobilized using a protein A-coated GLC sensor chip and soluble His-tagged TIGIT (Acro Biosystems, Cat. No. TIT-H52H3) was used as the analyte. Binding constants were determined at 25°C. As shown in Table 7, clone 24F8 has the highest binding affinity, as measured by the equilibrium dissociation constant (K _D ), among the seven recombinant chimeric anti-TIGIT antibodies tested.

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Table 7

Kinetics of binding of recombinant anti-TIGIT antibodies to His-tagged human TIGIT

Antibody to a-TIGIT ka (MV) k<i(s' ¹ ) Kd(M) Ch24F8 2D5E+06 7,5E-05 3,5E-11 Ch28O12 l,256E+06 1D35E-03 9,04E-10 Ch30M18 7.27E+05 l,730E-03 2,38E-09 Ch5J24 5.57E+06 2D4E-02 3,85E-09 Ch21B16 l,47E+06 9,29E-03 6,30E-09 Ch21B9 l,05E+07 7,4E-02 7DE-09 Ch22B22 l,44E+06 l,41E-02 9,78E-09

Example 2. Production of humanized antibodies to TIGIT.

Murine antibody 24F8 was chosen for humanization using the CDR grafting method (Queen et al, Proc. Natl. Acad. Sci. USA. 86:10029–10033, 1989). Murine 24F8 variable heavy (VH) and variable light (VL) region sequences were used to identify the two closest human germlines for each chain. For VH, IGHV434*09 were found with 70% sequence identity and IGHV4-4*02 with 66% identity. For VL, IGKV1-33*01 were found with 70% sequence identity and IGKV3-15*01 with 67% identity (Table 8).

Table 8

Identification of human germ lines and acceptors

Variable chain Human germline Identity of human and mouse sequences (%) Human acceptor VH1 IGHV4-34*09 70 AAV40102.1 VH2 IGHV4-4*02 66 ADX65334.1 VL1 IGKV1-3 3*01 70 ACY78416.1 VL2 IGKV3-15*01 67 ADU32611.1

The numbering of the positions of the three heavy-chain CDR sequences (HC-CDRs) in the VH chain and the three light-chain CDR sequences (LC-CDRs) in the VL chain were determined according to Kabat. Human acceptors for the VH and VL scaffolds were searched in the GenBank database (Benson et al., Nucleic Acids Res. 2005, 33, D34-D38), and human cDNA sequences encoding the VH and VL sequences were identified (see Table 8).

CDR grafting for each human acceptor was performed using HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50) for VH acceptors and LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53) for VL acceptors. The resulting sequences were examined for the introduction of any potential post-translational modifications or chemical degradation sites and were confirmed to be free of such deficiencies. Putative residues responsible for the mouse reverse mutation were also identified using antibody graphical homology modeling. Oligonucleotides were designed and synthesized to encode Fab fragments with human IgG1/kappa constant domains for human acceptors grafted with two VH and two VL and inserted into a vector system for high-throughput screening of expression levels and biophysical properties without the need for protein purification (Zhang & Hirama, Patent Application Publication US 2012/0178110). All four combinations of VH1 (SEQ ID NO: 76) or VH2 (SEQ ID NO: 78) or VL1 (SEQ ID NO: 79) or VL2 (SEQ ID NO: 77) (Figs. 1J-1M) were screened together with the chimeric Fab mVH+mVL constructed from the murine 24F8VH (SEQ ID NO: 5) and 24F8VL (SEQ ID NO: 6) domains and the constant domains of human IgG1/kappa Fab fragments. Supernatants of Fab secreted as SASA (single domain anti-serum albumin) fusion proteins were analyzed by surface plasmon resonance (SPR) using a Biacore 8K instrument, with the analysis performed with Fab capture using a BSA-coated chip and soluble His-tagged TIGIT as analyte. Kinetic binding data for the five Fab fragments with human TIGIT and cynomolgus macaque TIGIT are shown in Table 9.

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Table 9

Binding affinity kinetics data for humanized Fab fragments

Fab An alit ka (M ⁴ s ⁴ ) kd(s' ¹ ) Kd(M) mVH+mVL hTIGIT-His l,86E+06 2,37E-06 1,27E-12 VH1+VL2 (Hu24F8.1 Fab) 3.08E+06 1,11E-O4 3,61E-11 VH2+VL2 (Hu24F8.2 Fab) 2.08E+06 9,39E-05 4,50E-ll VH2+VL1 (Hu24F8.3 Fab) l,99E+06 4,81E-04 2,42E-10 VH1+VL1 (Hu24F8.4 Fab) 3.05E+06 8,55E-04 2,80E-10 mVH+mVL cTIGIT-His l,54E+06 7,28E-04 4.74E-10 VH1+VL2 (Hu24F8.1 Fab) 1,11E+O6 l,40E-03 l,26E-09 VH2+VL2 (Hu24F8.2 Fab) 7.42E+05 8,08E-04 l,09E-09 VH2+VL1 (Hu24F8.3 Fab) 9.74E+05 5,85E-03 6,00E-09 VH1+VL1 (Hu24F8.4 Fab) l,35E+06 l,33E-02 9,81E-09

These data demonstrated that the humanized VH1+VL2 and VH2+VL2 variable domain combinations bound most tightly to both human and cynomolgus monkey TIGIT as determined by equilibrium dissociation constant (KD) measurements and also retained most of the binding affinity of the chimeric mouse-human Fab fragment. Therefore, constructs containing back mutations of the mouse framework regions were not investigated. These two VH/VL combinations (Fig. 1J and Fig. 1K) were chosen to generate the full-length Hu24F8.1 and Hu24F8.2 IgG1/kappa antibodies, respectively.

Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA are IgG1/kappa antibodies that have a heavy chain constant region with the amino acid sequence of SEQ ID NO: 97 and a light chain constant region with the amino acid sequence of SEQ ID NO: 95. The designation IgG1.AA indicates that the heavy chain constant region has substitutions of the amino acid leucine for the amino acid alanine at positions 234 and 235 (EU numbering). Conversely, the designation IgG1 indicates the Fc region of wild-type IgG1. For example, Hu24F8.2-IgG1 is an IgG1/kappa antibody that has a heavy chain constant region with the amino acid sequence of SEQ ID NO: 94 and a light chain constant region with the amino acid sequence of SEQ ID NO: 95. The Hu24F8.1-IgG1.AA, Hu24F8.2-IgG1.AA, and Hu24F8.2-IgG1 antibodies used in these examples were produced recombinantly in HEK293 or CHO cells and purified by protein A affinity chromatography.

Purified full-length Hu24F8.1-IgG1.AA (containing VH1+VL2) and Hu24F8.2IgG1.AA (containing VH2+VL2) antibodies together with a full-length chimeric antibody consisting of mouse 24F8 VH+VL and human IgG1/kappa constant domains (Ch24F8) were analyzed by SPR using a Biacore T200 instrument with antibody capture using a chip coated with human anti-human IgG and soluble hTIGIT-His or cTIGIT-His as the analyte. Binding kinetics data for human and cynomolgus monkey TIGIT are shown in Table 10.

Table 10

Binding affinity kinetics data for humanized antibodies

Antibody Analyte ka (M ⁴ s ⁴ ) k<i( ^s4 ) Kd(M) Ch24F8 hTIGIT-His 8.53E+05 l,56E-04 l,83E-10 Hu24F8.1-IgGl.AA l,01E+06 l,45E-04 l,44E-10 Hu24F8.2-IgGl.AA 7.96E+05 l,40E-04 l,76E-10 Ch24F8 cTIGIT-His 6.58E+05 l,09E-03 l,66E-09 Hu24F8.1-IgGl.AA 8.06E+05 l,40E-03 l,74E-09 Hu24F8.2-IgGl.AA 6.79E+05 9,97E-04 l,47E-09

Binding affinity kinetics data for both full-length humanized antibodies confirmed that the binding affinity of the mouse antibody was fully retained and that there was no need to introduce any back mutations of the mouse framework residues.

Example 3. In vitro studies of binding of antibodies to TIGIT.

Binding of Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA antibodies to cell surface-expressed TIGIT was studied by flow cytometry. Cells expressing TIGIT on the cell surface were collected and incubated in 100 μl of HBSS buffer in the presence (or absence) of antibody at various concentrations for 1 h at 4°C. After washing with HBSS, binding

- 46,049,282 antibodies on cells were detected by incubation with A1exa488-labeled goat anti-human IgG at 2 μg/mL (ThermoFisher Scientific, Cat. #A-11013) for 30 min at 4°C. Cells were then washed and resuspended in PBS and flow cytometrically analyzed using an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA, USA). The geometric mean fluorescence intensity was obtained for the entire cell population, and for transient transfections, untransfected cells were used to gate the cell population on positive binding to determine the percentage of cells positive for binding. Data were calculated in GraphPad Prism using a standard 4-parameter curve fit.

Stable Cho-Kι cell lines expressing human TIGIT (clone 2A7) and cynomolgus TIGIT (clone C10) were used to test Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA for their binding to human TIGIT and cynomolgus TIGIT, respectively, as described above. Hu24F8.1IgG1.AA bound to human TIGIT with an EC ₅₀ of 0.447 ± 0.22 nM (n = 8) (Fig. 3A) and to cynomolgus TIGIT with an EC ₅₀ of 0.237 ± 0.33 nM (n = 6) (Fig. 3B). Hu24F8.2-IgG1.AA bound to human TIGIT with an _EC50 of 0.29±0.15 nM (n=8) (Fig. 3A) and to cynomolgus macaque TIGIT with an _EC50 of 0.35±0.16 nM (n=6) (Fig. 3B). These results indicate that both Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA strongly bind to cell surface-expressed human and cynomolgus macaque TIGIT.

Binding of Hu24F8.2-IgG1.AA to mouse TIGIT (Swiss-Prot Q86176; SEQ ID NO: 88) and rat TIGIT (Swiss-Prot D3ZTQ2; SEQ ID NO: 89) was examined using CHO-K1 cells transiently transfected with constructs expressing full-length mouse and rat TIGIT. Mouse TIGIT expression was confirmed using the GNE10A7 control antibody (US Patent No. 9,499,596 Clark et al., 2016) and rat TIGIT expression was confirmed using the eBioscience™ G1GD7 control antibody (Invitrogen, Cat. No. 12-9501-82). When tested using up to 30 nM antibody, Hu24F8.2-IgG1.AA did not bind to either mouse TIGIT (Fig. 4A) or rat TIGIT (Fig. 4B).

Binding of Hu24F8.2-IgG1.AA to isolated human CD8+ T cells was examined by flow cytometry. CD8+ T cells were isolated using RosetteSep™ Human CD8+ T cell Enrichment Cocktail (Stemcell, Cat. #15023) according to the manufacturer's recommendations. Cells were then activated with anti-CD3/CD28 beads supplemented with 20 U/mL rhIL-2 for 7-9 days. Activated or non-activated CD8+ T cells were blocked with human Fc Block (BD Biosciences, Cat. #564219) and analyzed for antibody binding by flow cytometry. As shown in Fig. 5A and Fig. 5B, Hu24F8.2-IgG1.AA bound to non-activated CD8+ cells with an _EC50 of 0.098±0.013 nM (n=2) and to activated CD8+ cells with an EC50 of 0.14±0.036 nM (n=2), respectively. Although the binding _EC50s are similar for activated and non-activated CD8+ cells, there is a significant difference in the maximal binding signals, consistent with the increased expression of TIGIT on the surface of CD8+ cells upon activation.

Example 4. In vitro blocking studies of antibodies to TIGIT.

The activity of Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA in blocking the interaction of TIGIT with CD155 was analyzed by flow cytometry using CHO cells stably overexpressing human TIGIT (CHO-hTIGIT) and a recombinant soluble protein derived from the fusion of human CD155 with the Fc fragment (hCD155-Fc) as described in Example 1. As shown in Fig. 6, both Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA blocked the interaction between hCD155Fc and CHO-hTIGIT on the cell surface in a dose-dependent manner. The IC ₅₀ for Hu24F8.1-IgG1.AA in blocking the interaction with TIGIT-CD155 was 0.68 nM, and for Hu24F8.2-IgG1.AA it was 0.67 nM.

Example 5. Characterization of TIGIT Antibodies in Relationship to the Jurkat Dual Reporter Cell Line The functional activity of Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA to block the human TIGIT receptor was analyzed using Promega's TIGIT/CD155 Blockade Bioassay (Promega, Cat. #J2205). In this assay, the effector cell line is the Jurkat cell line, which is characterized by overexpression of TIGIT as well as a luciferase reporter that is activated downstream of the T cell receptor (TCR). Another stable cell line is the CHO-K1 cell line, which overexpresses human CD155 in addition to T cell activator protein, which binds and activates the TCR. This CD155 aAPC/CHO-K1 cell line functions as artificial antigen-presenting cells. Co-culture of the two cell lines results in TCR activation by artificial antigen-presenting cells (aAPCs) on CHO-K1 cells, which activates the reporter construct, but activation of this pathway is inhibited by the TIGIT/CD155 interaction, resulting in a weak luciferase signal. The presence of anti-TIGIT antibody inhibits the TIGIT/CD155 interaction, abolishing the inhibitory effect of TIGIT, resulting in an enhanced luciferase signal. The assay was performed according to the manufacturer's protocol; briefly, Jurkat effector cells were plated overnight in a cell culture incubator in 96-well plates, test antibody was serially diluted and added to the effector cells, followed by the addition of CD155 aAPC/CHO-K1 cells. After 6 h,

- 47 049282 co-cultured at 37°C with 5% _CO2, Bio-Glo luciferase substrate reagent was added and the luminescent signal was read using Envision (PerkinElmer). As shown in Fig. 7, Hu24F8.1IgG1.AA was able to dose-dependently enhance the reporter activity with an EC50 of 3.35±0.26 nM (n=3), whereas the _{EC50 of} Hu24F8.2-IgG1.AA was 2.78±0.83 nM (n=3). Control human IgG1 did not show any effect.

Example 6. Molecular analysis of antibodies to TIGIT.

TIGIT and Fab expression, purification, crystallization.

Soluble protein of the mature extracellular domain of human TIGIT (aa residues 22130) was expressed recombinantly in HEK293 cells. The construct (SEQ ID NO: 90) contained a C-terminal hexahistidine tag with a (Gly) ₄ -Ala-(Gly) ₄ linker, and asparagine residues at positions 32 and 101 were replaced by glutamine to remove N-glycosylation sites. The clarified supernatant was purified by affinity chromatography using a Nickel Sepharose Excel column (GE Healthcare Life Sciences) followed by size exclusion chromatography (SEC) using a Superdex 200 pg column (GE Healthcare Life Sciences). TIGIT protein at a concentration of 8.4 mg/mL was placed in a final buffer composition of 20 mM Tris pH 7.0, 100 mM NaCl and flash frozen with liquid nitrogen. Soluble Fab fragment (Fab24F8) of Hu24F8.2IgG1.AA human IgG1 antibody was generated by papain digestion (Thermo Scientific, cat. #20341) in phosphate-buffered saline at 37 °C for 3 h followed by overnight digestion at room temperature. The cleaved Fc fragment was removed using a MabSelect SuRe Protein A column (GE Healthcare Life Sciences) and the eluate was further purified by SEC using a Superdex 200 pg column (GE Healthcare Life Sciences). Fab24F8 protein at a concentration of 28 mg/ml was placed in a final buffer composition containing 20 mM Tris pH 7.0, 100 mM NaCl and flash frozen with liquid nitrogen.

TIGIT was mixed with Fab24F8 protein at a 1:1 molar ratio by stirring for 60 min at 4°C to form the Fab-TIGIT complex, followed by final purification by SEC using a Superdex 200 pg column (GE Healthcare Life Sciences) to eluate the protein to 44 mg/mL. The purified complex was used in crystallization assays at 20°C using a standard screening of approximately 1500 different states. The states were initially optimized using standard strategies by systematically varying parameters critical to crystallization. These states were further refined by systematically varying pH or precipitant concentrations. Crystals of the Fab-TIGIT complex suitable for structure elucidation were prepared by mixing 0.1 μL of protein solution (15 mg/mL in 20 mM Tris, pH 7.0; 100 mM NaCl) with 0.1 μL of reservoir solution (20% (w/v) PEG3350; 0.20 M LiSO4) using the sessile drop vapor diffusion technique.

Data collection and structure decision.

The crystals were flash frozen and measurements were performed at 100 K. X-ray spectroscopy data were collected by studying the crystals of the Fab-TIGIT complex using the Canadian Light Source (CLS, Saskatoon, Canada) under cryogenic conditions. The crystals belong to the space group P 1. The data were processed using the autoPROC, XDS, and AIMLESS (The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763) computer software, see Table 11.

Table 11

Data collection and statistics processing for the Fab24F8/TIGIT complex

¹ Values in brackets refer to the interval with the highest resolution

The phase information required for structure determination and analysis was obtained by the molecular method85.11; 86.83; 87.83

94.3; 117.0; 116.1

126 (2657)

2.2(2.1)

84.8 (76.4)

6.2 (48.6)

8.2 (64.8)

9.9 (1.7)

Complex

X-ray source

Detector

Temperature (K)

Space group

0.9795

PILATUS 6M

Unique reflections

Completeness(%)

Rsym (96)

Rmeas (26)

Average (Ι)/σ

Hu24F8.2 Fab/human TIGIT extracellular domain (ECD)

CMCF-ID (08ID-1, CLS)

Wavelength (A)

Cell: a; b; c; (A)

Resolution (A)

2.24 (2.46-2.24) ¹

Magnification factor

- 48 049282 cular substitution. Previously determined structures of Fab (Bohrmann et al., J. Alzheimers Dis. 28:49–69, 2012) and TIGIT (Stengel et al., Proc. Natl. Acad. Sci. USA, 2012) were used as search models. There are three molecules of the Fab-TIGIT complex in the crystallographic asymmetric block. Subsequent model construction and refinement were performed according to standard protocols using the COOT program and the CCP4 software package, respectively. To calculate the free R-factor needed to cross-check the correctness of the final model, approximately 4.6% of the measured reflections were excluded from the refinement procedure. Refinements of the parameters describing possible solid-state root-mean-square (TLS) displacements were performed (using the CCP4 REFMAC5 program), which resulted in lower R-factors and higher quality of the electron density map. Automatically generated local non-crystallographic symmetry (NCS) constraints were applied. The water model was built using the Find waters algorithm of the COOT software by placing water molecules in the peaks of the Fo-Fc map simulated at 3.0 Å, followed by refinement with REFMAC5 and checking all waters with the COOT checker. The criteria for inclusion in the list of suspect water molecules were as follows: B-factor greater than 80 Å ² , 2Fo-Fc map less than 1.2 σ, nearest contact distance less than 2.3 Å or greater than 3.5 Å. Data in the range with a resolution of 73.5–2.24 Å were in the final refinement cycle, and the R-factors R _c r _{y s} t and R _free were 22.3 and 26.8%, respectively. The Ramachandran plot of the final model shows 89.8% of all residues in the most favored region, 8.8% in the extra-resolved region, and 0.7% in the highly resolved region. A summary of the refinements is presented in Table 12.

Table 12 Refinement statistics for Fab24F8/TIGIT

Hu24F8.2 Fab/extracellular domain (ECD) TIGIT complex

Resolution (A) person 73.52-2.24 Number of reflections (working/testing) 50 688/2467 Rcryst (%) 22.3 Rfree (%) ¹ 26.8 Total number of atoms: Squirrels 12 244 Water 427 30 Ligand - Sulfate 30 Deviation from ideal geometry: ² Lengths of connection (A) 0,010 Connection angles (°) 1.47 Related B (A ² ) ³ 17 Ramachandran's Graph: ⁴ Most preferred areas (%) 89.8 Additional permitted areas (%) 8,8 Widely resolved areas (%) 0,7 Unresolved areas (%) 0,7

¹ Test set contains 4.6% of measured reflections ² Standard deviations from target geometric values ³ Calculated with MOLEMAN ⁴ Calculated with PROCHECK

Structure of Fab24F8 bound to human TIGIT.

The crystallographic asymmetric block contains three independent molecules of the complex, whose atomic coordinates superpose pairwise with a standard deviation of 0.53-1.13 Å for all atoms other than hydrogen. The final model contains residues from Gln1 to Ser223 of the heavy chain of the Fab fragment, from Glu1 to Cys214 of the light chain of the Fab fragment, and from Met22 to Ser129 of TIGIT. Some short loop regions were not completely determined by electron density and were not included in the final model.

The heavy chain of the Fab fragment HC-CDR2 and HC-CDR3 and all three LC-CDRS of the light chain interact extensively with the large β-sheet structure of TIGIT, namely the β-strands C, C', C' and F and the loops C'C and C''D, consisting of the polypeptide chains ^{55TQVNWEQQDQLLAICNADLGWHISPSFK82} and ¹⁰⁹ IYH ⁿ¹ of SEQ ID NO: 80 (Fig. 8 and 9). This binding interaction results in a protein-protein interface with a surface area of 790±10 ^A2 (n = 3) between the Fab fragment and TIGIT (PISA, EMBL-EBI). The molecular nature of this interaction is both hydrophilic and hydrophobic. TIGIT residues Thr55, Gln56, Asn58, Glu60, Asp72, Ser80, and Lys82 (shown with the rod image in Fig. 9) form direct or water-mediated hydrogen bonding interactions (interatomic distance between donor and acceptor no more than 3.1 A) with residues of the CDR regions of the heavy and light chains of the Fab fragment.

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In addition, the TIGIT residue Glu60 forms a salt bridge with Arg30 on the light chain of the Fab fragment (Table 13). The hydrophobic TIGIT residues Leu65, Ile68, Leu73, Pro79, and Ile109 (represented by spheres in Fig. 9) form van der Waals contacts with residues of the heavy and light chains of the Fab fragment.

Table 13

TIGIT epitope residues that form hydrogen bonds and salt bridges with paratopic residues of the heavy and light chains of Fab24F8

Epitope TIGIT Paratope Fab24F8 (numbering no Kabat) Thr 55 LC-CDR3 Thr 94 Gin 56 LC-CDR3 Tyr 92 LC-CDR3 Trp* 96 HC-CDR3 Tyr* 97 Asn 58 LC-CDR3 Tyr* 92 Glu 60 LC-CDR1 Arg 30 LC-CDR3 Tyr 92 Asp 72 HC-CDR2 Tyr 50 Ser 80 LC-CDR2 Tyr 53 Lys 82 HC-CDR3 Asn 99

* Water-mediated hydrogen bonding

Fig. 10A shows a schematic of the complex structure of the N-terminal Ig-like domain (SEQ ID NO: 91) of human CD155 (depicted as a ribbon) bound to human TIGIT (depicted as a molecular surface), as reported in Stengel et al., 2012. Fig. 10B shows a superposition of CD155 in the same orientation as Fig. 10A on a schematic representation of the complex of the crystal structure of Fab24F8 bound to TIGIT (each represented by a molecular surface). It can be clearly demonstrated that Hu24F8.2 or other antibodies derived from the murine 24F8 antibody, by binding to the extracellular domain of TIGIT, will block the binding of CD155 to TIGIT.

Example 7: Anti-TIGIT antibody enhances T cell responses both alone and in combination with anti-PD-1 antibody.

This example demonstrates that Hu24F8.2-IgG1 enhances primary T cell responses in healthy subjects or subjects with cancer, either alone or in combination with an anti-PD-1 antibody such as AB122. PBMCs from healthy subjects and subjects with cancer treated with Hu24F8.2-IgG1 at 0.1, 1, or 10 μg/mL had significantly increased IL-2 concentrations compared to isotype controls. PBMCs from healthy subjects treated with a combination of Hu24F8.2-IgG1 at 10 μg/mL and anti-human PD-1 at 1 μg/mL (AB122, zimberelimab) had significantly higher IL-2 levels compared to cells treated with AB122 alone.

PBMCs from healthy subjects were isolated using Leukoreduction System (LRS) chambers, and PBMCs from cancer subjects were isolated in CPT tubes and cultured in vitro with either Hu24F8.2 at 0.1, 1, or 10 μg/ml, AB122 at 1 μg/ml, or a combination of Hu24F8.2-IgG1 at 10 μg/ml and AB122 at 1 μg/ml in the presence of SEA at 1 ng/ml. After four days, IL-2 concentration in the supernatant was measured by flow cytometric assay (FCA). IgG1 isotype was included as a negative control.

Methods

PBMCs from a healthy subject were isolated using LRS chambers and PBMCs from a subject with cancer were isolated in CPT tubes. PBMCs were resuspended at 2 x 106 cells/mL and aliquoted at 100 μL per well into a 96-well round-bottom plate. Fourfold antibody concentrates resuspended in CTS Optimizer medium were added to the appropriate wells at 50 μL per well: Hu24F8.2-IgG1 or human IgG1 isotype control was added to a final concentration of 0.1, 1, and 10 μg/mL and AB122 or human IgG4 isotype control was added to a final concentration of 1 μg/mL. The assay plates were incubated at 37°C, 5% CO2 for 1 h. Four-fold concentrates of staphylococcal enterotoxin A (SEA) resuspended in CTS Optimizer medium were added to appropriate wells at 50 μl per well to a final concentration of 1 ng/ml. Assay plates with a final well volume of 200 μl were incubated at 37°C, 5% CO2 for 4 days, after which the supernatant was collected for subsequent quantification of secreted IL-2. Supernatants were diluted 1:2 in the dilution solution from the Human Soluble Protein Master Buffer Kit. Assay performance, data collection, and quantification were performed using the Human IL-2 Flex Set with the Human Soluble Protein Master Buffer Kit according to the manufacturer's instructions.

Results.

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PBMCs from all donors used in the study were tested for TIGIT expression in the CD4+ nonregulatory T cell population, which responds to SEA stimulation, and CD155 (a ligand for TIGIT) in the CD14+ monocyte population. PBMCs isolated from 10 healthy subjects and 7 subjects with cancer were cultured with either Hu24F8.2-IgG1 alone at 0.1, 1, or 10 μg/ml, AB122 alone at 1 μg/ml, or a combination of Hu24F8.2-IgG1 at 10 μg/ml and AB122 at 1 μg/ml in the presence of SEA at 1 ng/ml. IL-2 concentration was measured in the supernatant after 4 days. IL-2 levels in the Hu24F8.2-treated group were compared with IL-2 levels in the corresponding IgG1 isotype control group, and IL-2 levels in the AB122 and Hu24F8.2-IgG1 combination group were compared with IL-2 levels in the AB122-only group (Table 14 and Table 15). An example of IL-2 levels for one healthy subject (#566) is shown in Fig. 11 for all Hu24F8.2-IgG1 concentrations tested. These two subjects showed statistically significant increases in IL-2 secretion at all Hu24F8.2-IgG1 concentrations tested compared to the corresponding IgG1 isotype control. In healthy subject #566, a statistically significant increase in IL-2 secretion was observed with the combination of AB122 and Hu24F8.2-IgG1 compared to AB122 alone. At the cohort level, a statistically significant increase in IL-2 secretion was observed in PBMCs from both healthy and cancer subjects with 10 μg/mL Hu24F8.2-IgG1 compared to the IgG1 isotype control in both healthy and cancer PBMCs (Figure 12A), and a statistically significant increase in IL-2 secretion was observed in PBMCs from healthy subjects with the combination of AB 122 and Hu24F8.2-IgG1 compared to AB122 alone (Figure 12B). Thus, treatment with Hu24F8.2-IgG1 at a concentration of 0.1 μg/ml significantly increased the concentration of IL-2 (compared with isotype control) in 6/10 PBMC samples from healthy subjects (by 1.1-3.4 times) and in 2/5 PBMC samples from subjects with cancer (by 1.6-1.7 times), treatment with Hu24F8.2-IgG1 at a concentration of 1 μg/ml significantly increased the concentration of IL-2 (compared with isotype control) in 7/10 PBMC samples from healthy subjects (by 1.4-4.0 times) and in 4/7 PBMC samples from subjects with cancer (1.6-2.0 times), treatment with Hu24F8.2-IgG1 at a concentration of 10 μg/ml significantly increased the concentration of IL-2 (compared with isotype control) in 7/10 PBMC samples from healthy subjects (1.2-4.0 times) and in 4/7 PBMC samples from subjects with cancer (1.3-2.0 times), and treatment with Hu24F8.2-IgG1 at a concentration of 10 μg/ml + AB122 increased the concentration of IL-2 (compared to treatment with AB122 alone) in 6/10 PBMC samples from healthy subjects (1.9-8.3 times).

Table 14

Mean IL-2 levels (pg/mL) in human PBMCs from healthy subjects

Subjects 223 225 226 229 272 273 566 967 568 969 0.1 mcg/ml Isotype 452.8 3282 1552 2629 n/t n/t 717.9 643,5 509.6 2625 Hu24F8.2-IgGl 377.8 3735 2353 3837 n/t n/t 1763 998.8 1725 6706 Significance of ^ь n/z * * * - - ** n/z *** **

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Subjects 223 225 226 229 272 273 566 967 568 969 Multiple change 0.83 1,13 1.51 1.45 - - 2.45 1.55 3.38 2.55 1.0 mcg/ml Isotype 385.2 3276 1920 2517 1035 321.4 735.9 565,1 510.6 2442 Hu24F8.2-IgGl 394.3 3671 2361 3588 1944 1296 2268 1270 1919 8256 Significance of ^ь n/z n/z n/z * *** **** *** * **** **** Multiple change 1,02 1D2 1,22 1.42 1.88 4.03 3.08 2.24 3.75 3.38 10.0 mcg/ml Isotype 380,1 3269 2017 3448 1385 673,1 804.4 854.9 519.9 2847 Hu24F8.2-IgGl 294.5 3775 2329 4175 1779 1006 2515 1649 1949 8510 Significance of ^ь n/z * n/z * n/z * **** * **** **** Multiple change 0.77 1,15 1,15 1,21 1.87 4.03 3,12 1.92 3.74 2.98 Isotype 430.2 3421 2070 3025 1154 785 928,1 733.3 593.4 n/t AB 122 1080 5628 3455 4841 1728 1453 2650 2074 1593 n/t 1 µg/ml AB 122+ 10 µg/ml Hu24F8.2-IgGl 1222 5762 3672 7077 2240 2067 7665 3614 4510 n/t AB 122 versus AB122+Hu24F8.2IgGl Significance of ^ь n/z n/z n/z * * *** **** **** **** - Multiple change 2.84 1.68 1.77 2.33 1.94 2.63 8.25 4.92 7.6 -

a _ __ „ „ ______ n - 3 technical replicates ^b One-way analysis of variance (ANOVA) with Šidák's multiple comparison correction (isotype versus Hu24F8.2 or AB122+Hu24F8.2 versus AB122); n/s - not significant; n/t - not tested; *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001

Table 15

Mean IL-2 levels (pg/mL) in human PBMCs from cancer subjects

Subjects 3 29 60 33 27 6 12 0.1 mcg/ml Isotype n/t 84.68 n/t 47,44 1097 3438 1349 Hu24F8.2-IgGl n/t 98,07 n/t 87,85 1874 5447 1871 Importance ⁶ - n/z - n/z *** **** n/z Multiple change - 1,16 - 1.85 1.71 1.58 1.38 1.0 mcg/ml Isotype 1583 95.57 792.3 170 1068 3396 1295 Hu24F8.2-IgGl 2074 91.4 1557 119.8 1746 5992 2221 Importance ⁶ n/z n/z * n/z ** **** ** Multiple change 1.31 0.95 1.96 0,7 1.63 1.76 1.71 10.0 mcg/ml Isotype 1600 95.34 674.7 129.7 1153 3885 1765 Hu24F8.2-IgGl 1897 53,54 1363 94.35 1934 5073 3089 Importance ⁶ n/z n/z * n/z ** ** **** Multiple change 1,18 0.56 2.02 0.72 1.67 1,3 1.75

a n - 3 technical replicates ^b One-way ANOVA with Šidák's multiple comparison correction (isotype versus Hu24F8.2); n/s - not significant; n/t - not tested; *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001

These data demonstrate that Hu24F8.2-IgG1, either alone or in combination with an anti-PD-1 antibody such as AB122, enhances primary human T cell responses from healthy subjects or subjects with cancer.

Example 8. Characterization of antibodies using in vitro complement-dependent cytotoxicity (CDC) assay.

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CDC is an immune response in which the complement system participates in antibody-dependent cell killing through the binding of complement component 1q (C1q) to the crystallizable fragment (Fc) region of the antibody. This initiates a complement cascade reaction resulting in the formation of a membrane attack complex that damages the cell membrane of target cells expressing target proteins recognized by the Fab region of the antibody. In this example, the CDC activity of Hu24F8.2-IgG1 was characterized using GS-J1/TIGIT cells (GenScript M00693) in the presence of Normal Human Serum Complement (NHSC, Quidel). Cell lysis by CDC was determined using the Cell Titer-Glo® Assay Kit (Promega), which determines the number of viable cells in culture based on ATP quantification. Briefly, GS-J1/TIGIT cells (5000 cells/well) were treated with 10 μg/ml Hu24F8.2-IgG1 or human IgG1 (Abcam) as a negative control in the presence of 5%, 10%, or 20% NHSC at 37°C with 5% _CO2 for 4 h (%NHSC optimization assay) or GSJ1/TIGIT cells (5000 cells/well) were treated with serial dilutions of Hu24F8.2-IgG1 (10 μg/ml) or human IgG1 (10 μg/ml) in the presence of 5% NHSC at 37°C with 5% _CO2 for 4 h (CDC concentration-dependent assay). As a positive control, Raji cells (ATCC CCL-86 (5000 cells/well)) were treated with serial dilutions of rituximab (10 μg/ml) in the presence of 5% NHSC at 37°C with 5% CO2 for 4 h. After incubation with test or control antibodies, Cell Titer-Glo® reagent was added, samples were incubated for an additional 10-30 min at room temperature and luminescence was read on a PHERAstar FSX (BMG LabTech).

Cell lysis due to CDC was calculated using the following formula:

% cell lysis = 100% x (1 - (RLU (luminescence signal intensity, relative light ^units) Samp|e ^{- RL} UNHSC ^{) / (RL} UCell + nhsc ^- RLUNHSC)).

The results of the systemic control (rituximab vs. Raji cells) were within the quality control standard in both assays. However, concentration-dependent CDC activity of the test sample (Hu24F8.2-IgG1) or the negative control (human IgG1) was not observed in either the %NHSC optimization assay or the concentration-dependent CDC assay. The results are presented in Tables 16 and 17, respectively. FACS analysis of Hu24F8.2-IgG1 binding to GS-J1/TIGIT cells was used to confirm that the absence of CDC was not due to lack of target cell binding (data not shown).

Table 16

Results of the %NHSC optimization analysis

Sample Conc, (mcg/ml) Target cells % NHSC Average % lysis of target cells ECso (ng/ml) Rituximab 10 Raji 5% 93.9 0.414 Hu24F8.2-IgGl 10 GS-J1/TIGIT 5% 5.77 N/A 10% 17,5 20% -15.5 Human IgGl 10 GS-J1/TIGIT 5% 5.39 N/A 10% 8.44 20% -12.3

Table 17

CDC Study Results by Concentration

Sample Highest conc. (mcg/ml) Target cell % NHSC ECso (ng/ml) Rituximab 10 Raji 5% 0.275 Hu24F8.2-IgGl 10 GS-J1/TIGIT 5% N/A Human IgGl 10 GS-J1/TIGIT 5% N/A

Example 9. Characterization of an antibody using SPR

Hu24F8.2-IgG1 was analyzed by SPR using a BioRad ProteOn XPR36 instrument.

- 53 049282 with antibody capture using an anti-human IgG or protein A chip coated with six different densities. The analyte was soluble hTIGIT-His (stock solution prepared at 33.3 μM) which was diluted to 33 nM as the highest concentration and tested in triplicate in a three-fold dilution series over the surface of Hu24F8.2-IgG1. The running buffer contained 10 mM HEPES, 150 mM NaCl, 0.05% Tween 20 and 0.2 mg/ml BSA. All data were collected at 25°C. Data obtained using all six surface densities were globally fitted to a 1:1 interaction model using local maxima of Rmax. The results are shown in Table 18.

Table 18

Kinetic constants of Hu24F8.2-IgG1 binding to human TIGIT

Method of capture ka (M'V ¹ ) kd(s' ¹ ) Kd (M) Anti-human IgG 2,425(6)E+06 5.97(3)E-05 2.46(1)E-11 Protein A 1,671(6)E+06 6.31(4)E-05 3.77(3)E-11

Note: Values in parentheses represent standard errors of the last reported values obtained from a global fit of the data to surfaces with 6 different densities.

Claims (28)

1. An antibody to a T-cell immunoreceptor with Ig and ITIM domains (TIGIT), or an antigen-binding fragment thereof, which specifically binds to human TIGIT and comprises a heavy chain variable region comprising an HC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 48, an HC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 49, and an HC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 50; and a light chain variable region comprising an LC-CDR1 having an amino acid sequence that comprises SEQ ID NO: 51, an LC-CDR2 having an amino acid sequence that comprises SEQ ID NO: 52, and an LC-CDR3 having an amino acid sequence that comprises SEQ ID NO: 53. 2. An antibody to TIGIT or an antigen-binding fragment thereof according to claim 1, comprising (a) a heavy chain variable region having an amino acid sequence that comprises SEQ ID NO: 5, and a light chain variable region having an amino acid sequence that comprises SEQ ID NO: 6; (b) a heavy chain variable region having an amino acid sequence that comprises SEQ ID NO: 76 and a light chain variable region having an amino acid sequence that comprises SEQ ID NO: 77; (c) a heavy chain variable region having an amino acid sequence that comprises SEQ ID NO: 78 and a light chain variable region having an amino acid sequence that comprises SEQ ID NO: 77; (d) a heavy chain variable region having an amino acid sequence that comprises SEQ ID NO: 76 and a light chain variable region having an amino acid sequence that comprises SEQ ID NO: 79; or (e) a heavy chain variable region having an amino acid sequence that comprises SEQ ID NO: 78 and a light chain variable region having an amino acid sequence that comprises SEQ ID NO: 79. 3. An antibody to TIGIT or an antigen-binding fragment thereof according to claim 1 or 2, wherein said antibody to TIGIT is a monoclonal antibody. 4. An antibody to TIGIT or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein said antibody to TIGIT is a chimeric, humanized or veneered antibody. 5. An antibody to TIGIT or an antigen-binding fragment thereof according to claim 4, wherein the chimeric antibody comprises a human IgG1/kappa Fab constant domain. 6. An antibody to TIGIT or an antigen-binding fragment thereof according to claim 1 or 2, wherein said antibody to TIGIT is a human antibody. 7. An anti-TIGIT antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said anti-TIGIT antibody or antigen-binding fragment thereof inhibits the binding of TIGIT to CD155. 8. An antibody to TIGIT or an antigen-binding fragment thereof according to any one of claims 1-4 or 6, 7, wherein said antibody further comprises a variant heavy chain constant region having an enhanced or weakened effector function compared to the wild-type heavy chain constant region, selected from a variant human IgG1, a variant human IgG2, - 54 049282 variant human IgG3 or variant human IgG4, and a human light chain constant region. 9. The TIGIT antibody or antigen-binding fragment thereof according to claim 8, wherein the variant constant region of the human IgG heavy chain comprises SEQ ID NO: 97, SEQ ID NO: 99 or SEQ ID NO: 101. 10. An antibody to TIGIT or an antigen-binding fragment thereof according to any one of claims 1-3 or 6-8, wherein said antibody additionally comprises a wild-type constant region of a human IgG heavy chain and a constant region of a human light chain. 11. An antibody to TIGIT or an antigen-binding fragment thereof according to claim 10, wherein the wild-type constant region of the human IgG heavy chain comprises SEQ ID NO: 94. 12. An antibody to TIGIT or an antigen-binding fragment thereof according to claim 10 or 11, comprising a human light chain kappa constant region, wherein the human light chain constant region comprises SEQ ID NO: 95. 13. The TIGIT antibody or antigen-binding fragment thereof according to claims 1-3 or 6-8, wherein said antibody has a heavy chain and a light chain, wherein (a) the heavy chain has an amino acid sequence comprising SEQ ID NO: 92, and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (b) the heavy chain has an amino acid sequence comprising SEQ ID NO: 96, and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (c) the heavy chain has an amino acid sequence comprising SEQ ID NO: 98, and the light chain has an amino acid sequence comprising SEQ ID NO: 93; or (d) the heavy chain has an amino acid sequence comprising SEQ ID NO: 100, and the light chain has an amino acid sequence comprising SEQ ID NO: 93. 14. The TIGIT antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein said antibody or binding fragment thereof (a) has an equilibrium binding constant (KD) of from 0.01x10’11 M to 100x10’11 M, from 0.1x10’11 M to 100x10’11 M, from 0.1x10’11 M to 10x10’14, from 1x10’11 M to 100x10’11 M, or from 1x10’11 M to 10x10’11 M, measured by surface plasmon resonance; (b) blocks the binding of soluble human CD155 ligand to human TIGIT on the cell surface with a half-maximal inhibitory concentration (IC50) of 0.2 nM to 2 nM, 0.2 nM to 0.8 nM, 0.4 nM to 0.8 nM, or 0.6 nM to 0.8 nM; (c) binds to an epitope that includes at least the following TIGIT residues: T55, Q56, N58, E60, D72, S80 and K82 of SEQ ID NO: 80; or (d) any combination of (a), (b) and (c). 15. An antibody to TIGIT or an antigen-binding fragment thereof that specifically binds to human TIGIT and comprises a heavy chain having an amino acid sequence comprising SEQ ID NO: 92 and a light chain having an amino acid sequence comprising SEQ ID NO: 93. 16. A method of inhibiting the binding of TIGIT to CD155, comprising contacting TIGIT with an anti-TIGIT antibody or antigen-binding fragment thereof according to any one of the preceding claims. 17. A method for treating or preventing cancer, comprising administering to a subject having cancer or at risk of developing cancer an effective regimen or a therapeutically effective amount of any of the TIGIT antibodies or antigen-binding fragments thereof according to any of the preceding claims, wherein said effective regimen designates a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration and/or alleviates at least one symptom of cancer. 18. The method according to claim 17, wherein the subject is further administered tumor-infiltrating T cells that are activated by said antibody or antigen-binding fragment thereof. 19. The method according to claim 17 or 18, wherein the subject is additionally administered natural killer cells whose anti-cancer cytotoxicity is enhanced by said antibody or antigen-binding fragment thereof. 20. The method according to any one of claims 17-19, wherein the subject is additionally administered a second antibody against an antigen expressed on the surface of cancer cells, as a result of which the effector-mediated anti-cancer cytotoxicity of the second antibody is enhanced by said antibody or antigen-binding fragment thereof. 21. The method according to any one of claims 17-19, wherein the subject is additionally administered a second antibody against an antigen expressed on the surface of an immune cell. 22. The method according to claim 21, wherein the immune cell is a T cell or a natural killer cell. 23. The method according to claim 20 or 21, wherein the antigen is CTLA-4, PD-1 or PD-L1. 24. The method according to any one of claims 17-23, wherein the subject is further administered one or more types of therapy selected from the group consisting of chemotherapy, radiation therapy, cell therapy, and - 55 049282 surgical intervention. 25. The method according to any one of claims 17-24, wherein the subject is further administered an inhibitor of one or more immune checkpoint receptors or ligands. 26. The method according to claim 25, wherein one or more receptors or ligands that are immune checkpoints are selected from the group consisting of CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, PVRIG, BTLA, VISTA, CD96, A _2a R, A _2b R, A _2a /A _2b R, arginase, CD39, CD73, IDO and TDO. 27. The method according to claim 25, wherein the inhibitor is selected from the group consisting of ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tislelizumab, zimberelimab, durvalumab and atezolizumab. 28. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.