Antibodies to tigit and use thereof

Abstract

This invention provides antibodies that specifically bind to TIGIT. These antibodies have the ability to substantially activate T cells and natural killer cells by inhibiting the binding of TIGIT to CD155. Among other applications, these antibodies can be used to treat cancer and infectious diseases.

Description

[Technical Field]

[0001] This article provides, in particular, antibodies that specifically bind to TIGIT, and their use, among other applications, in the treatment of cancer and infectious diseases. [Previous Technology]

[0002] In tumors, there exists a highly inhibitory microenvironment in which the function of T cells and NK cells is regulated by cell surface checkpoint receptors, allowing cancer cells to evade the immune system. Functional blockade of inhibitory checkpoint receptors, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and planned cell death 1 (PD-1), has yielded encouraging results for patients, thus generating substantial interest in seeking additional co-inhibitory molecules that can serve as potential interfering targets.

[0003] TIGIT (a T-cell immune receptor with Ig and ITIM domains) is a member of the immunoglobulin family with an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail region. It is a co-inhibitory receptor involved in the expression of regulatory T cells (Tregs), activated T cells, and natural killer (NK) cells. Increased TIGIT expression has been reported in CD8+ tumor-infiltrating lymphocytes (TILs) and Tregs in various tumors. Higher levels of TIGIT have also been reported in effector CD8+ T cells during HIV infection in the blood and SIV infection in lymphoid tissues. Furthermore, TIGIT blockade exhibits activating activity in human T-cell cultures and demonstrates therapeutic benefit in animal models of various tumors. Therefore, TIGIT plays an important role in anti-tumor immunity and could serve as a promising therapeutic target for controlling cancer and various other diseases and conditions. Therefore, molecules that can interfere with TIGIT binding are needed for beneficial therapeutic purposes. [Summary of the Invention]

[0004] This invention relates particularly to anti-TIGIT antibodies.

[0005] This document provides an anti-TIGIT antibody or an antigen-binding fragment thereof, comprising (a) a heavy chain variable region, the heavy chain variable region comprising a heavy chain (HC) complementarity determination region (CDR) 1 having at least 80% sequence identity with SEQ ID NO: 36, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 37, and an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 38; and a light chain variable region, the light chain variable region comprising a light chain (LC) CDR1 having at least 80% sequence identity with SEQ ID NO: 39, an LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 40, and an LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 41; (b) a heavy chain variable region, the heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 42, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 49, an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 40, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 41, and an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 42, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 49, an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 49 ... 43 HC-CDR2 with at least 80% sequence identity and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 44; and a light chain variable region comprising LC-CDR1 with at least 80% sequence identity to SEQ ID NO: 45, LC-CDR2 with at least 80% sequence identity to SEQ ID NO: 46, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 47; (c) a heavy chain variable region comprising HC-CDR1 with at least 80% sequence identity to SEQ ID NO: 48, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 49, and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 50; and a light chain variable region comprising LC-CDR1 with at least 80% sequence identity to SEQ ID NO: 51, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 44, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 45, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 46, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 47; 52 LC-CDR2 with at least 80% sequence identity and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 53; (d) Heavy chain variable region, the heavy chain variable region including HC-CDR1 with at least 80% sequence identity to SEQ ID NO: 54, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 55 and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 56;and a light chain variable region, the light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 57, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 58, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 59; (e) a heavy chain variable region, the heavy chain variable region comprising HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 60, HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 61, and HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 62; and a light chain variable region, the light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 63, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 64, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 69. 65 LC-CDR3 with at least 80% sequence identity; (f) heavy chain variable region, which includes HC-CDR1 with at least 80% sequence identity to SEQ ID NO: 60, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 66, and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 67; and light chain variable region, which includes LC-CDR1 with at least 80% sequence identity to SEQ ID NO: 63, LC-CDR2 with at least 80% sequence identity to SEQ ID NO: 68, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 65; (g) heavy chain variable region, which includes HC-CDR1 with at least 80% sequence identity to SEQ ID NO: 69, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 55, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 69. 70 HC-CDR3 having at least 80% sequence identity; and a light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 71, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 68, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 65; (h) a heavy chain variable region comprising HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 72, HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 73, and HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 67;and a light chain variable region, the light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 63, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 68, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 65; or (i) a heavy chain variable region, the heavy chain variable region comprising HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 74, HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 75, and HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 67; and a light chain variable region, the light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 63, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 68, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 65.

[0006] In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment includes (a) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 1; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 2; (b) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 3; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 4; (c) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 5; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 6; (d) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 7; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 8; (e) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 9; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 10; (f) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 10; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 10. NO: 11 is a heavy chain variable region with at least 80% sequence identity; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 12; (g) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 13; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 14; (h) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 15; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 16; (i) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 17; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 12; (j) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 76; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 77; (k) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 12; 78 is a heavy chain variable region with at least 80% sequence identity; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 77; (l) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 76; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 79; or (m) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 78; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 79.

[0007] This document provides an anti-TIGIT antibody or an antigen-binding fragment thereof bound to an antigenic determinant comprising 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 antigenic determinant comprising at least one of 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 antigenic determinant comprising at least the following TIGIT residues: E60 and D72 of SEQ ID NO: 80, and, if applicable, 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 antigenic determinant comprising at least the following TIGIT residues: D72 and K82 of SEQ ID NO: 80, and, if present, at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an antigenic determinant comprising at least the following TIGIT residues: E60, D72, and K82 of SEQ ID NO: 80, and, if present, at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80. In some embodiments, the antibody has structural features of the CDR and variable sequences as described herein.

[0008] The anti-TIGIT antibody of the present invention may be an isolated antibody. In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment is a monoclonal antibody. In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment is a chimeric, humanized, or faceted antibody. In some embodiments, the chimeric antibody includes a human IgG1 / κ Fab constant domain. In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment is a human antibody. In some embodiments, the anti-TIGIT antibody or its antigen-binding fragment inhibits the binding of TIGIT to CD155.

[0009] This document provides a pharmaceutical composition comprising an anti-TIGIT antibody as described herein and a pharmaceutically acceptable carrier.

[0010] This document provides a method for treating or preventing cancer, comprising administering to an individual with cancer or at risk of cancer an effective therapeutic or therapeutically effective dose of any anti-TIGIT antibody described herein. In some embodiments, the cancer is a blood cancer. 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 cancer, 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, tumor-infiltrating T cells are also administered to the individual. In some embodiments, a vaccine that induces an immune response against cancer is also administered to the individual. In some embodiments, the vaccine includes an antigen or fragment thereof expressed on the surface of cancer cells. In some embodiments, natural killer cells are also administered to the individual, the cytotoxicity against cancer being enhanced by the antibody. In some embodiments, the individual is further administered a second antibody that specifically binds to an antigen expressed on the surface of cancer cells, thereby enhancing the cytotoxicity mediated by the effector of the second antibody against cancer by means of the anti-TIGIT antibody of the present invention. In some embodiments, the individual is further administered a second antibody that specifically binds to an antigen expressed on the surface of immune cells. In some embodiments, the immune cell line is T cells or natural killer cells. In some embodiments, the antigen is CTLA-4, PD-1, or PD-L1. In some embodiments, the individual is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapies, and surgery. In some embodiments, the individual is further administered one or more inhibitors of immune checkpoint receptors or ligands. In some embodiments, 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, A2aR, A2bR, A2a / A2bR, arginase, CD39, CD73, IDO, and TDO. In some embodiments, 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, tislelizumab, zimberelimab, durvalumab, and atezolizumab.

[0011] This document provides a method for aiding in the treatment of cancer, comprising administering to an individual suffering from cancer a therapeutically effective amount of any of the anti-TIGIT antibodies described herein. In some embodiments, the cancer is a blood cancer. 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 cancer, 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 individual is also administered tumor-infiltrating T cells activated by the antibody. In some embodiments, the individual is also administered a vaccine that induces an immune response against cancer, the immune response being enhanced by the antibody. In some embodiments, the vaccine includes an antigen or fragment thereof expressed on the surface of cancer cells. In some embodiments, the individual is also administered natural killer cells whose cytotoxicity against cancer is enhanced by the anti-TIGIT antibody of the present invention. In some embodiments, an individual is also administered a second antibody that specifically binds to an antigen expressed on the surface of cancer cells, thereby enhancing the cytotoxicity mediated by the effector of the second antibody against cancer using the anti-TIGIT antibody of the present invention. In some embodiments, an individual is further administered a second antibody that specifically binds to an antigen expressed on the surface of immune cells. In some embodiments, the immune cell line is T cells or natural killer cells. In some embodiments, the antigen is CTLA-4, PD-1, or PD-L1. In some embodiments, an individual is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapies, and surgery. In some embodiments, an individual is further administered one or more inhibitors of immune checkpoint receptors or ligands. In some embodiments, 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, A2aR, A2bR, A2a / A2bR, arginase, CD39, CD73, IDO, and TDO. In some embodiments, 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, tramemumab, nivolumab, pembrolizumab, larizumab, cimiprizumab, tislelizumab, cepallimumab, durvalumab, and atezolizumab.

[0012] Unless explicitly or clearly excluded in the context of the embodiments or patterns, each of the patterns and embodiments described herein can be used together.

Implementation Method

[0013] This application claims priority to U.S. Provisional Application No. 63 / 033,609, filed June 2, 2020, the disclosure of which is incorporated herein by reference in its entirety, including any figures.

[0014] This application contains a sequence list, which is incorporated herein by reference in its entirety. The accompanying sequence list text file, named 050658_531001WO_Sequence_Listing_ST25, was created on May 25, 2021, and is 143 KB in size.

[0015] This invention particularly provides antibodies that specifically bind to the extracellular domain of TIGIT. The antibodies of this invention, also referred to herein as "anti-TIGIT antibodies," inhibit the binding of TIGIT to CD155 and thereby activate T cells and / or NK cells. These antibodies can also be used to treat cancer and infectious diseases, as well as for other applications. Further detailed descriptions of the structural and functional characteristics of the anti-TIGIT antibodies of this invention are provided below.

[0016] I. Definitions Unless otherwise defined, all technical terms, symbols, and other scientific terms or sets of terms used herein are intended to have the meanings commonly understood by those skilled in the art relating to this invention. In some cases, for clarity and / or convenience of reference, terms are defined herein with their commonly understood meanings, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the general understanding in this art. Those skilled in the art will readily understand and generally use known methodologies to employ many of the techniques and procedures described or referenced herein.

[0017] Unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include a plural reference. For example, the term “cell” includes one or more cells, including mixtures thereof. In this document, “A and / or B” is used to include all the following alternative forms: “A”, “B”, “A or B”, and “A and B”.

[0018] The term "antibody" includes complete antibodies and their binding fragments that specifically bind to a single antigen or specifically bind to multiple antigens (e.g., multispecific antibodies, such as bispecific antibodies, trispecific antibodies, etc.). Therefore, unless otherwise specified herein, any reference to antibody should be understood to refer to an antibody or its binding fragment in its complete form. Additional functionalities (e.g., antigen binding) covered in the context of this invention include anti-PD-1, anti-PD-1I, anti-TIM-3, anti-LAG-3, anti-PVRIG, anti-VISTA, anti-CTLA-4, anti-4-1BB, anti-BTLA, anti-CD39, anti-CD73, anti-OX40L, and anti-OX40 fragments.

[0019] The term "binding fragment," which may be used interchangeably with "antigen-binding fragment," refers herein to an antibody fragment formed by any other antibody fragment that is partially or specifically bound to an antigen but does not contain the complete structure of the native antibody, and which contains one or more CDRs. Examples of antigen-binding fragments include (but are not limited to) bifunctional antibodies, Fab, Fab', F(ab')2, F(ab)c, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), trifunctional antibodies, tetrafunctional antibodies, single-chain antibody molecules (scFv), scFv dimers, multispecific antibodies, camel single-domain antibodies, nanoantibodies, microantibodies, domain antibodies, bivalent domain antibodies, IgNAR, V-NAR, and hcIgG. Typically, the binding fragment competes for specific binding with the complete antibody from which it is derived. The binding fragments can be generated by recombinant DNA technology or by enzymatic or chemical separation of intact immunoglobulins.

[0020] The “Fab” in antibody refers to a portion of the antibody composed of a single light chain (variable region and constant region), which is bound to the variable region and the first constant region of the single heavy chain by disulfide bonds.

[0021] "Fab" refers to a Fab segment that includes a portion of the hinge area.

[0022] "F(ab')2" refers to the dimer of Fab'.

[0023] The "Fc" of an antibody refers to the portion of the antibody composed of the second and third constant regions of the first heavy chain, which binds to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc portion of the antibody is responsible for various effector functions, such as ADCC and CDC, but does not participate in antigen binding.

[0024] The "Fv" in antibody refers to the smallest fragment in the antibody that carries the complete antigen-binding site. The Fv fragment consists of a variable region of a single light chain that binds to a variable region of a single heavy chain.

[0025] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of light chain variable regions and heavy chain variable regions that are directly or indirectly linked together by peptide linker sequences (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0026] "Single-chain Fv-antibody" or "scFv-Fc" refers to an engineered antibody composed of scFvs linked to the Fc region of the antibody.

[0027] “Camel single-domain antibody”, “heavy chain antibody”, or “HCAb” refers to an antibody containing two VH domains and no light chain (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 originally came from the camelid family (camel, dromedary camel and llama). Although they do not contain light chains, camel antibodies possess a reliable antigen-binding spectrum (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 (VHH domain) of heavy chain antibodies represents 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)).

[0028] "Nano antibody" refers to an antibody fragment composed of the VHH domain and two constant domains CH2 and CH3 from a heavy chain antibody.

[0029] A "bifunctional antibody" comprises a small antibody fragment having two antigen-binding sites, wherein the fragment includes a VH domain linked to a VL domain (VH-VL or VL-VH) on the same polypeptide chain (see, for example, Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domain is forced to pair with a complementary domain on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or antigenic determinants).

[0030] "Domain antibody" refers to an antibody fragment containing only the heavy chain variable region or the light chain variable region. In some cases, two or more VH domains are covalently linked by peptide linkers to produce bivalent or multivalent domain antibodies. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0031] In some embodiments, "(dsFv)2" comprises three peptide chains: two VH moieties are linked by peptide linkers and are attached to two VL moieties by disulfide bridging bonds.

[0032] In some embodiments, the loaded bifunctional antibody contains VH1-VL2 (linked by a peptide linker), which is bound to VL1-VH2 (also linked by a peptide linker) via a disulfide bridge between VH1 and VL1.

[0033] In some embodiments, the "bispecific dsFv" or "dsFv-dsFv'" comprises three peptide chains: VH1-VH2 portions, wherein the heavy chains are linked by peptide linkers (e.g., long flexible linkers) and are respectively bound to the VL1 and VL2 portions via disulfide bridging bonds, wherein each disulfide pair of heavy and light chains has different antigen specificities.

[0034] In some embodiments, the "scFv dimer" is a bivalent bifunctional antibody or a bivalent scFv (BsFv) comprising a VH-VL moiety dimer such that one VH' coordinates to the other VL' and forms two binding sites (linked by peptide linkers) that can target the same antigen (or antigenic determinant) or different antigens (or antigenic determinants). In other embodiments, the "scFv dimer" is a bispecific bifunctional antibody comprising VH1-VL2 (also linked by peptide linkers) associated with VL1-VH2 (linked by peptide linkers), such that VH1 and VL1 coordinate and VH2 and VL2 coordinate, and each coordination pair has different antigen specificity. An "isolated" antibody is an antibody that has been isolated from its components in its natural environment. In some embodiments, the isolated antibody is purified to a purity greater than 95% or 99%, as determined by methods known in this art.

[0035] As used herein, “monoclonal antibody” means an antibody derived from a single copy or pure line (including any eukaryotic, prokaryotic, or phage pure line). “Monoclonal antibody” is not limited to antibodies produced by any particular method. For example, monoclonal antibodies can be produced using fusion tumor technology and recombinant technology, phage presentation technology, synthetic technology, or combinations of such technologies and other techniques well known in this field.

[0036] As used herein, the term "humanized antibody" refers to an antibody containing sequences derived from human and non-human (e.g., mouse or rat) antibodies.

[0037] As used herein, the term "human antibody" means an antibody having or consisting of one or more amino acid sequences, specifically antigen-binding residues, corresponding to the amino acid sequences of antibodies produced by humans or human immune cells, or derived from non-human sources, such as transgenic non-human animals utilizing human antibody lineages or other human antibody coding sequences. In some embodiments, a fully human antibody does not contain amino acid residues (specifically antigen-binding residues) derived from non-human antibodies.

[0038] For example, tetramers are subunits of the basic antibody structural unit exemplified by natural intact antibodies. Each tetramer comprises two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region having approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. This variable region initially appears to be bonded to a cleavable signal peptide. Variable regions without a signal peptide are sometimes referred to as mature variable regions. Thus, for example, a light chain mature variable region means a light chain variable region without a light chain signal peptide. The carboxyl-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0039] Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, and antibody isotypes are defined as IgG, IgM, IgA, IgD, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by “J” regions having about 12 or more amino acids, with the heavy chain also including “D” regions having about 10 or more amino acids. (See Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989), Ch. 7) (Incorporated in full for all purposes).

[0040] The maturation variable regions of each light / heavy chain pair form antibody binding sites. Therefore, a complete natural antibody has two identical binding sites; a bispecific antibody has two different binding sites; a trispecific antibody has three different binding sites; and so on. The maturation variable regions of both the heavy and light chains exhibit the same universal structure of relatively conserved framework regions (FRs) joined by three hypervariable regions (also known as complementarity-determining regions or CDRs). The CDRs of each pair are aligned via the framework regions, enabling them to bind to specific antigenic determinants. From the N-terminus to the C-terminus, both the light and heavy chains include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to their respective domains was based on the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues of different heavy chains or different light chains are assigned the same number.

[0041] The term "antigenic determinant" refers to the site on an antigen where an antibody binds. Antigenic determinants can be formed by linked or unlinked amino acids arranged side-by-side through the ternary folding of one or more proteins. Antigenic determinants formed by adjacent amino acids (also known as linear antigenic determinants) are generally preserved when exposed to denaturing solvents, while antigenic determinants formed by ternary folding (also known as conformational antigenic determinants) are generally unaffected by denaturing solvent treatment. Antigenic determinants typically include at least three, and more often at least five or eight to ten amino acids in a distinctive spatial conformation. Methods for determining the spatial conformation of antigenic determinants include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, ed. (1996).

[0042] Antibodies that identify identical or overlapping antigenic determinants can be identified in a simple immunoassay that shows the ability of one antibody to competitively bind to a target antigen. The antigenic determinant of an antibody can also be defined by X-ray crystallography of its binding to its antigen to identify contact residues. Alternatively, if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the two antibodies have identical antigenic determinants. If some, but not all, amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other antibody, then the two antibodies have overlapping antigenic determinants.

[0043] Competition between antibodies is determined by an analysis in which, under test conditions, the antibody inhibits the specific binding of the reference antibody to the common antigen (see, for example, Junghans et al., Cancer Res. 50:1495, 1990). If an excess of the test antibody (e.g., at least 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, 60×, 70×, 80×, 90×, 100× or more, including values ​​between these equivalents) inhibits the binding of the reference antibody by at least about 50%, such as at least about 75%, 90% or 99%, then the test antibody competes with the reference antibody. In other embodiments, as measured in a competitive binding assay, if an excess of the test antibody inhibits the binding of the reference antibody 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%, then the test antibody competes with the reference antibody. Antibodies identified by the competitive assay (competitive antibody) include antibodies that bind to the same antigenic determinant as the reference antibody and antibodies that bind to adjacent antigenic determinants sufficiently close to the antigenic determinant bound by the reference antibody to cause steric hindrance. The reference antibody may be a commercially available monoclonal antibody with function similar to a therapeutic antibody candidate, a polyclonal antibody that functionally interacts with a relevant target protein, or an antibody reconstructed from a sequence available in the public domain. For example (but not limited to), a reference antibody bound to TIGIT comprises a heavy chain having an amino acid sequence containing SEQ ID NO: 92 and a light chain having an amino acid sequence containing SEQ ID NO: 93.

[0044] As used herein, the terms "specific binding" and "specifically bound to" refer to measurable and reproducible interactions, such as the binding between an antigen (e.g., TIGIT) and an antibody. For example, an antibody that specifically binds to an antigen is an antibody that binds to the target with a higher affinity, affinity, ease, and / or longer duration than it binds to other antigens. The affinity of an antigen is inversely proportional to the equilibrium dissociation constant (KD) of the molecule. High affinity for an antigen is measured by a low KD value. As used herein, by means of surface plasma resonance, antibodies that specifically bind to an antigen have a KD (kilogram-distribution) against the antigen of 10⁻⁶ M or less, or 10⁻⁷ M or less, or 10⁻⁸ M or less, or 10⁻⁹ M or less, or 10⁻¹⁰ M or less, or 10⁻¹¹ M or less; 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. In one embodiment, the term "specific binding" means binding in which a molecule binds to a specific polypeptide or an antigenic determinant of a specific polypeptide and substantially does not bind to any other polypeptide or polypeptide antigenic determinant.

[0045] As used herein, "individual" or "subject" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, "individual" or "subject" is a patient under the care of a physician. Thus, an individual can be a human patient or individual who has or has, is at risk of having, or is suspected of having one or more symptoms of a related disease (e.g., cancer) and / or disease. An individual can also be an individual who is at risk of having a related condition at the time of diagnosis or later. The term "non-human animal" includes all vertebrates, such as mammals, such as rodents, such as mice, non-human primates and other mammals, such as sheep, dogs, cattle, chickens, and non-mammals, such as amphibians, reptiles, etc.

[0046] As used herein, "corresponds to" or "corresponding to" an amino acid residue in a reference amino acid sequence or "correspondence with" an amino acid residue in a related amino acid sequence indicates that the amino acid residue in the related sequence is located at a position homologous or equivalent to the residues counted in the reference amino acid sequence. Those skilled in this art can determine whether a specific amino acid residue position in a polypeptide such as the TIGIT polypeptide corresponds to an amino acid residue position in a homologous reference sequence. For example, the sequence of the TIGIT polypeptide can be aligned with a reference sequence using known techniques (e.g., the basic local alignment search tool (BLAST), ClustalW2, the structure-based sequence alignment program (STRAP), or similar). Additionally, the crystal structure coordinates of a reference sequence can be used to help determine the three-dimensional structure of homologous peptide residues (Stengel et al., Proc. Natl. Acad. Sci. USA, 109:5399-5404, 2012). In another approach, equivalent residues can be identified by determining homology at the tertiary level. Using such methods, the amino acid residues of TIGIT peptide variants can be numbered according to the corresponding amino acid residue positions in the reference sequence. For example, the amino acid sequence of SEQ ID NO: 80 can be used to determine the amino acid residue positions of each amino acid residue in the relevant TIGIT variant or antigenic determinant. In some embodiments, an amino acid sequence that shares 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 corresponds to another amino acid sequence.

[0047] For the purpose of classifying amino acid substitutions as conserved 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 substitution involves substitution between amino acids within the same category. Non-conservative substitution constitutes the replacement of one member of one of these categories with another member.

[0048] The sequence identity percentage is determined by maximizing the alignment of antibody sequences using the Kabat numbering protocol. After alignment, if the region of the original antibody (e.g., the entire mature variable region of the heavy or light chain) is compared with the same region of the reference antibody, the sequence identity percentage between the original antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acid in both regions by the total number of comparison positions in the two regions (excluding gaps), and multiplying by 100 to convert it to a percentage.

[0049] The word "comprising" or "including" or any grammatical variation thereof, a composition or method comprising one or more of the listed elements may include other elements not specifically listed. For example, a composition comprising an antibody may contain an antibody alone or in combination with other ingredients.

[0050] Where the term "approximately" precedes a numerical value, this document presents a specific range. The term "approximately" as used herein has its original meaning and provides textual support for both the exact numerical value preceding it and the numerical value preceding the term that is close to or approximates it. In determining whether a numerical value is close to or approximates a particular stated numerical value, a value that is close to or approximates an unstated numerical value may be a value that is substantially equivalent to the numerical value of the particular statement provided in the context in which it is presented. For example, if the degree of approximation is not otherwise explicitly stated from the context, "approximately" means within 10% of the provided value, or rounded to the nearest significant number, in all cases including the provided value. Where a range is provided, it includes boundary values.

[0051] As used herein, the term "substantially" and any grammatical variations thereof are broad terms and are used in their general sense, including (but not limited to) almost entirely or largely, but not entirely. For example, the term may refer to a value that is not 100% of the total value, wherein the 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 total value. For example, when the antibody or its antigen-binding fragment described herein has 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% sequence identity with the corresponding reference antibody or its antigen-binding fragment, the antibody or its antigen-binding fragment may be substantially derived from the corresponding reference antibody or its antigen-binding fragment. In another example, when the CDR in the antibody described herein has 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% sequence identity with the corresponding CDR in the reference antibody, the CDR in the antibody may be substantially derived from the corresponding CDR in the reference antibody. In another instance (but not limited to), when the corresponding CDR in the reference antibody is replaced, deleted, or added to the CDR of the antibody in this article by no more than two amino acids, the CDR in this article may be substantially derived from the corresponding CDR in the reference antibody.

[0052] It should be understood that certain features of the invention described in the context of individual embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination. All combinations of embodiments of the invention are specifically encompassed by the invention and disclosed herein, just as each combination is individually and explicitly disclosed herein. In addition, all sub-combinations of various embodiments and their elements are also specifically covered by the invention and disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0053] II. Unless otherwise specified, TIGIT means human TIGIT (hTIGIT). Cyno TIGIT or cTIGIT refers to cynomolgus monkey TIGIT.

[0054] An exemplary hTIGIT sequence is designated as Swiss-Prot register number Q495A1. The complete hTIGIT sequence has 244 amino acids (SEQ ID NO: 80), of which amino acids 1-21 are signal peptides and 22-244 constitute 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 hTIGIT is HIS-labeled (SEQ ID NO: 83). An exemplary cynomolgus macaque TIGIT sequence is designated as Swiss-Prot A0A2K5UW92. The complete cynomolgus macaque TIGIT sequence has 312 amino acids (SEQ ID NO: 84). In some embodiments, the extracellular domain of the cynomolgus monkey TIGIT is HIS-tagged (SEQ ID NO: 85).

[0055] Unless otherwise indicated, CD155 refers to the human form of this protein. An exemplary human sequence of human CD155 is designated as Swiss-Prot P15151, which is a 417-amino acid protein in which approximately residues 1-20 constitute 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.

[0056] Unless the context clearly differs, reference to one of the above proteins means at least the extracellular domain of the protein and is usually an intact protein other than the cleavable signal peptide.

[0057] III. Antibodies of the Invention A. Binding Specificity and Functional Properties The present invention provides antibodies that specifically bind to TIGIT, and more particularly to antigenic determinants within the extracellular domain of the TIGIT protein. In some embodiments, the anti-TIGIT antibodies of the present invention have a KD of 10⁻⁸ M or lower (e.g., 10⁻⁸, 10⁻⁹, 10⁻¹⁰, etc.) against TIGIT, as measured by surface plasma resonance (SPR). In various embodiments, the anti-TIGIT antibodies of the present invention have a KD (knockout) against TIGIT in the following ranges: about 1×10⁻⁹ M to about 1×10⁻¹³ M, or about 1×10⁻⁹ M to about 1×10⁻¹² M, or about 1×10⁻¹⁰ M to about 1×10⁻¹³ M, or about 1×10⁻¹⁰ M to about 1×10⁻¹² M, or about 1×10⁻¹¹ M to about 1×10⁻¹³ M, or about 1×10⁻¹⁰ M to about 1×10⁻¹¹ M, or about 1×10⁻¹¹ M to about 1×10⁻¹² M. Nine such exemplary mouse antibodies are named 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, and 28O12. The antibodies named Ch22B22, Ch21B16, Ch28O12, Ch5J24, Ch21B9, Ch24F8, and Ch30M18 are seven exemplary chimeric antibodies of this type. The antibodies named Hu24F8.1, Hu24F8.2, Hu24F8.3, and Hu24F8.4 are exemplary humanized antibodies. The sequences of the heavy and light chain maturation variable regions and the CDRs of the mouse and humanized antibodies are shown in Tables 1 and 2, respectively. Table 1. Sequences of the heavy and light chain maturation variable regions Antibody Heavy chain variable region Light chain variable region 21F8 SEQ ID NO: 1 SEQ ID NO: 2 30M18 SEQ ID NO: 3 SEQ ID NO: 4 24F8 SEQ ID NO: 5 SEQ ID NO: 6 5J24 SEQ ID NO: 7 SEQ ID NO: 8 21B9 SEQ ID NO: 9 SEQ ID NO: 10 22B22 SEQ ID NO: 11 SEQ ID NO: 12 28P24 SEQ ID NO: 13 SEQ ID NO: 14 21B16 SEQ ID NO: 15 SEQ ID NO: 16 28O12 SEQ ID NO: 17 SEQ ID NO: 12 Hu24F8.1 SEQ ID NO: 76 SEQ ID NO: 77 Hu24F8.2 SEQ ID NO: 78 SEQ ID NO: 77 Hu24F8.3 SEQ ID NO: 78 SEQ ID NO: 79 Hu24F8.4 SEQ ID NO: 76 SEQ ID NO: 79 Table 2. Sequences of CDRs for Heavy and Light Chains (Kabat Definition) 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 28O12 74 75 67 63 68 65

[0058] Some antibodies of the present invention bind to the same or overlapping antigenic determinants as antibodies named 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12, or antibodies named Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4. Other antibodies with such binding specificity can be generated by immunizing mice with TIGIT or a portion thereof, including the desired antigenic determinant, and screening for antibodies that bind to the extracellular domain of TIGIT, which may compete with 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, 28O12, Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4. Antibodies can also be screened against the mutagenic form of the TIGIT antigen to identify antibodies with the same or similar binding profile to the mutated set, such as 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, 28O12, Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4. Mutations can occur simultaneously or at intervals in the entire extracellular domain of the TIGIT antibody or in the portion where the known antigenic determinant is present, replacing a residue with an alanine (or serine if alanine is present) system. In some embodiments, some antibodies of the present invention bind to at least one of the following TIGIT antigenic determinant 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 two, three, four, five, or six of the antigenic determinant residues of the following TIGIT: 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 antigenic determinant residues of the following TIGIT: T55, Q56, N58, E60, D72, S80, and K82. In some embodiments, anti-TIGIT antibodies bind to an antigenic determinant comprising at least one of 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 antigenic determinant comprising at least the following TIGIT residues: E60 and D72 of SEQ ID NO: 80, and, if present, 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 antigenic determinant comprising at least the following TIGIT residues: D72 and K82 of SEQ ID NO: 80, and, if present, at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80. In some embodiments, the anti-TIGIT antibody binds to an antigenic determinant comprising at least the following TIGIT residues: E60, D72, and K82 of SEQ ID NO: 80, and, if present, at least one of T55, Q56, N58, and S80 of SEQ ID NO: 80.

[0059] Antibodies having binding specificity to selected murine antibodies (e.g., 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12) or selected humanized antibodies (e.g., Hu24F8.1, Hu24F8.2, Hu24F8.3, or Hu24F8.4) can also be generated using variants of the phage presentation method. See Winter, WO 92 / 20791. This method is particularly suitable for generating human antibodies. In this method, the heavy or light chain variable region of the selected murine antibody is used as the starting material. For example, if the light chain variable region is chosen as the starting material, a phage library is constructed in which each member exhibits the same light chain variable region (i.e., the murine starting material) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of self-rearranged human heavy chain variable regions. Phages exhibiting strong specific binding to TIGIT (e.g., at least 10⁸ or at least 10⁹ M⁻¹) are selected. The heavy chain variable region of this phage then serves as starting material for constructing another phage library. In this library, each phage presents the same heavy chain variable region (i.e., the region identified from the first presented library) and different light chain variable regions. The light chain variable region can be obtained, for example, from a library of rearranged human variable light chain regions. Phages exhibiting strong specific binding to TIGIT are again selected. The resulting antibodies typically have the same or similar antigenic determinant specificity as the murine starting material.

[0060] Some antibodies have mature heavy chain variable regions, including HC-CDR1, HC-CDR2, and HC-CDR3, and mature light chain regions, including LC-CDR1, LC-CDR2, and LC-CDR3, which are entirely or substantially derived from mAb 21F8. Some antibodies have mature heavy chain variable regions, including HC-CDR1, HC-CDR2, and HC-CDR3, and mature light chain regions, including LC-CDR1, LC-CDR2, and LC-CDR3, which are entirely or substantially derived from mAb 30M18. Some antibodies have mature heavy chain variable regions, including HC-CDR1, HC-CDR2, and HC-CDR3, and mature light chain regions, including LC-CDR1, LC-CDR2, and LC-CDR3, which are entirely or substantially derived from mAb 24F8. Some antibodies have mature heavy chain variable regions, including HC-CDR1, HC-CDR2, and HC-CDR3, and mature light chain regions, including LC-CDR1, LC-CDR2, and LC-CDR3, derived entirely or substantially from mAb 5J24. Some antibodies have mature heavy chain variable regions, including HC-CDR1, HC-CDR2, and HC-CDR3, and mature light chain regions, including LC-CDR1, LC-CDR2, and LC-CDR3, derived entirely or substantially from mAb 22B22. Some antibodies have mature heavy chain variable regions, including HC-CDR1, HC-CDR2, and HC-CDR3, and mature light chain regions, including LC-CDR1, LC-CDR2, and LC-CDR3, derived entirely or substantially from mAb 28P24. Some antibodies have mature heavy chain variable regions, including HC-CDR1, HC-CDR2, and HC-CDR3, and mature light chain regions, including LC-CDR1, LC-CDR2, and LC-CDR3, derived entirely or substantially from mAb 28O12. CDR can be defined by any known definition, including Kabat, Chothia, a combination of Kabat and Chothia, AbM, or Contact, as shown in Table 3 below: Table 3. ring Kabat AbM Chothia connect L1 L-24--L34 L24--34 L24--L34 L30--L36 L2 L50--L56 L50--l56 L50---L56 L46--L55 L3 L89--L97 L89--97 L89--L97 L89--L96 H1 H31--H35B<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0061] Other antibodies can be obtained by mutation of the cDNA encoding the heavy and light chains of the exemplary antibody, such antibodies being 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16 or 28O12. Antibodies that retain the functional properties of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, or 100% of the amino acid sequence of the mature heavy chain and / or light chain variable region corresponding to any of 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12, and / or those that differ from individual antibodies by means of a few functionally disjoint amino acid substitutions (e.g., conserved substitutions), deletions, or insertions, are also included in this invention. Amino acids in the variable region framework that may be important for binding can be identified as described below in the section on humanization. It also includes antibodies having at least one and, in some embodiments, all six CDRs as defined by Kabat, wherein the CDR has approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conformity to the corresponding CDR of 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12.

[0062] In some embodiments, the antibody has one or more of the following characteristics: (i) inhibiting the binding of human TIGIT to human CD155; (ii) inhibiting the binding of TIGIT to other ligands such as CD112 and CD113; (iii) increasing antigen-specific T cell responses; (iv) activating natural killer cells; (v) stimulating intrinsic T cell activation; and (vi) stimulating the production of one or more immunostimulatory cytokines of T cells and other cells of the immune system and / or reducing the production of one or more immunosuppressive cytokines.

[0063] In some embodiments, the antibodies described herein completely or partially inhibit the binding of TIGIT to CD155. As measured in Example 1, the anti-TIGIT antibody of the present invention can inhibit such interaction at a half-maximum inhibitory concentration (IC50) of about 0.1 nM to about 10 nM, or about 0.1 nM to about 8 nM, or about 0.1 nM to about 5 nM, or about 0.1 nM to about 4 nM, or about 0.1 nM to about 3 nM, or about 0.1 nM to about 2 nM, or about 0.1 nM to about 1 nM. In some embodiments, as measured in Example 1, some of the anti-TIGIT antibodies of the present invention can inhibit the binding of TIGIT to CD155 at an IC50 of about 0.1 nM to about 2 nM or about 0.2 nM to about 2 nM. In some embodiments, as measured in Example 1, some of the anti-TIGIT antibodies of the present invention can inhibit the binding of TIGIT to CD155 with an IC50 of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM. As measured in Example 1, some antibodies can be detected at concentrations of approximately 25 to 300 ng / ml, 25 to 75 ng / ml, 25 to 50 ng / ml, 40 to 75 ng / ml, 50 to 75 ng / ml, 50 to 90 ng / ml, 50 to 100 ng / ml, 75 to 100 ng / ml, 50 to 150 ng / ml, 75 to 175 ng / ml, 100 to 200 ng / ml, 125 to 225 ng / ml, 100 to 250 ng / ml, 150 to 300 ng / ml, 175 to 250 ng / ml, 200 to 300 ng / ml, 25 to 275 ng / ml, 250 to 300 ng / ml, 49+ / -10% ng / ml, 65+ / -10% ng / ml, or 76+ / -10%. Such interactions are inhibited at a half-maximal inhibitory concentration (IC50) of any of the following ng / ml values. In other embodiments, the antibody can completely or partially inhibit the binding of TIGIT to CD155 at an IC50 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 greater, including concentrations falling within these equivalent values. Additionally, some antibodies can increase antigen-specific T-cell responses by 1.5 to 3-fold, such as 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 can increase the production of 1, 2, 3, or all IL-2, IL-6, TNFα, and IFNγ by NK cells and / or T cells by 1.5 to 3 times, such as approximately 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 can increase innate T cell activation by 1.5 to 3 times, such as approximately 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 can inhibit cancer or infectious diseases, as demonstrated in animal models or clinical trials. Animal cancer models, in which human cancer cells are injected into immunodeficient laboratory animals such as mice or rats, are widely available.

[0064] In an exemplary embodiment, the antibody specifically binds to TIGIT and includes a mature heavy chain variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 and a mature light chain region comprising LC-CDR1, LC-CDR2 and LC-CDR3, which are entirely or substantially derived from antibody 24F8. In various embodiments, the antibody may (i) have an equilibrium binding constant (KD) of about 0.01 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, about 0.1 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, about 0.1 × 10⁻¹¹ M to about 10 × 10⁻¹¹ M, about 1 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, as measured by surface plasma resonance, and / or (ii) block the binding of soluble human CD155 ligand to human TIGIT on the cell surface at a half-maximum inhibitory concentration (IC50) of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, as measured in Example 1. Alternatively, in addition to the individual binding and blocking properties or combinations thereof described above, in some embodiments, the antibody binds to an antigenic determinant comprising at least the following TIGIT residues: (i) at least one of D72 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60, S80, and K82 of SEQ ID NO: 80; (ii) at least one of E60 and D72 of SEQ ID NO: 80 and, if present, at least one of T55, Q56, N58, S80, and K82 of SEQ ID NO: 80; (iii) at least one of D72 and K82 of SEQ ID NO: 80 and, if present, at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80; (iv) at least one of E60, D72, and K82 of SEQ ID NO: 80 and, if present, at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80. At least one of T55, Q56, N58 and S80 of 80; or (v) SEQ ID NO: T55, Q56, N58, E60, D72, S80 and K82 of 80.

[0065] Compared to the initial mouse antibody, humanized or chimeric antibodies prolong the in vivo half-life. For example, in humans, the resulting half-life can be 10 to 50 days. The half-life can be measured by pharmacokinetic studies, as described by Kim et al., Eur J of Immunol 24:542 (1994).

[0066] B. Other non-human antibodies against TIGIT (e.g., rodent, guinea pig, primate, rabbit, chicken, or rat) can be produced by immunizing animals, for example, with TIGIT or fragments thereof or cells carrying TIGIT. See Harlow and Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such immunogens can be obtained from natural sources by peptide synthesis or by recombinant expression. Immunogens can be administered by fusion with or otherwise conjugation to a carrier protein, depending on the circumstances. Immunogens can be administered with an adjuvant, depending on the circumstances. Several types of adjuvants can be used as described below. Complete Freund's adjuvant can be used, followed by incomplete adjuvant, for immunization of laboratory animals. Rabbits or guinea pigs are typically used to produce multiclonal antibodies. Mice are typically used to produce monoclonal antibodies. Antibodies that specifically bind to TIGIT are screened. Depending on the situation, antibodies that bind to specific regions of TIGIT may be further screened. This screening can be achieved by measuring the binding of the antibody to a range of TIGIT deletion mutants and identifying which deletion mutants bind to the antibody. Binding can be assessed, for example, using Western blotting, FACS, or ELISA.

[0067] C. The reduction or elimination of HAMA (human anti-mouse (also applicable to human anti-rat or human anti-rabbit or human anti-hamster, etc.) antibody) response by humanized antibodies is a significant trend in the clinical development of suitable therapeutic agents. See, for example, 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 that have been humanized to reduce or eliminate the HAMA response. Variants of these antibodies can be further obtained using conventional methods known in this technique, some of which are further described below.

[0068] Humanized antibody systems are genetically engineered antibodies in which a CDR derived from a non-human "donor" antibody is transplanted into a human "recipient" antibody sequence (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 recipient antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a common sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all of the CDRs, entirely or substantially derived from the donor antibody, as well as a variable region framework sequence and a constant region (if present, entirely or substantially derived from the human antibody sequence). Similarly, the humanized heavy chain has at least one, two, and usually all three CDRs that are entirely or substantially derived from the donor antibody heavy chain, as well as the heavy chain variable region framework sequence and the heavy chain constant region (if present, substantially derived from the human heavy chain variable region framework and constant region sequence). Similarly, the humanized light chain has at least one, two, and usually all three CDRs that are entirely or substantially derived from the donor antibody light chain, as well as the light chain variable region framework sequence and the light chain constant region (if present, substantially derived from the human light chain variable region framework and constant region sequence). As elsewhere in this application, when at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the corresponding residues (as defined by Kabat) are identical among the individual CDRs, the CDRs in this antibody are substantially derived from the corresponding CDRs in the reference antibody; however, when at least approximately 65%, 66%, or 100% of the corresponding residues (as defined by Kabat) are identical among the individual CDRs, the CDRs in this antibody are substantially derived from the corresponding CDRs in the reference antibody. When the corresponding residues (as defined by Kabat) of 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% are identical, the CDR H2 in the antibody described herein, as defined by Kabat, is essentially derived from the corresponding CDR in the reference antibody. When 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% of the corresponding residues as defined by Kabat are identical, the variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially derived from the human variable region framework sequence or the human constant region, respectively.

[0069] Although humanized antibodies often incorporate all six CDRs from non-human (e.g., mouse) antibodies (as defined by Kabat), they can also be made to contain less than all of the CDRs from non-human antibodies (e.g., at least 3, 4, or 5) (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).

[0070] In some antibodies, only a subset of the CDRs is required for binding in humanized antibodies; that is, a subset of the CDR residues required for binding, referred to as the SDR. CDR residues that do not contact the antigen and are not in the SDR can be identified based on previous studies (e.g., residues H60-H65 in CDR H2 are often not required), according to the region of the Kabat CDR outside the Chothia hypervariable ring (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 locations where one or more donor CDR residues are absent or the entire donor CDR is omitted, the amino acid occupying that position can be the amino acid occupying the corresponding position in the acceptor antibody sequence (by Kabat numbering). The number of such substitutions of the acceptor and donor amino acids included in the CDR reflects a balance of competitive considerations. Such substitutions may be beneficial in reducing the number of mouse amino acids in humanized antibodies, and therefore may be beneficial in reducing potential immunogenicity. However, substitutions can also lead to changes in affinity, and a significant reduction in affinity can be avoided. The position of the substitution within the CDR and the amino acid to be substituted can also be selected empirically.

[0071] Although the receptor sequence may be identical to the selected human framework sequence, regardless of whether it originates from human immunoglobulins or the human common framework, the present invention covers receptor sequences that may include pre-existing amino acid substitutions relative to the human immunoglobulin sequence or the human common framework sequence. Such pre-existing substitutions may be minimal; typically only four, three, two, or one amino acid difference relative to the human immunoglobulin sequence or the common framework sequence.

[0072] The human receptor antibody sequence may be selected from many known human antibody sequences, thereby achieving a high degree of sequence identity (e.g., 65-85% identity) between the variable region framework of the human receptor sequence and the corresponding variable region framework of the donor antibody chain.

[0073] Certain amino acids from human variable region framework residues can be selected based on their potential effects on CDR conformation and / or binding to antigens. Such potential effects are studied by modeling, examining the characteristics of amino acids at specific positions, or empirically observing the effects induced by substitution or mutation of specific amino acids.

[0074] For example, when the amino acids between the non-human variable region framework residues and the selected human variable region framework residues are different, the human framework amino acids may be replaced by equivalent framework amino acids from the non-human antibody when it is reasonably expected that the amino acids are: (1) directly and non-covalently bound to the antigen, (2) adjacent to the CDR region, (3) otherwise interacting with the CDR region (e.g., within about 6 Å of the CDR region).

[0075] Other substitution candidates at that position are receptor human framework amino acids that are unusual for human immunoglobulins. These amino acids may be substituted by amino acids from the same position derived from non-human donor antibodies or amino acids from the same position derived from more typical human immunoglobulins. Other substitution candidates at that position are receptor human framework amino acids that are unusual for human immunoglobulins.

[0076] In some embodiments, the humanized anti-TIGIT antibody has a mature heavy chain variable region comprising a CDR1 containing an amino acid sequence of SEQ ID NO: 48 having zero to two amino acid substitutions or deletions, a CDR2 containing an amino acid sequence of SEQ ID NO: 49 having zero to two amino acid substitutions or deletions, and a CDR2 containing an amino acid sequence of SEQ ID NO: 49 having zero to two amino acid substitutions or deletions. The CDR3 of the amino acid sequence 50, and a framework region having 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 region of GenBank registration number AAV40102.1 or GenBank registration number ADX65334.1; and having a mature light chain variable region, the mature light chain variable region including a CDR1 containing the amino acid sequence of SEQ ID NO: 51 with zero to two amino acid substitutions or deletions, a CDR2 containing the amino acid sequence of SEQ ID NO: 52 with zero to two amino acid substitutions or deletions, and a CDR2 containing the amino acid sequence of SEQ ID NO: 52 with zero to two amino acid substitutions or deletions. The CDR3 of the amino acid sequence 53, and a framework region having 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% similarity to the framework region of GenBank registration number ACY78416.1 or GenBank registration number ADU32611.1. The framework regions of AAV40102.1, ADX65334.1, ACY78416.1, and ADU32611.1 are determined according to the Kabat definition, see Example 2 or see SEQ ID NO: 76-79 containing the framework regions of AAV40102.1, ADX65334.1, ACY78416.1, and ADU32611.1 and the donor CDR. In some embodiments, the mature heavy chain variable region is connected to at least a portion of the heavy chain constant region and the mature light chain variable region is connected to at least a portion of the light chain constant region. In some embodiments, for the performance of the full-length antibody, the mature heavy chain variable region is connected to the heavy chain constant region and the mature light chain variable region is connected to the light chain constant region. Suitable constant regions are further described in detail in section III(F). In some of the above embodiments, the heavy chain constant region has functional FcγR binding capability. In still other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 94, and the light chain constant region comprises or is composed of SEQ ID NO: 95.In some of the above embodiments, the heavy chain constant region has reduced functional FcγR binding capacity. In other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 97, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In still other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 101, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In some of the above embodiments, the heavy chain constant region has enhanced functional FcγR binding capacity. In other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 99, and the light chain constant region comprises or is composed of SEQ ID NO: 95.

[0077] 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 variation occurs at variable region framework residues other than residues identified as potentially important for binding. In some embodiments, any variation is a conserved amino acid substitution. In some embodiments, the antibody includes a mature heavy chain variable region having the sequence of SEQ ID NO: 76 and a mature light chain variable region having the sequence of SEQ ID NO: 77. The Hu24F8.1 antibody of the present invention comprises a mature heavy chain variable region having the sequence of SEQ ID NO: 76 and a mature light chain variable region having the sequence of SEQ ID NO: 77. In some of the above 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, for the performance of the 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 are further described in detail in section III(F). In some of the above embodiments, the heavy chain constant region can induce Fcγ receptor (FcγR)-mediated signaling, which is measured in a commercially available antibody-dependent cell-mediated toxicity report bioassay kit according to the manufacturer's instructions. In yet another embodiment, the heavy chain constant region comprises or is composed of SEQ ID NO: 94, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcγ receptor (FcγR)-mediated signaling and is measured in a commercially available antibody-dependent cell-mediated toxicity report bioassay kit according to the manufacturer's instructions.

[0078] 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: 77, 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 variation occurs at variable region framework residues other than residues identified as potentially important for binding. In some embodiments, any variation is a conserved amino acid substitution. In some embodiments, the antibody includes a mature heavy chain variable region having the sequence of SEQ ID NO: 78 and a mature light chain variable region having the sequence of SEQ ID NO: 77. The Hu24F8.2 antibody of the present invention comprises a mature heavy chain variable region having the sequence of SEQ ID NO: 78 and a mature light chain variable region having the sequence of SEQ ID NO: 77. In some of the above 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, for the performance of the 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 are further described in detail in section III(F). In some of the above embodiments, the heavy chain constant region can induce Fcγ receptor (FcγR)-mediated signaling, which is measured in a commercially available antibody-dependent cell-mediated toxicity report bioassay kit according to the manufacturer's instructions. In other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 94, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcγ receptor (FcγR)-mediated signaling and is measured in a commercially available antibody-dependent cell-mediated toxicity report bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 97, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 101, and the light chain constant region comprises or is composed of SEQ ID NO: 95.In other embodiments, the heavy chain constant region induces enhanced Fcγ receptor (FcγR)-mediated signaling, which is measured in a commercially available antibody-dependent cell-mediated toxicity reporting bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 99, and the light chain constant region comprises or is composed of SEQ ID NO: 95.

[0079] 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 variation occurs at variable region framework residues other than residues identified as potentially important for binding. In some embodiments, any variation is a conserved amino acid substitution. In some embodiments, the antibody includes a mature heavy chain variable region having the sequence of SEQ ID NO: 76 and a mature light chain variable region having the sequence of SEQ ID NO: 79. The Hu24F8.3 antibody of the present invention includes a mature heavy chain variable region having the sequence of SEQ ID NO: 78 and a mature light chain variable region having the sequence of SEQ ID NO: 79. In some of the above 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, for the performance of the 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 are further described in detail in section III(F). In some of the above embodiments, the heavy chain constant region can induce Fcγ receptor (FcγR)-mediated signaling, which is measured in a commercially available antibody-dependent cell-mediated toxicity report bioassay kit according to the manufacturer's instructions. In other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 94, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcγ receptor (FcγR)-mediated signaling and is measured in a commercially available antibody-dependent cell-mediated toxicity report bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 97, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 101, and the light chain constant region comprises or is composed of SEQ ID NO: 95.In other embodiments, the heavy chain constant region induces enhanced Fcγ receptor (FcγR)-mediated signaling, which is measured in a commercially available antibody-dependent cell-mediated toxicity reporting bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 99, and the light chain constant region comprises or is composed of SEQ ID NO: 95.

[0080] 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: 79, 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 variation occurs at variable region framework residues other than residues identified as potentially important for binding. In some embodiments, any variation is a conserved amino acid substitution. In some embodiments, the antibody includes a mature heavy chain variable region having the sequence of SEQ ID NO: 78 and a mature light chain variable region having the sequence of SEQ ID NO: 79. The Hu24F8.4 antibody of the present invention includes a mature heavy chain variable region having the sequence of SEQ ID NO: 76 and a mature light chain variable region having the sequence of SEQ ID NO: 79. In some of the above 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, for the performance of the 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 are further described in detail in section III(F). In some of the above embodiments, the heavy chain constant region can induce Fcγ receptor (FcγR)-mediated signaling, which is measured in a commercially available antibody-dependent cell-mediated toxicity report bioassay kit according to the manufacturer's instructions. In other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 94, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In other embodiments, the heavy chain constant region does not induce Fcγ receptor (FcγR)-mediated signaling and is measured in a commercially available antibody-dependent cell-mediated toxicity report bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 97, and the light chain constant region comprises or is composed of SEQ ID NO: 95. In other embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 101, and the light chain constant region comprises or is composed of SEQ ID NO: 95.In other embodiments, the heavy chain constant region induces enhanced Fcγ receptor (FcγR)-mediated signaling, which is measured in a commercially available antibody-dependent cell-mediated toxicity reporting bioassay kit according to the manufacturer's instructions. In some embodiments, the heavy chain constant region comprises or is composed of SEQ ID NO: 99, and the light chain constant region comprises or is composed of SEQ ID NO: 95.

[0081] In other embodiments of each of the above, the humanized anti-TIGIT antibody may (i) have an equilibrium binding constant (KD) of about 0.01 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, about 0.1 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, about 0.1 × 10⁻¹¹ M to about 10 × 10⁻¹¹ M, about 1 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, or about 1 × 10⁻¹¹ M to about 10 × 10⁻¹¹ M, as measured by surface plasma resonance, and / or (ii) a KD of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM. The half-maximum inhibitory concentration (IC50) of nM blocks the binding of soluble human CD155 ligand to human TIGIT on the cell surface, as measured in Example 1. Alternatively, in addition to the individual binding and blocking properties or combinations thereof described above, in some embodiments, the humanized anti-TIGIT antibody binds to an antigenic determinant comprising 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, if present, 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, if present, 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, if present, at least one of T55, Q56, N58, E60, and S80 of SEQ ID NO: 80. At least one of T55, Q56, N58 and S80 of 80; or (v) SEQ ID NO: T55, Q56, N58, E60, D72, S80 and K82 of 80.

[0082] D. Chimeric and Face-Decorated Antibodies The present invention further provides chimeric and face-decorated forms of non-human antibodies, particularly examples of antibodies 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16 and 28O12.

[0083] Chimeric antibodies are antibodies that combine the mature variable regions of the light and heavy chains of non-human antibodies (e.g., mouse antibodies) with the constant regions of the light and heavy chains of humans. These antibodies essentially or completely retain the binding specificity of non-human antibodies and are approximately two-thirds human sequences.

[0084] A face-modified antibody system is a type of humanized antibody that retains some, and usually all, CDRs and some of the non-human variable region framework residues of the non-human antibody, but replaces other variable region framework residues (e.g., exposed residues) that may contribute to B-cell or T-cell antigenic determinants with residues from corresponding positions in the human antibody sequence (Padlan, Mol. Immunol. 28:489, 1991). The result is an antibody in which the CDRs are entirely or substantially derived from the non-human antibody and the variable region framework of the non-human antibody becomes more human-like through substitution. Face-modified forms of antibodies such as 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, or 28O12 are included in this invention.

[0085] In some embodiments, the TIGIT chimeric antibody system has a mouse-human chimera comprising a mouse variable domain and a human IgG1 / κ constant domain. In one embodiment, the TIGIT chimeric antibody is a chimera Fab mVH+mVL constructed from mouse 21F8VH (SEQ ID NO: 1) and 21F8VL (SEQ ID NO: 2) domains and a human IgG1 / κ Fab constant domain (Ch21F8). In one embodiment, the TIGIT chimeric antibody is a chimera Fab mVH+mVL constructed from mouse 30M18VH (SEQ ID NO: 3) and 30M18VL (SEQ ID NO: 4) domains and a human IgG1 / κ Fab constant domain (Ch30M18). In one embodiment, the TIGIT chimeric antibody is a chimeric Fab mVH+mVL constructed from mouse 24F8VH (SEQ ID NO: 5) and 24F8VL (SEQ ID NO: 6) domains and a human IgG1 / κ Fab constant domain (Ch24F8). In another embodiment, the TIGIT chimeric antibody is a chimeric Fab mVH+mVL constructed from mouse 5J24VH (SEQ ID NO: 7) and 5J24VL (SEQ ID NO: 8) domains and a human IgG1 / κ Fab constant domain (Ch5J24). In yet another embodiment, the TIGIT chimeric antibody is a chimeric Fab mVH+mVL constructed from mouse 21B9VH (SEQ ID NO: 9) and 21B9VL (SEQ ID NO: 10) domains and a human IgG1 / κ Fab constant domain (Ch21B9). In one embodiment, the TIGIT chimeric antibody is a chimeric Fab mVH+mVL constructed from mouse 22B22VH (SEQ ID NO: 11) and 22B22VL (SEQ ID NO: 12) domains and a human IgG1 / κ Fab constant domain (Ch22B22). In one embodiment, the TIGIT chimeric antibody is a chimeric Fab mVH+mVL constructed from mouse 28P24VH (SEQ ID NO: 13) and 28P24VL (SEQ ID NO: 14) domains and a human IgG1 / κ Fab constant domain (Ch28P24). In one embodiment, the TIGIT chimeric antibody is a chimeric Fab mVH+mVL constructed from mouse 21B16VH (SEQ ID NO: 15) and 21B16VL (SEQ ID NO: 16) domains and a human IgG1 / κ Fab constant domain (Ch21B16).In one embodiment, the TIGIT chimeric antibody is a chimera Fab mVH+mVL constructed from the mouse 28O12VH (SEQ ID NO: 17) and 28O12VL (SEQ ID NO: 12) domains and the human IgG1 / κ Fab constant domain (Ch28O12).

[0086] E. Human antibodies against TIGIT are provided by the various techniques described below. Human antibodies are selected to have the same antigenic determinant specificity as specific mouse antibodies, such as one of the mouse monoclonal antibodies described in the examples, by means of competitive binding assays, by means of the phage presentation method described above for Winter, or by other means. Human antibodies can also be screened for specific antigenic determinant specificity by using only fragments of TIGIT as the target antigen, and / or by screening for antibodies against a series of deletion mutants of TIGIT.

[0087] Methods for generating human antibodies include Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Patent No. 4,634,664, a trioma method; and Engleman et al., U.S. Patent No. 4,634,666, using transgenic mice, including human immunoglobulin genes (see, for example, 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)) and phage presentation 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).

[0088] F. Selection of Constant Regions: The heavy and light chain variable regions of chimeric antibodies, humanized (including faceted) antibodies, or human antibodies may each be linked to at least a portion of the human constant region. In some embodiments, the heavy chain variable domain described in the preceding sections is linked to a portion of the human heavy chain constant region, and the light chain variable domain described in the preceding sections is linked to a portion of the human light chain constant region. In some embodiments, the heavy chain variable domain described in the preceding sections is linked to a portion of the human heavy chain constant region, and the light chain variable domain described in the preceding sections is linked to a portion of the full-length human light chain constant region. The heavy chain constant region includes an Fc (fragment crystallizable) region, which is the tail region of an antibody that interacts with cell surface receptors (Fc receptors) and some proteins of the complement system. In some embodiments, the heavy chain variable domain described in the preceding sections is linked to a portion of the full-length human heavy chain constant region, and the light chain variable domain described in the preceding sections is linked to a portion of the full-length human light chain constant region.

[0089] The selection of the constant region (or its truncation) depends in part on whether effector function is required, or even whether enhancement is required. "Effector function" refers to biological activity attributable to the Fc region of the antibody and varying depending on the antibody isotype. Non-limiting examples of antibody effector functions include: C1q binding to C1 complexes and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; de-modulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Human antibodies are classified into five isotypes (IgM, IgD, IgG, IgA, and IgE) based on their heavy chains, each providing different functions. IgG consists of four human subtypes (IgG1, IgG2, IgG3, and IgG4), each containing a different heavy chain. They are highly homologous and differ primarily in their hinge regions and the degree to which they activate the host immune system. For example, human isotopes IgG1 and IgG3 can mediate complement-mediated cytotoxicity, while human isotypes IgG2 and IgG4 may mediate or not mediate complement-mediated cytotoxicity at very low concentrations. The light chain constant region can be subclass λ or κ. For immunotherapy against cancers or pathogens that do not express TIGIT, in addition to human IgG1 and IgG3, human IgG2 or IgG4, or attenuated forms of human IgG1 with reduced effector function, may be used. Regarding human IgG4, mutations engineered with S228P (Eu number) on the heavy chain can be used to prevent Fab arm crossing. However, for the elimination of TIGIT-expressing cancer cells (e.g., tumors of T cells or NK cells) for immunosuppression, human IgG1 or IgG3 may be used. For example, for the direct killing or immunosuppression of TIGIT-expressing cancer cells (e.g., some hematological malignancies), antibodies with Fc effector function (e.g., human IgG1 or IgG3) may be used. Suitable sequences for human IgG1 or IgG3 are known in this art and include, for example, SEQ ID NO: 94 and human IgG3 disclosed in US 5,624,821.

[0090] Human constant regions exhibit allotropic and isoallotypic variations among individuals, meaning that constant regions can differ in one or more polymorphic sites in different individuals. The difference between isoallotypic and isomorphic regions lies in the binding of serum identifying the isoallotypic to one or more other isomorphic non-polymorphic regions. The reference to human constant regions includes constant regions possessing any natural allotropy or any arrangement of residues occupying polymorphic sites within natural allotropy.

[0091] One or more amino acids at the amino or carboxyl terminus of the light chain and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be omitted or derived in proportion to or in whole of the molecule. The N-terminal glutamic acid of the heavy or light chain may be substituted with glutamic acid residues to prevent the formation of pyroglutamic acid. Substitution may be made in the constant region to reduce or increase effector functions, such as complement-mediated cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) (see, for example, Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA103:4005, 2006), or to prolong the half-life in humans (see, for example, Hinton et al., J. Biol. Chem.279:6213, 2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (Eu number) to increase the half-life of the antibody.

[0092] In some embodiments, the antibody described herein includes the 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 exemplary heavy chain amino acid sequence comprising the wild-type constant region of SEQ ID NO: 94. In other embodiments, the antibody described herein has a variant selected from the wild-type heavy chain constant region (or a truncated version thereof) of variant human IgG1, variant human IgG2, variant human IgG3, or variant human IgG4. In some embodiments, the variant heavy chain constant region is SEQ ID NO: 97, SEQ ID NO: 99, or SEQ ID NO: 101. SEQ ID NO: 96, 98, and 100 are exemplary heavy chain amino acid sequences comprising the variant heavy chain constant region.

[0093] Compared to the same antibody without one or more mutations, some antibodies of the present invention are engineered by introducing one or more constant region mutations to have reduced effector functions, such as CDC and ADCC or antibody-dependent phagocytosis (ADCP). In some embodiments, each or all of these effector functions are reduced by at least 50%, 75%, 90%, or 95% compared to the antibody without the mutation. Other analyses are described by Shields et al., 2001 J. Biol. Chem., Vol. 276, pp. 6591-6604; Chappel et al., 1993 J. Biol. Chem., Vol. 268, pp. 25124-25131; Lazar et al., 2006 PNAS, 103; 4005-4010.

[0094] Substitution at any or all of positions 234, 235, 236, and / or 237 reduces affinity for the Fcγ receptor, particularly the FcγRI receptor (see, for example, US 6,624,821). In some embodiments, alanine residues are used for substitution, such as the L234A / L235A double mutation, to reduce effector function. Other combinations of mutations with reduced effector function include L234A / L235A / G237A, E233P / L234V / L235A / ΔG236, A327G / A330S / P331S, K322A, L234A and L235A, L234F / L235E / P331S (Eu number). Depending on the circumstances, positions 234, 236, and / or 237 in human IgG2 are substituted with alanine, and position 235 is substituted with glutamine. (See, for example, US 5,624,821.) Substitution of two amino acids at complement C1q binding sites at EU index positions 330 and 331 reduces complement binding (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution to IgG2 residues of human IgG1 at positions 233-236 and IgG4 residues at positions 327, 330, and 331 significantly reduces ADCC and CDC (see, for example, Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shields RL. et al., 2001 J Biol Chem. 276(9):6591-604). N297A, N297Q, or N297G (Eu number) mutations reduce glycosylation and thus reduce effector function.

[0095] In some embodiments, the antibodies of the present invention may be engineered to enhance Fc effector function. For example, FcγR binding may be enhanced by amino acid engineering. In some embodiments, this may be achieved by substituting one or more amino acids in the Fc region. Desired mutations may be determined by, for example, alanine screening or rational design and library screening. These techniques may be used to identify IgG variants with enhanced FcR binding and enhanced effector function. Alternatively, certain mutations in the Fc receptor region may be known in this art, for example as described in Smith P. et al. (2012) PNAS 6181-6186.

[0096] In some embodiments, the antibodies described herein include a modified IgG1 constant domain that increases the antibody's ability to mediate ADCC compared to unmodified wild-type IgG1. The modified IgG1 domain may be characterized by amino acid substitutions at one or more of L235V, S239D, F243L, R292P, A330L, I332E, and P396L (Eu number). In other embodiments, the modified IgG1 domain is characterized by substitutions at S239D, A330L, and I332E (Eu number). In some embodiments, a therapeutically effective amount of the antibody as described herein can induce 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 assessed by the methods described in this art.

[0097] Alternatively, glycoform perturbation can be used to enhance FcR-mediated therapeutic antibody function. N-linked Fc glycosylation on IgG1 antibodies is crucial for effector function. Sialination, galactosylation, diglycosylation, and fucosylation can all affect the binding and activity of IgG molecules. Glycosylation patterns on therapeutic antibodies can be controlled in many different ways. The cell type and culture conditions for producing recombinant antibodies can affect the glycosylation and activity of therapeutic antibodies. Furthermore, bioreactor conditions and downstream processing can also affect glycan microheterogeneity. Low- or no-fucosylated antibodies have shown enhanced Fc-mediated properties. Many ways to achieve this reduction in fucose levels through glycosylation engineering are well known in this art. One approach involves manipulating enzymes involved in antibody post-translational modifications. This can involve the overexpression of glucosidases, such as β-1-4-N-acetylglucosyltransferase III, gene knockout fucosyltransferase, or the use of cell lines that are naturally lacking in fucose or have been mutated to express low levels of fucosylation. Alternatively, N-linked glucosidase inhibitors, such as spermine, can be used to obtain low-fucoose cells with IgG molecules.

[0098] In some embodiments, amino acid-engineered variants may have a broader and enhanced affinity for a variety of FcγRs, while glycoform-engineered antibodies may generally have a more specific affinity for enhanced FcγRIIIa binding. Different glycoform structures can be produced by the interaction of the glycoform with the proximal amino acid on the Fc moiety and by the substitution of the amino acid in contact with the Ig oligosaccharide.

[0099] G. Recombinant antibodies are typically generated through recombinant expression of chimeric antibodies, humanized (including face-modified) antibodies, and human antibodies. Therefore, the present invention also provides polynucleotides encoding anti-TIGIT antibodies of Sections IIIA-G, vectors containing polynucleotides, and host cells containing such vectors.

[0100] The polynucleotide encoding the anti-TIGIT antibody of the present invention can be inserted into a vector for amplification, expression, or further optimization. Many vectors are available. In some embodiments, the vector system includes mammalian, bacterial, yeast, and other systems, and contains 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, and 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-1α), and a transcription termination sequence. For expression, the recombinant polynucleotide construct typically includes an expression control sequence operatively linked to the coding sequence of the antibody chain, including a naturally associated or heterologous promoter region. In some embodiments, the expression control sequence is a eukaryotic promoter system in the vector capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression nucleotide sequences, and the recombinant antibody is collected and purified.

[0101] A vector containing a polynucleotide sequence encoding the anti-TIGIT antibody of the present invention can be introduced into host cells for selection or gene expression. Host cells suitable for selection or expression of the polynucleotide sequence in the vector, as described herein, include prokaryotic and eukaryotic cells. Non-limiting examples suitable for prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as bacilli, such as Bacillus subtilis and Bacillus licheniformis, and Pseudomonas, such as Pseudomonas aeruginosa. Aeruginosa and Streptomyces. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable colonization or expression hosts for vectors encoding anti-TIGIT antibodies.Non-limiting examples include *Saccharomyces cerevisiae* and *Schizosaccharomyces pombe*; hosts of the genus *Kluyveromyces*, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wickeramii* (ATCC 24,178), *Kluyveromyces waltii* (ATCC 56,500), *Kluyveromyces drosophilarum* (ATCC 36,906), *Kluyveromyces thermotolerans*, and *Kluyveromyces marxianus*; and *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 Neurospora, Penicillium, Tolypocladium, and Aspergillus, such as Aspergillus nidulans and Aspergillus niger. Suitable host cells can also be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Various baculovirus strains and variants have been identified, along with corresponding permitted insect host cells from hosts such as the armyworm (Spodoptera frugiperda), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Several virus strains for transfection are publicly available, such as the L-1 variant of the alfalfa looper (Autographa californica) NPV and the Bm-5 strain of the silkworm NPV, and according to the present invention, such viruses can be used as the viruses described herein, particularly for transfecting armyworm cells.Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts. In some embodiments, mammalian cells are host cells used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting complete heterologous proteins have been developed in this technology, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myeloma, including Sp2 / 0 and NSO. In some embodiments, the cells are non-human. Expression vectors for these cells may include expression control sequences such as origin of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and essential processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. In some embodiments, the expression control sequence is derived from the promoter of an endogenous gene, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0102] Host cells are transformed with the aforementioned expression or selection vector for producing anti-TIGIT antibodies and cultured in a conventional nutrient medium, regulated as appropriate, to induce promoters, select the transformant, or amplify the gene encoding the desired sequence. Once expressed, the antibody can be purified according to standard procedures of this technique, including HPLC purification, column chromatography, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0103] IV. Therapeutic Applications The anti-TIGIT antibody of the present invention can be used to enhance the immune response in the treatment of cancer and infectious diseases. Conditions that can be treated by the antibody of the present invention include (but are not limited to) cancers, including hematologic malignancies, solid tumors, Merkel cell carcinoma, urothelial carcinoma, squamous cell carcinoma of the head and neck, B-cell lymphoma, uterine cancer, cervical cancer, testicular cancer, gastrointestinal cancers (e.g., esophageal cancer, gastroesophageal junction cancer, oropharyngeal cancer, gastric cancer, small or large intestine cancer, colon cancer or rectal cancer), bladder cancer, bone cancer, bone marrow cancer, skin cancer, gallbladder cancer, heart cancer, lung cancer, salivary gland cancer, adrenal cancer, thyroid cancer, ganglion cancer, cancers of the central nervous system (CNS) and peripheral nervous system (PNS), and cancers of the hematopoietic and immune systems (e.g., spleen or thymus).

[0104] The present invention also provides methods for treating or preventing other cancer-related diseases, conditions, or illnesses, including, for example, immunogenic tumors, non-immunogenic tumors, dormant tumors, virus-induced cancers (e.g., epithelial carcinoma, endothelial carcinoma, squamous cell carcinoma, and papillomavirus), adenocarcinoma, teratoma, chemically induced cancer, cancer metastasis, and angiogenesis. In certain embodiments, the tumor or cancer is colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, glioblastoma, or leukemia. The terms cancer-related conditions, diseases, and illnesses are used broadly to refer to conditions directly or indirectly related to cancer, and include, for example, angiogenesis and precancerous conditions such as dysplasia. In some embodiments, the cancer may be metastatic or at risk of becoming metastatic, or may occur in diffuse tissues, including blood or bone marrow cancers (e.g., leukemia).

[0105] These types of cancer may or may not exhibit TIGIT or CD155. Antibodies against TIGIT are effective against cancers that do not exhibit TIGIT because inhibiting the interaction between TIGIT and CD155 stimulates an immune response against these cancers. Examples of hematologic malignancies include leukemia, lymphoma, and myeloma, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin's and 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 or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, stomach cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, and brain tumors (such as gliomas, such as glioblastoma).

[0106] The method of the present invention can be practiced in an adjuvant setting. "Adjuvant setting" refers to a clinical setting in which an individual has a history of proliferative disease, particularly cancer, and generally (but not necessarily) has responded to therapy, including (but not limited to) surgery, radiation therapy, and / or chemotherapy. However, due to the history of proliferative disease, such individuals are considered to be at risk of developing the disease. Treatment or administration in an "adjuvant setting" refers to a subsequent treatment modality. In some embodiments, this document provides a method for treating cancer or achieving cancer prevention, comprising administering a therapeutically effective amount of any of the antibodies disclosed herein to an individual with cancer or at risk of developing cancer in an adjuvant setting.

[0107] The methods provided herein can also be practiced in a neoadjuvant setting, i.e., the method can be performed prior to primary / definitive therapy. In some cases, the individual has been previously treated. In other cases, the individual has not been previously treated. In some cases, the treatment is a first-line therapy. In some embodiments, this document provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of any of the antibodies disclosed herein to an individual with cancer or at risk of cancer in a neoadjuvant setting.

[0108] Other conditions that can be treated with the antibodies of the present invention include infectious diseases caused by viruses, bacteria, fungi, protozoa, and other pathogens, such as hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenoviruses, influenza viruses, flaviviruses, echoviruses, rhinoviruses, coxsackieviruses, coronaviruses, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, poxvirus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, HIV, SIV and vector-borne encephalitis virus, chlamydia, and rickettsial bacteria. Bacteria), mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, Salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lymes disease.

[0109] A. Antibody Administration The antibody system described herein is administered with an effective therapy, meaning a dose, route of administration, and frequency that delays the onset of symptoms, reduces severity, inhibits further deterioration, and / or improves at least one sign or symptom. If an individual has already developed the disease, the therapy may be called a therapeutically effective therapy. If an individual has a high risk of developing the disease relative to the general population but has not yet experienced symptoms, the therapy may be called a preventatively effective therapy. In some cases, therapeutic or preventative efficacy can be observed in an individual relative to a historical control or past experience of the same individual. In other cases, therapeutic or preventative efficacy can be demonstrated in a preclinical or clinical trial in a group of treated individuals relative to a control group of untreated individuals.

[0110] In some cases, individuals are identified as PD-L1 positive, CD155 positive, TIGIT positive, high MSI, having infiltrative T cells, having activated T cells, having high levels of molecules associated with antigen processing and presentation, high TMB, or any combination thereof. In some embodiments, patients are selected for treatment with the antibodies described herein based on, for example, high TIGIT expression relative to the control population on CD8+ cells and / or CD4+ cells and / or NK cells. In some cases, an individual is identified as having oncogene-driven cancer and has mutations in at least one of the following groups of genes: 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, EPA. S1, PAK4, RHOB, TBL1XR1, KEAP1, ZFP36L2, FGFR3, FOXA1, FLT3, TRAF3, RNF111, PPP2R1A, TXNIP, STAG2, RIT1, TGIF1, FOXQ1, ATR, CYSLTR2, PCBP1, PIK3R2, ASXL1, HIS T1H1C, KLF5, PIK3CB, SPOP, MECOM, CACNA1A, CTNND1, DACH1, XPO1, ZNF750, FBXW7, MUC6, KDM6A, GATA3, ZBTB20, PIK3CA, RB1, SOX17, SMARCA4, KIT, CHD8, CHD4, and APOB.

[0111] In some embodiments, any of the methods described herein includes administering a therapeutically effective amount of one or more of the anti-TIGIT antibodies described herein to an individual in need. As used herein, a "therapeutically effective amount" or "therapeutic dose" of an anticancer therapy (such as any of the anti-TIGIT antibodies described herein) is an amount sufficient to achieve a favorable or desired outcome. For therapeutic purposes, favorable or desired outcomes include (but are not limited to) clinical outcomes such as: reduction of one or more symptoms caused by cancer, improvement of the quality of life of an individual with cancer, reduction of the dosage of other drugs required to treat cancer, enhancement of the effect of another drug (such as via targeting), delay of disease progression, and / or prolongation of survival. The effective dose may be administered in one or more doses. For the purposes of this invention, the effective dose of an anticancer therapy is an amount sufficient to directly or indirectly achieve therapeutic or preventative treatment. As understood in a clinical context, the therapeutically effective dose of an anticancer therapy may or may not be combined with another anticancer therapy.

[0112] An illustrative dose of any of the antibodies described herein is approximately 0.1-20 mg or 0.5-5 mg per kilogram of body weight (e.g., approximately 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., 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). A fixed dose may be administered at a dose of 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 (inclusive) as described herein. In one embodiment, the antibody described herein is administered at a dose of approximately 300 to 1500 mg every three weeks. In another embodiment, the antibody described herein is administered at a dose of approximately 300 to 1800 mg every four weeks. Regardless of whether the treatment is prophylactic or therapeutic and regardless of whether the condition is acute or chronic, the dosage is determined based on the individual's condition and response to prior treatment (if present) and other factors.

[0113] Administration may be non-intestinal, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, intratumoral, local, intranasal, or intramuscular. In some embodiments, administration into the systemic circulation is by intravenous or subcutaneous administration. Intravenous administration may be, for example, by infusion over a period of time such as 30-90 minutes.

[0114] The dosing frequency depends on the half-life of the circulating antibody, the individual's condition, the route of administration, and other factors. In response to changes in the individual's condition or the progression of the disease to be treated, the frequency can be daily, weekly, monthly, quarterly, or at irregular intervals. In one embodiment, the frequency may be a two-week cycle. In another embodiment, the frequency may be a three-week cycle. In another embodiment, the frequency is a four-week cycle. In another embodiment, the frequency is a six-week cycle. During continuous treatment, the exemplary intravenous dosing frequency is between weekly and quarterly, but more frequent or less frequent dosing is also possible. For subcutaneous dosing, the exemplary dosing frequency is daily to monthly, but more frequent or less frequent dosing is also possible.

[0115] The number of doses administered depends on whether the condition is acute or chronic and the condition's response to treatment. For acute conditions or acute exacerbations of chronic conditions, one to ten doses are often sufficient. Sometimes, a single bolus dose in a split form may be sufficient for acute conditions or acute exacerbations of chronic conditions. Treatment may be repeated for relapses of acute conditions or acute exacerbations. For chronic conditions, antibodies may be administered at regular intervals, such as weekly, bi-weekly, monthly, quarterly, or semi-annually, for at least 1, 5, or 10 years, or for an individual's lifetime.

[0116] Compared with control individuals, treatment including anti-TIGIT antibody can increase median progression-free survival or overall survival in individuals with cancer by at least approximately 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%, 6 4%, 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% to alleviate the disease, or to extend these periods by 2 weeks, 1 month, 2 months or 3 months, or 4 months, 6 months or even 9 months or a year. Alternatively, compared with control individuals, treatment including with anti-TIGIT antibodies may increase the complete response rate, partial response rate, or objective response rate (complete + partial) by at least approximately 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%, and 57%, respectively. 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%. Except for the anti-TIGIT antibody, the control individuals received the same treatment as those receiving the anti-TIGIT antibody. Therefore, if the individuals receiving the anti-TIGIT antibody received a placebo alone or a combination of placebo and some chemotherapy agent other than the anti-TIGIT antibody, the control individuals could also receive the same treatment.

[0117] The anti-TIGIT antibodies disclosed herein can enhance the amount of NK cell-mediated cytotoxicity of CD155-expressing cells (such as, but not limited to, K562 cells) relative to the amount of NK cell-mediated cytotoxicity of CD155-expressing cells in the absence of one of the anti-TIGIT antibodies disclosed herein by any of the following: approximately 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 greater.

[0118] Typically, in clinical trials (e.g., phase II, II / III, or III trials), the increase in median progression-free survival and / or response rate in individuals treated with anti-TIGIT antibodies relative to control individuals is statistically significant, for example at the p=0.05, 0.01, or even 0.001 level. Complete and partial response rates are determined by objective criteria commonly used in cancer clinical trials, such as those listed or approved by the National Cancer Institute and / or the Food and Drug Administration, and may particularly include, for example, tumor volume, number of tumors, metastasis, survival time, and quality of life.

[0119] Pharmaceutical compositions intended for non-enteral administration may be sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., for a single administration dose). The pharmaceutical composition may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. Formulation depends on the chosen route of administration. For injection, antibodies may be formulated in aqueous solutions, such as in physiologically compatible buffers, such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce injection site discomfort). The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, antibodies may be in lyophilized form and reconstituted with a suitable medium, such as sterile pyrogen-free water, prior to use. The concentration of antibodies in liquid formulations may vary, for example, in the range of about 10-150 mg / ml. In some formulations, the concentration is about 20-80 mg / ml.

[0120] B. Combination Therapy This invention covers the use of anti-TIGIT antibodies alone or in combination with one or more active therapeutic agents. Other active therapeutic agents may be small chemical molecules; macromolecules, such as proteins, antibodies, peptides, DNA, RNA, or fragments of such macromolecules; or cell or gene therapies. Combination therapies can target different but complementary mechanisms of action, thereby providing synergistic therapeutic or preventative effects against underlying diseases, symptoms, or conditions. Alternatively or additionally, combination therapies may allow for a reduction in the dosage of one or more of the agents, thereby improving, reducing, or eliminating adverse effects associated with one or more of the agents.

[0121] The active therapeutic agents in such combination therapies can be formulated as single compositions or individual compositions. If administered separately, the individual therapeutic agents in the combination can be administered at the same or approximately the same time or at different times. Furthermore, the "combination" administration of therapeutic agents may involve different forms of administration (e.g., oral capsules and intravenous), administration at different dose intervals, administration of one therapeutic agent at a constant dosing regimen, titration increase, titration decrease, or discontinuation of another therapeutic agent, or independent titration increase, titration decrease, increase, or decrease, or discontinuation and / or resumption of treatment of the individual therapeutic agents in the combination during the course of treatment for the patient. If the combination is formulated as individual compositions, in some embodiments, the individual compositions are provided together in the kit.

[0122] In some embodiments, any of the anti-TIGIT antibodies disclosed herein is sequentially administered or applied to one or more other active therapeutic agents, for example, one or more of the other active therapeutic agents are administered before or after administration of the anti-TIGIT antibody according to the invention. In other embodiments, the antibody is administered simultaneously with one or more other active therapeutic agents, for example, the anti-TIGIT antibody is administered simultaneously or approximately simultaneously with one or more other therapeutic agents; the anti-TIGIT antibody and one or more other therapeutic agents may be present in two or more individual formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether the other agents are administered sequentially or simultaneously with the anti-TIGIT antibody, they are considered to be administered in combination for the purposes of the invention.

[0123] The antibody of the present invention can be used in combination with at least one other (active) agent in any manner suitable in each case. In one embodiment, treatment with at least one active agent and at least one anti-TIGIT antibody of the present invention is maintained for a period of time. In another embodiment, treatment with at least one active agent is reduced or interrupted (e.g., when the individual is stable), while treatment with the anti-TIGIT antibody of the present invention is maintained at a constant dosing regimen. In another embodiment, treatment with at least one active agent is weakened or interrupted (e.g., when the individual is stable), while treatment with the anti-TIGIT antibody of the present invention is weakened (e.g., lower dose, less frequent dosing, or shorter treatment duration). In another embodiment, treatment with at least one active agent is weakened or interrupted (e.g., when the individual is stable), while treatment with the anti-TIGIT antibody of the present invention is increased (e.g., higher dose, more frequent dosing, or longer treatment duration). In another embodiment, treatment with at least one active agent is maintained, while treatment with the anti-TIGIT antibody of the present invention is weakened or interrupted (e.g., lower dose, less frequent dosing, or shorter treatment duration). In another embodiment, treatment with at least one active agent and treatment with the anti-TIGIT antibody of the present invention are reduced or interrupted (e.g., at a lower dose, with less frequent administration, or for a shorter course of treatment).

[0124] Treatment with the antibody of the present invention can be combined with other treatments that are effective against the condition to be treated. When used to treat proliferative conditions, cancer, tumors or precancerous lesions, symptoms or conditions, the antibody of the present invention can be combined with chemotherapy, radiation (e.g., local or total radiation therapy), stem cell therapy, surgery or treatment with other biological products.

[0125] The antibodies of the present invention can be administered together with vaccines that induce an immune response against cancer. Such an immune response is enhanced by the antibodies of the present invention. The vaccine may include antigens expressed on the surface of cancer cells and / or tumors, or fragments thereof that can effectively induce an immune response, and may be linked to a carrier molecule, as appropriate.

[0126] In some embodiments, one or more of the additional therapeutic agents are immunomodulators. Suitable immunomodulators that may be used in this invention include CD40L, B7, and B7RP1; activated monoclonal antibodies (mAbs) that stimulate receptors, such as anti-CD40, anti-CD38, anti-ICOS, and 4-IBB ligands; dendritic cell antigen loads (in vitro or in vivo); anticancer vaccines, such as dendritic cell cancer vaccines; intercytokines / chemokines, such as IL1, IL2, IL12, IL18, ELC / CCL19, SLC / CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC, IFNα / β, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacterial lipopolysaccharide (LPS); indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors; and immunostimulatory oligonucleotides.

[0127] In some embodiments, the present invention provides a method for inhibiting tumor growth, comprising administering a combination of an anti-TIGIT antibody and a signal transduction inhibitor (STI) described herein to achieve additional or synergistic inhibition of tumor growth. As used herein, the term "signal transduction inhibitor" means an agent that selectively inhibits one or more steps in a signal transduction pathway. The signal transduction inhibitors (STIs) covered by this invention include: (i) bcr / abl kinase inhibitors (e.g., imatinib mesylate (GLEEVEC®)); (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) Akt family kinase or Akt pathway inhibitors (e.g., rapamycin); (v) circulating kinase inhibitors (e.g., flavopiridol); and (vi) phosphatidylinositol kinase inhibitors. Immunomodulatory agents may also be used in combination with the anti-TIGIT antibodies described herein to inhibit tumor growth in cancer patients.

[0128] In some embodiments, one or more of the additional therapeutic agents are chemotherapeutic agents. 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; ethyleneimine and methyl melamine, including hexamethyl melamine, tratamine, triethylphosphamide, triethylthiophosphamide, and trimethylolpropionic acid; nitrogen mustard, such as chlorambucil chlorbutazone, naphthiamethoxam, chlorambucil, estradiol, and ifosfamide. Ifosfamide, dichloromethyldiethylamine, dichloromethyldiethylamine oxide hydrochloride, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;Antibiotics, such as aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin C, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, actinomycin D, donomycin, detorubicin, 6-diazo-5-sideoxy-L-leucine, cranberry, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, and mycophenolic acid. The following are listed as potential drug names: nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozotocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, pteroxate (pt...). Eropterin), trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, fluxuridine, and 5-FU; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone.Anti-adrenergic drugs, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; acetylglucuronide; aldoxycycline; aminoacetylpropionic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate); etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazine; procarbazine; razoxane; sizofuran; spirogermanium; tenuazonic acid acid); triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; piperobroman; gacytosine; Ara-C; cyclophosphamide; thiotepa; taxoids, for example Examples include taxanes, nab-paclitaxel, and docetaxel; chlorambucil chlorbutate; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes such as cisplatin, carboplatin, and oxaliplatin; vincristine; etoposide / VP-16; ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; and navelbine.Novotrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and any pharmaceutically acceptable salt, acid, or derivative of the above.

[0129] Chemotherapy agents also include anti-hormonal agents used to modulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone and toremifene; and anti-androgens, such as abiraterone, enzalutamide, apalutamide, darolutamide, flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and any medically acceptable salts, acids or derivatives of the above. In some embodiments, the combination therapy includes a chemotherapy regimen comprising one or more chemotherapeutic agents. In some embodiments, the combination therapy includes administration of hormones or related hormonal agents.

[0130] Additional therapeutic modalities that can be used in combination with anti-TIGIT antibodies include radiotherapy, monoclonal antibodies against tumor antigens, complexes of antibodies and toxins, T-cell adjuvants, bone marrow transplantation, or antigen-presenting cells (e.g., dendritic cell therapy), including TLR agonists for stimulating such antigen-presenting cells.

[0131] In some embodiments, the present invention covers the use of a combination of anti-TIGIT antibodies described herein with RNA interference-based therapies to silence gene expression. RNAi begins by breaking a long double-stranded RNA into small interfering RNAs (siRNAs). One strand of the siRNA is incorporated into a ribonucleoprotein complex called an RNA-induced silencing complex (RISC), which then recognizes an mRNA molecule that is at least partially complementary to the incorporated siRNA strand. The RISC can bind to or break the mRNA, both of which inhibit translation.

[0132] In some embodiments, the present invention covers the use of combinations of anti-TIGIT antibodies described herein with agents that regulate adenosine levels. These therapeutic agents act on extracellular nucleotides that catalyze the conversion of ATP to adenosine, including extracellular nucleoside triphosphate diphosphate hydrolase 1 (ENTPD1, ​​also known as CD39 or Cluster of Differentiation 39), which hydrolyzes ATP to ADP and ADP to AMP, and extracellular 5'-nucleotidase (NT5E or 5NT, also known as CD73 or Cluster of Differentiation 73), which converts AMP to adenosine. In one embodiment, the present invention covers combinations with 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, adenosine A2a and A2b receptors are targeted. It also covers combinations with antagonists of A2a and / or A2b receptors. In one embodiment, the invention covers combinations with adenosine receptor antagonists described in WO / 2018 / 136700 or WO 2018 / 204661. In one embodiment, the adenosine receptor antagonist is AB928 (etrumadenant).

[0133] In some embodiments, the present invention covers the use of combinations of anti-TIGIT antibodies described herein with inhibitors of phosphatidylinositol 3-kinase (PI3K), specifically PI3Kγ isoforms. PI3Kγ inhibitors can stimulate anti-cancer immune responses by modulating bone marrow cells, such as by inhibiting repressive bone marrow cells, suppressing immunosuppressive tumor-infiltrating macrophages, or by stimulating macrophages and dendritic cells, to produce cytokines that contribute to an effective T-cell response, thereby leading to reduced cancer development and spread. Exemplary PI3Kγ inhibitors that can be combined with the anti-TIGIT antibodies described herein include those described in WO 2020 / 0247496A1. In one embodiment, the PI3Kγ inhibitor is IPI-549.

[0134] In some embodiments, the present invention covers the use of combinations of anti-TIGIT antibodies described herein with inhibitors of arginase, which have been shown to cause or participate in inflammatory-triggered immune dysfunction, tumor immune escape, immunosuppression, and immunopathology of infectious diseases. Exemplary arginase compounds may be found, for example, in PCT / US2019 / 020507 and WO / 2020 / 102646.

[0135] In some embodiments, the present invention covers the use of anti-TIGIT antibodies and HIF-2α inhibitors according to the invention, which act holistically in cellular responses to hypoxia availability. Under hypoxic conditions, hypoxia-inducible factor (HIF) transcription factors can activate gene expression that regulates metabolism, angiogenesis, cell proliferation and survival, immune evasion, and inflammatory responses. HIF-2α overexpression has been associated with poor clinical outcomes in patients with various cancers; hypoxia is also prevalent in many acute and chronic inflammatory conditions, such as inflammatory bowel disease and rheumatoid arthritis.

[0136] This invention also covers combinations of the anti-TIGIT antibody described herein with one or more RAS signaling inhibitors. Oncogenic mutations in RAS gene families, such as HRAS, KRAS, and NRAS, are associated with a variety of cancers. For example, G12C, G12D, G12V, G12A, G13D, Q61H, G13C, and G12S, as well as other mutations, have been observed in multiple tumor types. Direct and indirect inhibition strategies for inhibiting mutant RAS signaling have been investigated. Indirect inhibitors target effectors in the RAS signaling pathway other than RAS, including (but not limited to) inhibitors of RAF, MEK, ERK, PI3K, PTEN, SOS (e.g., SOS1), mTORC1, SHP2 (PTPN11), and AKT. Non-limiting examples of indirect inhibitors under development include RMC-4630, RMC-5845, RMC-6291, RMC-6236, JAB-3068, JAB-3312, TNO155, FLY-1971, and BI1701963. Direct inhibitors of RAS mutants have also been investigated, generally targeting KRAS-GTP or KRAS-GDP complexes. Exemplary direct RAS inhibitors under development include (but are not limited to) sotorasib (UNK G510), MRTX849, mRNA-5671, and ARS1620. In some embodiments, one or more RAS signaling 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, one or more RAS signaling inhibitors directly inhibit the RAS mutant.

[0137] In some embodiments, the present invention relates to a combination of an anti-TIGIT antibody according to the disclosure with one or more anexelekto (i.e., AXL) inhibitors. The AXL signaling pathway is associated with tumor growth and cancer metastasis and is believed to mediate resistance to various cancer therapies. Several AXL inhibitors under development also inhibit other kinases in the TAM family (i.e., TYRO3, MERTK), as well as other receptor tyrosine kinases, including MET, FLT3, RON, and AURORA, and others. Exemplary multi-kinase inhibitors include gilteritinib, merestinib, cabozantinib, BMS777607, and foretinib. AXL-specific inhibitors have also been developed, such as SGI-7079, TP-0903 (i.e., dubermatinib), BGB324 (i.e., bemcentinib), and DP3975.

[0138] In some embodiments, the present invention covers the use of the anti-TIGIT antibody described herein in combination with adoptive cell therapy (a novel and promising form of personalized immunotherapy in which immune cells with anti-tumor activity are delivered to a cancer patient). Adoptive cell therapy is being explored 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 typically involves collecting T cells from an individual, genetically modifying them to target specific antigens or enhance their anti-tumor effects to expand them to a sufficient number, and infusing the genetically modified T cells into a cancer patient. T cells may be collected from a patient to whom the expanded cells will subsequently be re-infused (e.g., autologous) or from a donor patient (e.g., allogeneic).

[0139] T-cell-mediated immunity involves multiple sequential steps, each regulated by a balance of stimulatory and inhibitory signals to optimize the response. While almost all inhibitory signals in the immune response ultimately regulate intracellular signaling pathways, many initiate via membrane receptors whose ligands are membrane-bound or soluble (cytokines). Although costimulatory and inhibitory receptors and ligands regulating T-cell activation are often not overexpressed in cancer relative to normal tissues, inhibitory ligands and receptors regulating T-cell effector function in tissues are typically overexpressed on tumor cells or on untransformed cells associated with the tumor microenvironment. The function of soluble and membrane-bound receptors (ligand immune checkpoints) can be modulated using agonist antibodies (for costimulatory pathways) or antagonist antibodies (for inhibitory pathways). Therefore, unlike most antibodies currently approved for cancer therapy, antibodies that block or agonize immune checkpoints do not directly target tumor cells but rather target lymphocyte receptors or their ligands to enhance endogenous antitumor activity. [See Pardoll, (April 2012) Nature Rev. Cancer 12:252-64].

[0140] Examples of immune checkpoints (ligands and receptors) that are candidates for blocking (some of which are selectively upregulated in various types of tumor cells) include PD-1 (planned cell death protein 1); PD-L1 (planned cell death 1 ligand 1); BTLA (B and T lymphocyte attenuators); CTLA4 (cytotoxic T lymphocyte-associated antigen 4); TIM-3 (T cell immunoglobulin mucin 3); LAG-3 (lymphocyte activation gene 3); TIGIT (T cell immune receptor with Ig and ITIM domains); and killer inhibitory receptors, which can be divided into two categories based on their structural characteristics: i) killer cell immunoglobulin-like receptors (KIRs), and ii) type C lectin receptors (members of the type II transmembrane receptor family). Other less well-defined immune checkpoints have been described in the literature, including receptors (e.g., the 2B4 (also known as CD244) receptor) and ligands (e.g., certain B7 family inhibitory ligands, 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].

[0141] This invention encompasses the use of the anti-TIGIT antibodies described herein, inhibitors of the aforementioned immune checkpoint receptors and ligands, and combinations of immune checkpoint receptors and ligands to be described. Certain modulators of immune checkpoints are currently approved, and many others are under development. The fully humanized CTLA4 monoclonal antibody ipilimumab (e.g., YERVOY®; Bristol-Myers Squibb) became the first regulatoryly approved immune checkpoint inhibitor in the United States when it was approved in 2011 for the treatment of melanoma. Fusion proteins including CTLA4 and antibodies (CTLA4-Ig; abatcept (ORENCIA®; Bristol-Myers Squibb)) have been used to treat rheumatoid arthritis, and other fusion proteins have shown efficacy in kidney transplant patients sensitive to Epstein-Barr virus. Another class of regulatoryly approved immune checkpoint inhibitors target PD-1 and its ligands PD-L1 and PD-L2. Approved anti-PD-1 antibodies include nivolumab (OPDIVO®; Bristol Myers Squibb) and pelizumab (KEYTRUDA®; Merck) for various cancers, including squamous cell carcinoma, classic Hodgkin lymphoma, and urothelial carcinoma. Approved anti-PD-L1 antibodies include avelumab (BAVENCIO®, EMD Serono & Pfizer), atezolizumab (TECENTRIQ®; Roche / Genentech), and durvalumab (IMFINZI®; AstraZeneca) for certain cancers, including urothelial carcinoma. In some of the combinations provided herein, the immune checkpoint inhibitor is selected from MEDI-0680 nivolumab, pelizumab, avelumab, atezolizumab, budigalimab, BI-754091, camrelizumab, cosibelimab, durvalumab, dostarlimab, cimiprimab, sintilimab, tislelizumab, 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 is a recombinant protein, termed AMP-224, formed by fusing the extracellular domain of PD-L2 (B7-DC) with the Fc portion of IgG1. In one embodiment, the invention covers the use of the anti-TIGIT antibody and PD-1 antibody according to the invention. In one particular embodiment, the PD-1 antibody is cepalimumab. In some embodiments, the anti-TIGIT antibody is provided at a dose of approximately 200 to 1500 mg every three weeks and the anti-PD-1 antibody at a dose of approximately 100 to 1200 mg every three weeks. In another embodiment, the anti-TIGIT antibody described herein is provided at a dose of approximately 300 to 1800 mg every four weeks and the anti-PD-1 antibody at a dose of approximately 200 to 1500 mg every four weeks. In yet another embodiment, the anti-PD-1 antibody is cepalimumab and is provided at a dose of approximately 360 mg or 480 mg every three or four weeks.

[0142] In another embodiment, the present invention covers combinations of interleukins that inhibit T cell activation (e.g., IL-6, IL-10, TGF-B, VEGF and other immunosuppressive interleukins) or interleukins that stimulate T cell activation in order to stimulate an immune response.

[0143] In another phenotype, the T cell response can be stimulated by the revealed anti-TIGIT antibody in combination with one or more of the following: (i) antagonists of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, PVRIG, galactoglobulin 9, CEACAM-1, BTLA, CD69, galactoglobulin-1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4; and / or (ii) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, and 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 this 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 antagonists of KIR such as lirilumab.

[0144] Other agents used in combination therapy 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 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249, WO13169264, WO14 / 036357).

[0145] In another variant, the disclosed anti-TIGIT antibody may be combined with one or more of the following: activators that bind to positive co-stimulatory receptors; blockers that attenuate signal transduction via inhibitory receptors; antagonists; and one or more agents that systemically increase the occurrence of anti-tumor T cells; agents that overcome different immunosuppressive pathways in the tumor microenvironment (e.g., blocking inhibitory receptor binding (e.g., PD-L1 / PD-1 interaction), depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by depletion of ex vivo anti-CD25 beads) or reversing / preventing T cell dysfunction or depletion); and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0146] In one category, the immuno-oncology agent is a CTLA-4 antagonist, such as an antagonistic CTLA-4 antibody. Suitable CTLA-4 antibodies include, for example, ipilimumab (e.g., YERVOY®; Bristol Myers Squibb) or tramemumab. In another category, the immuno-oncology agent is a PD-L1 antagonist, such as an antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include, for example, atezolizumab (MPDL3280A; WO2010 / 077634) (e.g., TECENTRIQ®; Roche / Genentech), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), and MSB0010718C (WO2013 / 79174). In yet another category, the immuno-oncology agent is a LAG-3 antagonist, such as an antagonistic LAG-3 antibody. Suitable LAG-3 antibodies include, for example, BMS-986016 (WO10 / 19570, WO14 / 08218) or IMP-731 or IMP-321 (WO08 / 132601, WO09 / 44273). In another category, immuno-oncology agents are CD137 (4-1BB) agonists, such as agonist CD137 antibodies. Suitable CD137 antibodies include, for example, urelumab and PF-05082566 (WO12 / 32433). In yet another category, immuno-oncology agents are GITR agonists, such as agonist GITR antibodies. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06 / 105021, WO09 / 009116), and MK-4166 (WO11 / 028683). In another embodiment, the immuno-oncology agent is an OX40 agonist, such as an agonist OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383 or MEDI-6469. In another embodiment, the immuno-oncology agent is an OX40L antagonist, such as an antagonist OX40 antibody. Suitable OX40L antagonists include, for example, RG-7888 (WO06 / 029879). In another embodiment, the immuno-oncology agent is a CD40 agonist, such as an agonist CD40 antibody. In yet another embodiment, the immuno-oncology agent is a CD40 antagonist, such as an antagonist CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab or dacetuzumab. In another category, immuno-oncology agents are CD27 agonists, such as agonist CD27 antibodies. Suitable CD27 antibodies include, for example, varlilumab.In another embodiment, the immuno-oncology agent is MGA271 (targeting B7H3) (WO11 / 109400). In yet another embodiment, this includes a combination of the anti-TIGIT antibody according to the invention with an agent targeting Trop-2, such as an antibody-drug conjugate, sacituzumab govitecan-hziy. In yet another embodiment, this includes a combination of the anti-TIGIT antibody described herein with an agent inhibiting the CD47-SIRPα pathway. An example of an anti-CD47 antibody is magrolimab.

[0147] Examples of therapeutic agents suitable for combination therapy in the treatment of cardiovascular and / or metabolic-related diseases, conditions and illnesses include statins (e.g., CRESTOR®, LESCOL®, LIPITOR®, MEVACOR®, PRAVACOL®, and ZOCOR®) that inhibit the enzymatic synthesis of cholesterol; bile acid resins (e.g., COLESTID®, LO-CHOLEST®, PREVALITE®, QUESTRAN®, and WELCHOL®) that chelate cholesterol and prevent its absorption; ezetimibe (ZETIA®) that blocks cholesterol absorption; fibrinolytic acid (e.g., TRICOR®) that reduces triglycerides and may appropriately increase HDL; niacin (e.g., NIACOR®) that appropriately lowers LDL cholesterol and triglycerides; and / or combinations thereof (e.g., VYTORIN®). (Ezetimibe and Simvastatin). Alternative cholesterol treatment agents that may be used in combination with the anti-TIGIT antibody described herein include various supplements and herbs (e.g., garlic, policosanol, and gum arabic).

[0148] Examples of therapeutic agents suitable for combination therapy of immune and inflammatory diseases, conditions or illnesses include (but are not limited to) the following: nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen and other propionic acid derivatives (alminoprofen, benzoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprafen, tiaprofenic acid). Acetic acid derivatives (including tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, firofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), and fennamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid, and tolfenamic acid). (acid), biphenyl carboxylic acid derivatives (diflunisal and flufenisal), oxicam.(Isoxicam, piroxicam, sudoxicam, and tenoxican) salicylates (acetylsalicylic acid, sulfasalazine) and dihydropyrazolone (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, and phenylbutazone)). Other combinations include cyclooxygenase-2 (COX-2) inhibitors.

[0149] Other active agents used in the combination include steroids such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone, or hydrocortisone. This combination can be particularly advantageous because one or more adverse effects of steroids can be reduced or even eliminated by gradually reducing the required dose of steroids.

[0150] Additional examples of active agents that can be used in combination for the treatment of, for example, rheumatoid arthritis include cytokine-inhibiting anti-inflammatory drugs (CSAIDs); antibodies 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.

[0151] Specific combinations of active agents can interfere at different sites in the autoimmune and subsequent inflammatory cascade responses, and include TNF antagonists such as chimeric, humanized, or human TNF antibodies, REMICADE®, HUMIRA®, anti-TNF antibody fragments (e.g., CDP870), and soluble p55 or p75 TNF receptors, their derivatives, p75TNFRIgG (ENBREL®) or p55TNFR1gG (lenercept), soluble IL-13 receptor (sIL-13), and TNFα-converting enzyme (TACE) inhibitors; similarly, IL-1 inhibitors (e.g., interleukin-1-converting enzyme inhibitors) can be effective. Other combinations include interleukin-11, anti-P7s, and p-selectin glycoprotein ligands (PSGL). Other examples of agents suitable for combination with the A2AR / A2BR inhibitors described herein include interferon-131a (AVONEX®); interferon-13lb (BETASERON®); copaxone; hyperbaric oxygen; intravenous immunoglobulin; clabribine; and antibodies against other human interferons or growth factors, or antagonists of other human interferons or growth factors (e.g., antibodies against CD40 ligand and CD80).

[0152] In some embodiments, the combination consists of the antibody of the present invention and a second antibody, the second antibody targeting a surface antigen that expresses better on cancer cells than on control normal tissue. Some examples of antibodies that can be administered in combination therapy with the antibody of the present invention to treat cancer include Herceptin® (trastuzumab) targeting the HER2 antigen, Avastin® (bevacizumab) targeting VEGF, or antibodies targeting the EGF receptor, such as Erbitux® (cetuximab) and Vectibix® (panitumumab). Other agents that can 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 downstream signaling inhibitors, such as mTOR and GSK3β inhibitors; and interleukins, such as interferon-γ, IL-2, and IL-15. Other specific examples of agents include: ipilimumab, pazopanib, sunitinib, dasatinib, pembrolizumab, INCR024360, dabrafenib, trametinib, atezolizumab (MPDL3280A), erlotinib (e.g., TARCEVA®), cobimetinib, nivolumab, and cepalimumab. The choice of a secondary antibody or other agent for combination therapy depends on the cancer being treated. Depending on the situation, the cancer may be tested against antigenic expression or preferred expression to guide the selection of an appropriate antibody. In some embodiments, the secondary antibody isotyped as human IgG1 to promote effector functions such as ADCC, CDC, and phagocytosis.

[0153] Similar combination therapies can be used to treat or prevent infectious diseases, such as viral, bacterial, fungal, and parasitic diseases, symptoms and conditions, and related conditions. For example, the antibody of the present invention can be combined with an antibody against a pathogen or a vaccine against a pathogen (such as palivizumab against rous sarcoma virus). The vaccine can be a protein of the pathogen or a fragment thereof that can effectively induce an immune response. The antibody of the present invention enhances the immune response of the antibody or vaccine against the pathogen. The antibody of the present invention can also be administered together with in vitro expanded T cells or natural killer cells.

[0154] These combination therapies include antiviral agents that target various viral life-cycle stages and have different mechanisms of action, including (but not limited to) the following: inhibitors of viral uncoating (e.g., amantadine and rimantidine); reverse transcriptase inhibitors (e.g., acyclovir, zidovudine, and lamivudine); agents targeting integrase; agents blocking the binding of transcription factors to viral DNA; and agents affecting translation (e.g., antisense molecules). (e.g., fomivirsen); agents that modulate translation / ribonuclease function; protease inhibitors; viral assembly modulators (e.g., rifampicin); antiretroviral agents, such as nucleoside analog reverse transcriptase inhibitors (e.g., azidothymidine (AZT), ddl, ddC, 3TC, d4T); non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz, nevirapine); nucleotide analog reverse transcriptase inhibitors; and agents that prevent viral particle release (e.g., zanamivir and oseltamivir). Treatment and / or prevention of certain viral infections (e.g., HIV) often require a combination of antiviral agents ("mixtures").

[0155] Other antiviral agents intended for use in combination with any of the anti-TIGIT antibodies disclosed herein include (but are not limited to) the following: abacavir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, ATRIPLA®, boceprevirertet, cidofovir, combivir, darunavir, and delavirin. rdine, didanosine, docosanol, edoxudine, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquid Imiquimod, indinavir, inosine, various interferons (e.g., pegylated interferon alpha-2a), lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nexavir, penciclovir, peramivir, pleconaril, podophyllotoxin, and rabexin. Teglavin, Ribavirin, Ritonavir, Pyramidine, Saquinavir, Stavudine, Telaprevir, Tenofovir, Tipranavir, Trifluridine, Trizivir, Tromantadine, TRUVADA®, Valaciclovir, ValganciclovirVicriviroc, vidarabine, viramidine, and zalcitabine.

[0156] This invention covers the use of any of the anti-TIGIT antibodies disclosed herein in combination with antiparasitic agents. These agents include, but are not limited to, thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide, thiamethoxam, oxamniquine, metrifonate, ivermectin, albendazole, eflornithine, melarsoprol, pentamidine, benzyl benzoate, nifurtimox, and nitroimidazole. Those skilled in the art will recognize other agents that can be used to treat parasitic diseases.

[0157] Embodiments of the present invention cover the use of any of the anti-TIGIT antibodies disclosed herein in combination with agents for the treatment or prevention of bacterial diseases. Antibacterial agents can be classified in various ways, including based on mechanism of action, chemical structure, and activity spectrum. Examples of antibacterial agents include those that target the bacterial cell wall (e.g., cephalosporins and penicillins) or cell membrane (e.g., polymyxins) or interfere with essential bacterial enzymes (e.g., sulfadiazine, rifamycin, and quinoline). Most antibacterial agents that target protein synthesis (e.g., tetracyclines and macrocyclic lactones) are bacteriostatic, while agents such as aminoglycosides are bactericidal. Another way to classify antibacterial agents is based on their targeting specificity; "narrow-spectrum" agents target specific types of bacteria (e.g., Gram-positive bacteria, such as streptococci), while "broad-spectrum" agents have activity against a wider range of bacteria. Those skilled in the art will understand the types of antibacterial agents suitable for use in specific bacterial infections.

[0158] Embodiments of the present invention cover the use of any of the anti-TIGIT antibodies disclosed herein in combination with medicaments for the treatment or prevention of fungal diseases. Antifungal agents include polyenes (e.g., amphotericin, nystatin, and pimaricin); azoles (e.g., fluconazole, itraconazole, and ketoconazole); allylamines (e.g., naftifine and terbinafine); morpholines (e.g., amorolfine); and antimetabolites (e.g., 5-fluorocytosine).

[0159] This invention includes any of the medically acceptable salts, acids or derivatives of the above.

[0160] V. Other Applications The anti-TIGIT antibody of the present invention can be used to detect TIGIT in clinical diagnostic or therapeutic situations or in research. For example, the antibody can be used to detect the presence of TIGIT on T cells, natural killer cells, and cancer cells as an indicator that an individual has a treatable cancer or infectious disease. The expression of TIGIT on T cells, natural killer cells, and / or cancer cells in an individual with cancer or an infectious disease also provides an indication that the cancer or infectious disease can be treated with the antibody of the present invention. The antibody can also be sold as a research reagent for laboratory studies to detect T cells, natural killer cells, and cancer cells and their responses to various stimuli. In such uses, the antibody can be labeled with one or more detectable signals, including (but not limited to) fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in kit form with all the necessary reagents for TIGIT analysis. The anti-TIGIT antibody of the present invention can also be used to purify TIGIT, for example by affinity chromatography.

[0161] VI. The antibody against TIGIT in the kit can be combined with any of the second antibody or pharmaceutical agent described as a component of the kit for use in a co-therapy. The disclosure herein provides one or more kits containing one or more of the antibodies disclosed herein and one or more pharmaceutically acceptable excipients or carriers (such as (but not limited to) phosphate-buffered saline solutions, water, sterile water, polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil, emulsions such as oil / water emulsions or water / oil emulsions, microemulsions, nanocarriers, and various types of wetting agents). Additives such as alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like may also be included in the kits of the present invention along with the carriers, diluents, or excipients. In one embodiment, the pharmaceutically acceptable carrier suitable for use in the antibody compositions disclosed herein is sterile, pathogen-free, and / or otherwise safe for individual administration, posing no risk of associated infection or other excessive adverse side effects. In the kit, each agent may be provided in a separate vial containing instructions for combination, subsequent administration, or separate administration. The kit may also include written instructions for the proper handling and storage of any of the anti-TIGIT antibodies disclosed herein.

[0162] VII. Examples Example 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 (HC) complementarity determination region (CDR) 1 having at least 80% sequence identity with SEQ ID NO: 36, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 37, and an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 38; and a light chain variable region comprising a light chain (LC) CDR1 having at least 80% sequence identity with SEQ ID NO: 39, an LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 40, and an LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 41; (b) a heavy chain variable region comprising an HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 42, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 49, an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 49, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 40, and an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 41; and a light chain variable region comprising an HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 49, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 40, an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 49, an HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 49, an HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 49, an HC-CDR2 having at least 43 HC-CDR2 with at least 80% sequence identity and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 44; and a light chain variable region comprising LC-CDR1 with at least 80% sequence identity to SEQ ID NO: 45, LC-CDR2 with at least 80% sequence identity to SEQ ID NO: 46, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 47; (c) a heavy chain variable region comprising HC-CDR1 with at least 80% sequence identity to SEQ ID NO: 48, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 49, and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 50; and a light chain variable region comprising LC-CDR1 with at least 80% sequence identity to SEQ ID NO: 51, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 44, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 45, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 46, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 47; 52 LC-CDR2 with at least 80% sequence identity and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 53; (d) Heavy chain variable region comprising HC-CDR1 with at least 80% sequence identity to SEQ ID NO: 54, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 55, and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 56;(e) a light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 57, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 58, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 59; (e) a heavy chain variable region comprising HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 60, HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 61, and HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 62; and a light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 63, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 64, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 69. 65 LC-CDR3 with at least 80% sequence identity; (f) heavy chain variable region comprising HC-CDR1 with at least 80% sequence identity to SEQ ID NO: 60, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 66, and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 67; and light chain variable region comprising LC-CDR1 with at least 80% sequence identity to SEQ ID NO: 63, LC-CDR2 with at least 80% sequence identity to SEQ ID NO: 68, and LC-CDR3 with at least 80% sequence identity to SEQ ID NO: 65; (g) heavy chain variable region comprising HC-CDR1 with at least 80% sequence identity to SEQ ID NO: 69, HC-CDR2 with at least 80% sequence identity to SEQ ID NO: 55, and HC-CDR3 with at least 80% sequence identity to SEQ ID NO: 69. 70 HC-CDR3 having at least 80% sequence identity; and a light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 71, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 68, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 65; (h) a heavy chain variable region comprising HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 72, HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 73, and HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 67;and a light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 63, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 68, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 65; or (i) a heavy chain variable region comprising HC-CDR1 having at least 80% sequence identity with SEQ ID NO: 74, HC-CDR2 having at least 80% sequence identity with SEQ ID NO: 75, and HC-CDR3 having at least 80% sequence identity with SEQ ID NO: 67; and a light chain variable region comprising LC-CDR1 having at least 80% sequence identity with SEQ ID NO: 63, LC-CDR2 having at least 80% sequence identity with SEQ ID NO: 68, and LC-CDR3 having at least 80% sequence identity with SEQ ID NO: 65.

[0163] Example 2. The anti-TIGIT antibody or its antigen-binding fragment as described in Example 1, comprising (a) a heavy chain variable region comprising HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 36, HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 37, and HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 38; and a light chain variable region comprising LC-CDR1 having an amino acid sequence identical to that of SEQ ID NO: 39, LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 40, and LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 41; (b) a heavy chain variable region comprising HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 42, HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 43, and HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 44. (c) A heavy chain variable region comprising HC-CDR1 containing the amino acid sequence of SEQ ID NO: 44, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 46, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 47; (d) a heavy chain variable region comprising HC-CDR1 containing the amino acid sequence of SEQ ID NO: 48, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 49, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 50; and a light chain variable region comprising LC-CDR1 containing the amino acid sequence of SEQ ID NO: 51, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 51. (d) A heavy chain variable region comprising HC-CDR1 containing the amino acid sequence of SEQ ID NO: 54, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 55, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 56; and a light chain variable region comprising LC-CDR1 containing the amino acid sequence of SEQ ID NO: 57, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 58, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 59;(e) a heavy chain variable region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 60, HC-CDR2 having the amino acid sequence of SEQ ID NO: 61, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 62; and a light chain variable region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 63, LC-CDR2 having the amino acid sequence of SEQ ID NO: 64, and LC-CDR3 having the amino acid sequence of SEQ ID NO: 65; (f) a heavy chain variable region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 60, HC-CDR2 having the amino acid sequence of SEQ ID NO: 66, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 67; and a light chain variable region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 60, HC-CDR2 having the amino acid sequence of SEQ ID NO: 61, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 62; and a light chain variable region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 60, HC-CDR2 having the amino acid sequence of SEQ ID NO: 64, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 65; (g) A heavy chain variable region comprising HC-CDR1 containing the amino acid sequence of SEQ ID NO: 63, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 68, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 65; and a light chain variable region comprising LC-CDR1 containing the amino acid sequence of SEQ ID NO: 69, HC-CDR2 containing the amino acid sequence of SEQ ID NO: 55, and HC-CDR3 containing the amino acid sequence of SEQ ID NO: 70; and a light chain variable region comprising LC-CDR1 containing the amino acid sequence of SEQ ID NO: 71, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 68, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 65; and (h) a heavy chain variable region comprising LC-CDR1 containing the amino acid sequence of SEQ ID NO: 63, LC-CDR2 containing the amino acid sequence of SEQ ID NO: 68, and LC-CDR3 containing the amino acid sequence of SEQ ID NO: 65; The HC-CDR1 having the amino acid sequence of SEQ ID NO: 72, the HC-CDR2 having the amino acid sequence of SEQ ID NO: 73, and the HC-CDR3 having the amino acid sequence of SEQ ID NO: 67; and a light chain variable region having the LC-CDR1 having the amino acid sequence of SEQ ID NO: 63, the LC-CDR2 having the amino acid sequence of SEQ ID NO: 68, and the LC-CDR3 having the amino acid sequence of SEQ ID NO: 65;Or (i) a heavy chain variable region comprising HC-CDR1 having an amino acid sequence comprising SEQ ID NO: 74, HC-CDR2 having an amino acid sequence comprising SEQ ID NO: 75, and HC-CDR3 having an amino acid sequence comprising SEQ ID NO: 67; and a light chain variable region comprising LC-CDR1 having an amino acid sequence comprising SEQ ID NO: 63, LC-CDR2 having an amino acid sequence comprising SEQ ID NO: 68, and LC-CDR3 having an amino acid sequence comprising SEQ ID NO: 65.

[0164] Example 3. The anti-TIGIT antibody or its antigen-binding fragment as described in Example 1 or 2 comprises (a) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 1; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 2; (b) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 3; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 4; (c) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 5; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 6; (d) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 7; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 8; (e) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 9; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 10; and (f) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 10; and a light ... NO: 11 is a heavy chain variable region with at least 80% sequence identity; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 12; (g) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 13; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 14; (h) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 15; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 16; (i) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 17; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 12; (j) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 76; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 77; (k) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 12; 78 is a heavy chain variable region with at least 80% sequence identity; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 77; (l) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 76; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 79; or (m) a heavy chain variable region with at least 80% sequence identity to SEQ ID NO: 78; and a light chain variable region with at least 80% sequence identity to SEQ ID NO: 79.

[0165] Example 4. An anti-TIGIT antibody or its antigen-binding fragment as described in any of Examples 1 to 3, wherein the anti-TIGIT antibody or its antigen-binding fragment is a monoclonal antibody.

[0166] Example 5. An anti-TIGIT antibody or antigen-binding fragment thereof as described in any of Examples 1 to 4, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a chimeric, humanized, or faceted antibody.

[0167] Example 6. An anti-TIGIT antibody or its antigen-binding fragment as described in Example 5, wherein the chimeric antibody contains a human IgG1 / κ Fab constant domain.

[0168] Example 7. An anti-TIGIT antibody or its antigen-binding fragment as described in any of Examples 1 to 3, wherein the anti-TIGIT antibody or its antigen-binding fragment is a human antibody.

[0169] Example 8. An anti-TIGIT antibody or antigen-binding fragment thereof as described in any of the preceding examples, wherein the anti-TIGIT antibody or antigen-binding fragment thereof inhibits the binding of TIGIT to CD155, wherein, as appropriate, the anti-TIGIT antibody or antigen-binding fragment thereof inhibits the binding with an IC50 of about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.4 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM, as measured in Example 1.

[0170] Example 9. An anti-TIGIT antibody or its antigen-binding fragment as described in any of Examples 1 to 5 or 7 to 8, wherein the antibody further comprises a variable heavy chain constant region selected from variant human IgG1, variant human IgG2, variant human IgG3 or variant human IgG4, and a human light chain constant region as appropriate.

[0171] Example 10. An anti-TIGIT antibody or its antigen-binding fragment as in Example 9, wherein the variant heavy chain constant region has enhanced or reduced effector function relative to the wild-type heavy chain constant region.

[0172] Example 11. The anti-TIGIT antibody or its antigen-binding fragment as in Example 10, wherein the constant region of the human IgG heavy chain of the variant contains SEQ ID NO: 97, SEQ ID NO: 99 or SEQ ID NO: 101.

[0173] Example 12. An anti-TIGIT antibody or antigen-binding fragment thereof as described in any of Examples 1 to 5 or 7 to 8, wherein the antibody further comprises a constant region of the wild-type human IgG heavy chain and, where applicable, a constant region of the human light chain.

[0174] Example 13. The anti-TIGIT antibody or its antigen-binding fragment as in Example 12, wherein the constant region of the wild-type human IgG heavy chain contains SEQ ID NO: 94.

[0175] Example 14. An anti-TIGIT antibody or antigen-binding fragment thereof, as in Example 12 or 13, comprising a human light chain κ constant region, wherein, where applicable, the human light chain constant region comprises SEQ ID NO: 95.

[0176] Example 15. An anti-TIGIT antibody or its antigen-binding fragment as described in Examples 1 to 5 or 7 to 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.

[0177] Example 16. An anti-TIGIT antibody or its antigen-binding fragment as described in any of the preceding examples, wherein the antibody or its binding fragment (a) has an equilibrium binding constant (KD) of about 0.01 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, about 0.1 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, about 0.1 × 10⁻¹¹ M to about 10 × 10⁻¹¹ M, about 1 × 10⁻¹¹ M to about 100 × 10⁻¹¹ M, or about 1 × 10⁻¹¹ M to about 10 × 10⁻¹¹ M as measured by surface plasma resonance; and (b) has a KD of about 0.2 nM to about 2 nM, about 0.2 nM to about 0.8 nM, about 0.6 nM to about 0.8 nM, or about 0.6 nM to about 0.8 nM. The half-maximum inhibitory concentration (IC50) of nM blocks the binding of soluble human CD155 ligands to human TIGIT on the cell surface, as measured in Example 1; (c) binding to an antigenic determinant comprising 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 at least one of T55, Q56, N58, S80 and K82 of SEQ ID NO: 80, if present, (iii) D72 and K82 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60 and S80 of SEQ ID NO: 80, if present, (iv) SEQ ID NO: At least one of E60, D72 and K82 of SEQ ID NO: 80 and, as appropriate, 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).

[0178] Example 17. An anti-TIGIT antibody or antigen-binding fragment thereof as described in Example 16, wherein the antibody or antigen-binding fragment thereof competes with an antibody or antigen-binding fragment thereof as described in any one of Examples 1 to 17 for binding to TIGIT.

[0179] Example 18. An anti-TIGIT antibody or antigen-binding fragment thereof as in Example 16 or 17, wherein an excess of the antibody or antigen-binding fragment thereof competitively binds to TIGIT 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 measured in a competitive 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.

[0180] Example 19. An anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, comprising 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.

[0181] Example 20. A method for inhibiting the binding of TIGIT to CD155, comprising contacting TIGIT with an anti-TIGIT antibody or its antigen-binding fragment as described in any of the foregoing examples.

[0182] Example 21. A method for treating an individual infected with a pathogen, comprising administering to the individual an effective therapeutic or therapeutically effective amount of an antibody as described in any of the foregoing examples.

[0183] Example 22. The method as in Example 21, wherein the pathogen is a virus, bacteria, fungus or protozoa.

[0184] Example 23. The method as in Example 22, wherein the pathogen is HIV, SIV, hepatitis virus, herpesvirus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, poxvirus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, vector-borne encephalitis virus, chlamydia, or rickettsial bacteria. Bacteria), mycobacteria, staphylococci, streptococci, pneumococci, meningococci, gonococci, klebsiella, Proteus, Serratia, Pseudomonas, Legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease.

[0185] Example 24. The method of any one of Examples 21 to 23, wherein the system is treated with a vaccine that induces an immune response against the pathogen, the immune response being enhanced by the antibody.

[0186] Example 25. The method of Example 24, wherein the vaccine contains a protein or fragment thereof of the pathogen.

[0187] Example 26. The method of any one of Examples 21 to 25, wherein the individual is further administered a second antibody against the pathogen, wherein the effector-mediated cytotoxicity of the second antibody against the pathogen is enhanced by the antibody.

[0188] Example 27. The method of any one of Examples 21 to 26, wherein the individual is further administered one or more of an antiviral agent, an antiparasitic agent, an antibacterial agent or an antifungal agent.

[0189] Example 28. A method for treating or preventing cancer, comprising administering to an individual suffering from or at risk of cancer an effective therapeutic or therapeutically effective amount of any of the anti-TIGIT antibodies or antigen-binding fragments thereof as described in any of the foregoing examples.

[0190] Example 29. The method of Example 28, wherein the cancer is a hematological malignancy, solid tumor, Merkel cell carcinoma, urothelial carcinoma, head and neck squamous cell carcinoma, 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, ganglion cancer, cancer of the central nervous system (CNS) and peripheral nervous system (PNS), as well as cancers of the hematopoietic system and cancers of the immune system.

[0191] Example 30. The method of Example 28 or 29, wherein the individual is given tumor-infiltrating T cells activated by the antibody or its antigen-binding fragment.

[0192] Example 31. The method of any one of Examples 28 to 30, wherein the individual is given a vaccine that induces an immune response against the cancer, the immune response being enhanced by the antibody or an antigen-binding fragment thereof.

[0193] Example 32. The method of Example 31, wherein the vaccine contains an antigen or fragment thereof expressed on the surface of cancer cells.

[0194] Example 33. The method of any one of Examples 28 to 32, wherein the individual is given natural killer cells whose cytotoxicity against the cancer is enhanced by the antibody or its antigen-binding fragment.

[0195] Example 34. The method of any one of Examples 28 to 33, wherein the individual is further administered a second antibody against an antigen expressed on the surface of cancer cells, whereby the effector-mediated cytotoxicity of the second antibody against the cancer is enhanced by the antibody or its antigen-binding fragment.

[0196] Example 35. The method of any one of Examples 28 to 33, wherein the individual is further administered a second antibody against an antigen expressed on the surface of immune cells.

[0197] Example 36. The method as in Example 35, wherein the immune cell line is T cells or natural killer cells.

[0198] Example 37. The method as in Example 35 or 36, wherein the antigen is CTLA-4, PD-1, or PD-L1.

[0199] Example 38. The method of any one of Examples 28 to 37, wherein the individual is further given one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapy and surgery.

[0200] Example 39. The method of any one of Examples 28 to 38, wherein the individual is further administered an inhibitor of one or more immune checkpoint receptors or ligands.

[0201] Example 40. The method of Example 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, A2aR, A2bR, A2a / A2bR, arginase, CD39, CD73, IDO, and TDO.

[0202] Example 41. The method of Example 39, wherein the inhibitor is selected from the group consisting of: ipilimumab, tramemumab, nivolumab, pembrolizumab, larizumab, cimipril, tislelizumab, cepallimumab, durvalumab, and atezolizumab.

[0203] Example 42. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of Examples 1 to 19 and a pharmaceutically acceptable carrier.

[0204] Example 43. An anti-TIGIT antibody or an antigen-binding fragment thereof that binds to an antigenic determinant of human TIGIT, the antigenic determinant comprising at least one amino acid residue from the following SEQ ID NO 80: T55, Q56, N58, E60, D72, S80, and K82.

[0205] Example 44. An anti-TIGIT antibody or antigen-binding fragment thereof as described in Example 43, wherein the antibody or antigen-binding fragment thereof binds to an antigenic determinant comprising 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 at least one of T55, Q56, N58, S80 and K82 of SEQ ID NO: 80, if present; (iii) D72 and K82 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60 and S80 of SEQ ID NO: 80, if present; (iv) E60, D72 and K82 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60 and S80 of SEQ ID NO: 80, if present. At least one of T55, Q56, N58 and S80 of 80; or (v) SEQ ID NO: T55, Q56, N58, E60, D72, S80 and K82 of 80.

[0206] Example 45. An anti-TIGIT antibody or antigen-binding fragment thereof as described in Example 43 or 44, wherein the antibody or antigen-binding fragment thereof competes with an antibody or antigen-binding fragment thereof as described in any one of Examples 1 to 19 for binding to TIGIT.

[0207] Example 46. An anti-TIGIT antibody or antigen-binding fragment thereof as described in Examples 43 or 44, wherein an excess of the antibody or antigen-binding fragment thereof competitively binds to TIGIT 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 measured in a competitive 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.

[0208] Example 47. An anti-TIGIT antibody or antigen-binding fragment thereof as described in any of Examples 1 to 19, wherein the antibody or antigen-binding fragment thereof binds to an antigenic determinant of human TIGIT, the antigenic determinant comprising at least one amino acid residue of the following SEQ ID NO 80: T55, Q56, N58, E60, D72, S80, and K82.

[0209] It is intended that each maximum numerical limit given throughout this specification includes each lower numerical limit as if such lower numerical limit were expressly written herein. Each minimum numerical limit given throughout this specification includes each higher numerical limit as if such higher numerical limit were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range as if such narrower numerical range were all expressly written herein.

[0210] All patent applications, websites, other publications, registration numbers, and the like cited above or below are incorporated herein by reference in their entirety for all purposes, as if each individual item specifically and individually indicated that it was incorporated in this manner. Where different forms are associated with a registration number in different time series, it means the form associated with the registration number at the effective filing date of this application. Effective filing date means, if applicable, earlier than the actual filing date or filing date of the priority application that mentions the registration number. Similarly, where different forms of publications, websites, or the like are published at different times, it means, unless otherwise specified, the most recently published version at the effective filing date of this application. Unless otherwise specifically indicated, any feature, step, element, embodiment, or pattern of the invention may be used in any other combination.

[0211] Although the invention has been described in considerable detail by means of illustration and examples 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.

[0212] Examples The following examples illustrate the generation, characterization and humanization of antibodies against human TIGIT, and also provide exemplary methods for determining the binding characteristics of the antibodies described in this application.

[0213] Example 1. Generation of anti-TIGIT antibody. The anti-TIGIT system was obtained from immunized mice. The extracellular domains of the His-labeled human TIGIT protein (hTIGIT-His) with SEQ ID NO: 83 and the cynomolgus monkey TIGIT protein (cTIGIT-His) with SEQ ID NO: 85 were transiently expressed in HEK293 cells and purified by anti-His affinity chromatography. BALB / c mice were immunized with RIMMS using a mixture of recombinant hTIGIT-His and cTIGIT-His proteins. Plasma titers against the immunogen were analyzed by ELISA prior to the final booster immunization to confirm good titers. After the final booster immunization, terminal hemorrhages along with the spleen, groin, upper arm, axillary, and cervical lymph nodes were collected. The collected material was purified by B cells and then fused to generate fusion tumors for initial screening.

[0214] Ten days post-fusion, preliminary screening of fusion tumors was performed using ELISA. 384-well ELISA discs were coated with 1 µg / mL hTIGIT-His protein. After blocking the discs, 20 µL of fusion tumor supernatant was added and allowed to bind to the TIGIT-coated discs. After incubation at room temperature, the discs were washed, and HRP-conjugated goat anti-mouse IgG antibody was used to detect the antibody bound to the TIGIT-coated discs.

[0215] Positive fusion tumor cells were then expanded into 48-well plates, and the supernatant was collected for antibody specificity testing in an ELISA assay. The 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 control counter to deselect antibodies that recognize the His tag on the immunogenic protein. The functional blockade of TIGIT / CD155 binding was then tested using antibodies that showed positive binding to both human and cynomolgus monkey TIGIT while being negative for the His-tagged control protein.

[0216] The extracellular domain of human TIGIT was fused into a mouse Fc sequence, and this hTIGIT-mFc (SEQ ID NO: 87) protein was expressed in HEK293 cells and purified by protein A affinity chromatography. A CD155 / TIGIT interaction blocking assay was established using recombinant human CD155 (including the extracellular domain fused with a human Fc sequence (hCD155-hFc) from R&D Systems (Cat# 9174-CD-01M)). To test the functional blocking activity of the antibody, 0.5 µg / mL hTIGIT-mFc protein was spread onto ELISA plates. After blocking, the supernatant of the fusion tumor was added together with 0.5 µg / mL hCD155-hFc protein. After incubation, the ELISA plates were washed, and the bound hCD155-hFc was detected using an HRP-bound goat anti-human IgG antibody. The antibody was identified as a pure line that can bind to both human and cynomolgus monkey TIGIT and can block CD155 / TIGIT interaction.

[0217] These pure lines were further expanded, and the antibodies were purified using a protein G column. The binding of these purified antibodies to human and cynomolgus monkey TIGIT expressed on the cell surface was tested by flow cytometry. Stable CHO-K1 cell lines expressing the full-length human TIGIT pure line 2A7 (Swiss-Prot Q495A1; SEQ ID NO: 80) or the full-length cynomolgus monkey TIGIT pure line C10 (Swiss-Prot A0A2K5UW92; SEQ ID NO: 84) were obtained. For flow analysis, cells were collected and incubated for 1 hour at 4°C in 100 μL HBSS buffer with various antibody concentrations (or without). After washing with HBSS, antibody binding on the cells was detected at 4°C for 30 minutes using 2 μg / mL of Alexa488-labeled goat anti-mouse IgG antibody (ThermoFisher Scientific, Cat#A-11001). Cells were then washed and resuspended in PBS, and flow cytometry was performed using an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA). The geometric mean of the fluorescence intensity of the entire single-cell population was obtained. Further testing was conducted to determine the blocking activity of antibodies binding to human and cynomolgus monkey TIGIT on the cell surface against recombinant human CD155 binding to human TIGIT on the surface of CHO cells. At room temperature, CHO-hTIGIT cells (10⁵ cells) were incubated with 2.5 µg / mL hCD155-Fc protein for 1 hour in the presence of different antibody concentrations. After washing with HBSS buffer, the binding of hCD155-Fc to hTIGIT-CHO cells was detected using a PerCP-eFluor 710-bound anti-CD155 antibody (ThermoFisher, Cat# 1550-42). Cells were then washed and analyzed by flow cytometry. Table 4 shows the half-maximal effective concentration (EC50) of the antibody binding to TIGIT in humans and cynomolgus monkeys (n=2) and the IC50 for inhibiting CD155 binding to TIGIT. Table 4: Binding of purified mouse anti-TIGIT antibody to TIGIT expressed on cell surface. α-TIGIT pure line hTIGIT EC 50 (nM) cTIGIT EC 50 (nM) hTIGIT / hCD155 IC 50 (nM) 22B22 0.507 0.580 0.83 21B16 0.498 1.201 1.15 28O12 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

[0218] The top few fusion tumor cell lines were selected based on their binding affinity for human and cynomolgus monkey TIGIT and their ability to block the binding of CD155 to TIGIT. These purebred fusion tumors were amplified, and the heavy and light chain variable regions (VH and VL) sequences of the mouse anti-TIGIT antibodies were determined according to standard procedures. The amino acid sequences of the mature VH and VL of antibodies 21F8, 30M18, 24F8, 5J24, 21B9, 22B22, 28P24, 21B16, and 28O12 are shown in Figures 1A-1I, with their CDR bands underlined. The allocation of CDR sequences and the numbering of amino acid positions were based on the Kabat definition.

[0219] The seven antibodies shown in Figures 1A-1I were recombined and expressed as mouse-human chimeras with mouse variable domains and human IgG1 / κ constant domains. The recombinant proteins were expressed in HEK293 cells and purified by protein A affinity chromatography.

[0220] The binding ability of these chimeric anti-TIGIT antibodies to human and cynomolgus monkey TIGIT overexpressed on the cell surface was confirmed using flow cytometry analysis as previously described. After incubation of the chimeric anti-TIGIT antibodies with hTIGIT-CHO or cTIGIT-CHO, the binding antibodies were detected using an Alexa488-conjugated goat anti-human IgG antibody (ThermoFisher Scientific, Cat# A-11013). The functional activity of the antibodies inhibiting the binding of recombinant hCD155-hFc to hTIGIT-CHO-K1 cells was also determined using flow cytometry as previously described. The EC50 for binding of the anti-TIGIT chimeric antibodies to human and cynomolgus monkey TIGIT and the IC50 for inhibiting the binding of hCD155 to hTIGIT-expressing cells are presented in Table 5. Table 5: The recombinant anti-TIGIT mouse / human chimeric antibody binds to TIGIT overexpressed on the surface of CHO-K1 cells and exhibits blocking activity against the binding of human CD155 to human TIGIT. α-TIGIT antibody hTIGIT-CHO EC 50 (nM) cTIGIT-CHO EC 50 (nM) hTIGIT-CHO / hCD155 IC 50 (nM) Ch22B22 0.16 0.24 1.16 Ch21B16 0.12 0.42 0.20 Ch28O12 0.159 0.358 0.48 Ch5J24 0.131 0.212 0.70 Ch21B9 0.102 0.18 0.80 Ch24F8 0.15 0.199 0.62 Ch30M18 0.336 6.138 2.18

[0221] Flow cytometry was used to analyze the binding ability of the chimeric anti-TIGIT antibody to endogenously expressed TIGIT on isolated human CD4+ and CD8+ cells. Human CD4+ or CD8+ T cells were isolated from whole blood using RosetteSep™ human CD4+ T cell enrichment mixture (Stemcell, Cat# 15022) or human CD8+ T cell enrichment mixture (Stemcell, Cat# 15022), respectively. As shown in Table 6, comparable EC50s of the recombinant anti-TIGIT antibody binding to human CD4+ or CD8+ cells were observed, with binding affinity similar to that of the overexpressed full-length human TIGIT on CHO-K1 cells. However, differences in maximum binding activity (MFImax) were observed between pure lines. Table 6: Binding of recombinant anti-TIGIT mouse / human chimeric antibody to isolated human T cells. α-TIGIT antibody CD4 + EC 50 (nM) CD4 + MFI max CD8 + EC 50 (nM) CD8 + MFI max 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

[0222] The ability of the recombinant anti-TIGIT chimeric antibody to bind to whole blood of cynomolgus monkeys was tested to confirm its ability to bind to endogenous cynomolgus monkey TIGIT protein on CD4+ and CD8+ cells. Whole blood of cynomolgus monkeys was incubated with the recombinant anti-TIGIT chimeric antibody at concentrations of 20 µg / mL, 5 µg / mL, 1 µg / mL, and 0.2 µg / mL. After incubation at 4°C for 30 min, RBC lysis was performed at room temperature for 15 min. Cells were then collected by centrifugation and washing, and blocked with a mixture containing the Fc block (BD Biosciences, Cat# 564219) and Live-dead fixable Aqua (Invitrogen, Cat# L34957). Cells bound to anti-TIGIT antibody were detected at 4°C for 30 minutes using anti-human IgG Fc-biotin (Southern Biotech, Cat# 9040-08), followed by washing and centrifugation, and a second incubation at 4°C for 30 minutes with streptavidin bound to PE (Invitrogen, Cat# 12-4317-87). Wild-type human IgG1 antibody was used as an isotype control, and directly bound anti-human TIGIT-PE (eBiosciences, Cat# 12-9500-42) was used as a positive control. Figure 2 shows that Ch24F8, Ch28O12, and Ch22B22 can bind to cynomolgus macaque TIGIT expressed on CD4+ and CD8+ cells. The geometric mean of fluorescence intensity (gMFI) was obtained, and the data are presented as a multiple of gMFI relative to the isotype control.

[0223] The kinetics of binding between these recombinant anti-TIGIT antibodies and human TIGIT were determined by surface plasma resonance (SPR) using a Bio-Rad ProteOn XPR36 instrument. The recombinant antibodies were immobilized on a protein A-coated GLC sensor chip, and soluble His-labeled TIGIT (Acro Biosystems, Cat# No. TIT-H52H3) was used as the analyte. The binding constant was determined at 25°C. As shown in Table 7, pure 24F8 exhibited the highest binding affinity among the seven recombinant anti-TIGIT chimeric antibodies tested, as measured by the equilibrium dissociation constant (KD). Table 7: Binding kinetics of recombinant anti-TIGIT antibodies to His-labeled human TIGIT. α-TIGIT antibody k a (M -1 s -1 ) k d (s -1 ) K D (M) Ch24F8 2.15E+06 7.5E-05 3.5E-11 Ch28O12 1.256E+06 1.135E-03 9.04E-10 Ch30M18 7.27E+05 1.730E-03 2.38E-09 Ch5J24 5.57E+06 2.14E-02 3.85E-09 Ch21B16 1.47E+06 9.29E-03 6.30E-09 Ch21B9 1.05E+07 7.4E-02 7.1E-09 Ch22B22 1.44E+06 1.41E-02 9.78E-09

[0224] Example 2. Generation of Humanized Anti-TIGIT Antibodies: Mouse antibody 24F8 was selected for humanization using CDR transplantation technology (Queen et al., Proc. Natl. Acad. Sci. USA. 86:10029-10033, 1989). The mouse variable heavy chain (VH) and variable light chain (VL) sequences of 24F8 were used to identify the two closest human germlines for each chain. For VH, IGHV4-34*09 with 70% sequence identity and IGHV4-4*02 with 66% identity were found. For VL, IGKV1-33*01 with 70% sequence identity and IGKV3-15*01 with 67% identity were found (Table 8). Table 8. Identification of Human Germlines and Recipients Variable chain Human reproductive system Human / mouse concordance (%) Human receptors VH1 IGHV4-34*09 70 AAV40102.1 VH2 IGHV4-4*02 66 ADX65334.1 VL1 IGKV1-33*01 70 ACY78416.1 VL2 IGKV3-15*01 67 ADU32611.1

[0225] The positioning of the three heavy chain CDR (HC-CDR) sequences in the VH chain and the three light chain CDR (LC-CDR) sequences in the VL chain is based on the Kabat definition.

[0226] Search the GenBank database for human receptors with VH and VL structures (Benson et al., Nucleic Acids Res. 2005, 33, D34-D38) and identify the VH and VL sequences encoding human cDNA (see Table 8).

[0227] CDR transplantation of each human receptor was performed using HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49), and HC-CDR3 (SEQ ID NO: 50), as well as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52), and LC-CDR3 (SEQ ID NO: 53) of the VL receptor. The resulting sequences were examined for any potential post-translational modifications or chemical degradation sites, and it was confirmed that they did not have such a tendency. Antibody homology modeling was also used to identify putative residues of mouse reversion mutations.

[0228] Oligonucleotides were designed and synthesized into Fab fragments containing two VH and two VL domains of human IgG1 / κ constant domains for transplanting human receptors, and inserted into vector systems for high-throughput screening of performance levels and biophysical properties without protein purification (Zhang and Hirama, Patent Application Publication US 2012 / 0178110). All four combinations of VH1 (SEQ ID NO: 76) or VH2 (SEQ ID NO: 78) and VL1 (SEQ ID NO: 79) or VL2 (SEQ ID NO: 77) (Figures 1J-1M) were screened together with chimeras Fab mVH+mVL constructed from mouse 24F8VH (SEQ ID NO: 5) and 24F8VL (SEQ ID NO: 6) domains and human IgG1 / κ Fab constant domains. Using a Biacore 8K instrument with surface plasma resonance (SPR), Fab was captured on a BSA-coated wafer, and soluble His-labeled TIGIT was used as the analyte to analyze the supernatant of Fab secreted by the SASA (a single-domain antibody against serum albumin) fusion protein. Kinetic binding data of human and cynomolgus macaque TIGIT for five Fab fragments are shown in Table 9. Table 9. Kinetic binding affinity data of humanized Fab fragments. Fab Analytes k a (M -1 s -1 ) k d (s -1 ) K D (M) mVH+mVL hTIGIT-His 1.86E+06 2.37E-06 1.27E-12 VH1+VL2 (Hu24F8.1 Fab) 3.08E+06 1.11E-04 3.61E-11 VH2+VL2 (Hu24F8.2 Fab) 2.08E+06 9.39E-05 4.50E-11 VH2+VL1 (Hu24F8.3 Fab) 1.99E+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 1.54E+06 7.28E-04 4.74E-10 VH1+VL2 (Hu24F8.1 Fab) 1.11E+06 1.40E-03 1.26E-09 VH2+VL2 (Hu24F8.2 Fab) 7.42E+05 8.08E-04 1.09E-09 VH2+VL1 (Hu24F8.3 Fab) 9.74E+05 5.85E-03 6.00E-09 VH1+VL1 (Hu24F8.4 Fab) 1.35E+06 1.33E-02 9.81E-09

[0229] These data confirm that, as measured by the equilibrium dissociation constant (KD), the humanized variable domain combinations of VH1+VL2 and VH2+VL2 bind most tightly to human and cynomolgus monkey TIGIT, while also retaining most of the binding affinity of the mouse / human chimera Fab. Therefore, constructs containing mouse framework residue reversion mutations were not studied. These two VH / VL combinations (Figure 1J and Figure 1K) were selected for the construction design of full-length Hu24F8.1 and Hu24F8.2 IgG1 / κ antibodies, respectively.

[0230] Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA are IgG1 / κ antibodies having a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 97 and a light chain constant region containing the amino acid sequence of SEQ ID NO: 95. The name "IgG1.AA" indicates that the heavy chain constant region has leucine to alanine amino acid substitutions at positions 234 and 235 (Eu number). In contrast, the name "IgG1" indicates the wild-type IgG1 Fc region. For example, Hu24F8.2-IgG1 is an IgG1 / κ antibody having a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 94 and a light chain constant region containing 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 in a recombinant manner in HEK293 or CHO cells and purified by protein A affinity chromatography.

[0231] Using a Biacore T200 instrument with SPR, the purified full-length antibodies Hu24F8.1-IgG1.AA (containing VH1+VL2) and Hu24F8.2-IgG1.AA (containing VH2+VL2), along with the full-length chimeric antibodies of 24F8 mouse VH+VL and human IgG1 / κ constant domain (Ch24F8), were analyzed using anti-human IgG wafers coated with anti-human IgG and soluble hTIGIT-His or cTIGIT-His as analytes. Kinetic binding data for human and cynomolgus monkey TIGIT are shown in Table 10. Table 10. Kinetic binding affinity data of humanized antibodies. Antibody Analytes k a (M -1 s -1 ) k d (s -1 ) K D (M) Ch24F8 hTIGIT-His 8.53E+05 1.56E-04 1.83E-10 Hu24F8.1-IgG1.AA 1.01E+06 1.45E-04 1.44E-10 Hu24F8.2-IgG1.AA 7.96E+05 1.40E-04 1.76E-10 Ch24F8 cTIGIT-His 6.58E+05 1.09E-03 1.66E-09 Hu24F8.1-IgG1.AA 8.06E+05 1.40E-03 1.74E-09 Hu24F8.2-IgG1.AA 6.79E+05 9.97E-04 1.47E-09

[0232] The kinetic binding affinity data of the two full-length humanized antibodies confirmed that the binding affinity of the mouse antibody was completely preserved and no mouse structural residues were introduced to revert to the mutation.

[0233] Example 3. In vitro binding study of anti-TIGIT antibodies: The binding of antibodies Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA to TIGIT expressed on the cell surface was detected by flow cytometry. Cells expressing TIGIT were collected and incubated at 4°C in 100 μL HBSS buffer for 1 hour in the presence (or absence) of different antibody concentrations. After washing with HBSS, antibody binding on the cells was detected at 4°C for 30 minutes using 2 μg / mL of Alexa488-labeled goat anti-human IgG antibody (ThermoFisher Scientific, Cat# A-11013). The cells were then washed and resuspended in PBS, and flow cytometry was performed using an Attune NxT flow cytometer (ThermoFisher Scientific, Waltham, MA). The geometric mean of fluorescence intensity for the entire single-cell population was obtained, and for transient transfection, the percentage of positive cells was obtained by using untransfected cells to gate the positive-binding cell population. Data were calculated using a standard 4-parameter curve fitting method with GraphPad Prism.

[0234] As described above, stable CHO-K1 cell lines representing human TIGIT (pure line 2A7) and cynomolgus monkey TIGIT (pure line C10) were used to test the binding of Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA to human TIGIT and cynomolgus monkey TIGIT, respectively. Hu24F8.1-IgG1.AA bound to human TIGIT with an EC50 of 0.447±0.22 nM (n=8) (Figure 3A) and to cynomolgus monkey TIGIT with an EC50 of 0.237±0.33 nM (n=6) (Figure 3B). Hu24F8.2-IgG1.AA bound to human TIGIT at an EC50 of 0.29 ± 0.15 nM (n=8) (Fig. 3A) and to cynomolgus monkey TIGIT at an EC50 of 0.35 ± 0.16 nM (n=6) (Fig. 3B). These results indicate that Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA bind tightly to human and cynomolgus monkey TIGIT expressed on the cell surface.

[0235] The 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 detected using CHO-K1 cells transiently transfected with full-length mouse or rat TIGIT constructs. Mouse TIGIT expression was confirmed using the control antibody GNE10A7 (US Patent 9,499,596 Clark et al., 2016), and rat TIGIT expression was confirmed using the control antibody eBioscience™ G1GD7 (Invitrogen, Cat# 12-9501-82). When tested with antibodies up to 30 nM, Hu24F8.2-IgG1.AA did not bind to either mouse TIGIT (Fig. 4A) or rat TIGIT (Fig. 4B).

[0236] The 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 Concentration Mixture (Stemcell, Cat# 15023) as recommended by the manufacturer. Cells were then activated for 7 to 9 days with anti-CD3 / CD28 beads supplemented with 20 U / mL rhIL-2. Activated or inactivated CD8+ cells were blocked with human Fc blocks (BD Biosciences, Cat# 564219), followed by flow cytometry antibody binding analysis. As shown in Figures 5A and 5B, Hu24F8.2-IgG1.AA binds to CD8+ cells, with an EC50 of 0.098 ± 0.013 nM (n=2) for inactive cells and 0.14 ± 0.036 nM (n=2) for activated CD8+ cells. Although the binding EC50 values ​​are similar for activated and inactive CD8+ cells, the maximum binding signal differs significantly, consistent with the increased TIGIT expression on the cell surface of activated CD8+ cells.

[0237] Example 4. In Vitro Blocking Study of Anti-TIGIT Antibodies As described in Example 1, CHO cells stably overexpressing human TIGIT (CHO-hTIGIT) and human CD155-Fc fusion recombinant soluble protein (hCD155-Fc) were used. Flow cytometry was used to analyze the activity of Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA in blocking the interaction between TIGIT and CD155. As shown in Figure 6, both Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA blocked the interaction between hCD155-Fc and CHO-TIGIT on the cell surface in a dose-dependent manner. The IC50 for Hu24F8.1-IgG1.AA blocking the TIGIT-CD155 interaction was 0.68 nM, and for Hu24F8.2-IgG1.AA it was 0.67 nM.

[0238] Example 5. Characterization of Jurkat Dual Reporter Cell Lines with Anti-TIGIT Antibody The functional activity of Hu24F8.1-IgG1.AA and Hu24F8.2-IgG1.AA in blocking the human TIGIT receptor was determined using Promega's TIGIT / CD155 blocking bioassay (Promega, Cat# J2205). In this analysis, the effector cell lines were Jurkat cells overexpressing TIGIT and luciferase reporter cells activated downstream of the T cell receptor (TCR). Another stable cell line was the CHO-K1 cell line overexpressing human CD155 and containing T cell activation protein that binds to and activates the TCR. This CD155 aAPC / CHO-K1 cell line served as artificial antigen-presenting cells. Co-culture of these two cell lines induced TCR activation via aAPCs on CHO-K1 cells, which would activate the reporter construct; however, this pathway activation was inhibited by the TIGIT / CD155 interaction, resulting in low luciferase signaling. The presence of anti-TIGIT antibody inhibits TIGIT / CD155 interaction, releasing TIGIT inhibition and generating increased luciferase signaling. Analysis was performed according to the manufacturer's protocol. In short, effector Jurkat cells were recovered overnight in a cell culture incubator in 96-well plates, serially diluted with the test antibody, and added to the effector cells, followed by antigen presentation of CD155 aAPC / CHO-K1 cells. After co-culturing at 37°C and 5% CO2 for 6 hours, the luciferase substrate Bio-Glo reagent was added, and the luminescence signal was read on an Envision (PerkinElmer) microscope. As shown in Figure 7, Hu24F8.1-IgG1.AA enhanced reporter activity in an EC50-dependent manner of 3.35 ± 0.26 nM (n=3), while Hu24F8.2-IgG1.AA showed an EC50 of 2.78 ± 0.83 nM (n=3). The human IgG1 control showed no effect.

[0239] Example 6. Molecular Analysis of Anti-TIGIT Antibody TIGIT and Fab Expression, Purification, and Crystallization Soluble proteins (residues 22-130) of the mature extracellular domain of human TIGIT were recombinantly expressed in HEK293 cells. The construct (SEQ ID NO: 90) contained a C-terminal hexahistamine tag with a (Gly)4-Ala-(Gly)4 linker, and aspartic acid residues 32 and 101 were mutated to glutamine to remove the N-glycosylation site. The clarified supernatant was purified by affinity chromatography using a Nickel Sepharose Excel (GE Healthcare Life Sciences) column, followed by size exclusion chromatography (SEC) purification using a Superdex 200 pg (GE Healthcare Life Sciences) column. The TIGIT protein at a concentration of 8.4 mg / mL was prepared in a final buffer of 20 mM Tris pH 7.0 and 100 mM NaCl and then rapidly frozen in liquid nitrogen.

[0240] The soluble Fab fragment (Fab24F8) of the human IgG1 antibody Hu24F8.2-IgG1.AA was prepared as follows: Papain (Thermo Scientific, Cat. #20341) was used to break down the fragments in phosphate-buffered saline at 37°C for 3 hours, followed by overnight breakdown at room temperature. The lysed Fc fragments were removed using a MabSelect SuRe Protein A (GE Healthcare Life Sciences) column, and the flow-through was further purified by SEC using a Superdex 200 pg (GE Healthcare Life Sciences) column. Fab24F8 protein at a concentration of 28 mg / mL was prepared in a final buffer of 20 mM Tris pH 7.0 and 100 mM NaCl and then rapidly frozen in liquid nitrogen.

[0241] TIGIT and Fab24F8 protein were mixed at a 1:1 molar ratio at 4°C with stirring for 60 minutes to form a Fab-TIGIT complex. Final SEC purification was then performed using a Superdex 200 pg (GE Healthcare Life Sciences) column, and the protein dissociation concentration was adjusted to 44 mg / mL. The purified complex was used for crystallization experiments under approximately 1500 different conditions at 20°C using standard sieves. The initially obtained conditions were optimized using standard strategies that systematically varied key parameters affecting crystallization. These conditions were further purified by systematically varying pH or precipitate concentration. Crystals of Fab-TIGIT complexes suitable for structural elucidation were obtained 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 storage solution (20% (w / v) PEG3350; 0.20 M LiSO4) using a drop-down vapor diffusion technique.

[0242] Data Collection and Structural Design: The crystals were rapidly frozen and measured at 100 K. X-ray diffraction data were collected from the Fab-TIGIT complex crystals under cryogenic conditions at the Canadian Light Source (CLS, Saskatoon, Canada). The crystals belong to space group P1. Data were processed using the computer software autoPROC, XDS, and AIMLESS (The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763), see Table 11. Table 11. Statistics on Data Collection and Processing for Fab24F8 / TIGIT Complexes Wrong Combination Hu24F8.2 Fab / Human TIGIT ECD X-ray source CMCF-ID(08ID-1,CLS) Wavelength [Å] 0.9795 Detector PILATUS 6M Temperature [K] 100 Space Group P1 Cell: a; b; c; [Å] 85.11; 86.83; 87.83 α; β; γ; [°] 94.3; 117.0; 116.1 Resolution [Å] 2.24 (2.46-2.24) 1 only reflection 53126 (2657) Multiplicity 2.2 (2.1) Completeness [%] 84.8 (76.4) R sym [%] 6.2 (48.6) R maes [%] 8.2 (64.8) Average value (I) / σ 9.9 (1.7) 1 The value in parentheses refers to the highest resolution.

[0243] Phase information necessary for determining and analyzing the structure was obtained through molecular substitution. Previously resolved 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. Three Fab-TIGIT complex molecules were found within the asymmetric crystal unit. Subsequent model building and improvement were performed using the COOT program and the CCP4 software suite, respectively, according to standard procedures. Approximately 4.6% of the measured reflections were excluded from the improvement process in the calculation of the free R-factor (a measure of the correctness of the final model through cross-validation). TLS improvement (using the CCP4 program REFMAC5) was performed, which produced a lower R-factor and a higher-quality electron density map. Automatically generated local NCS constraints were applied. A water model was built using COOT's "Find waters" algorithm, which places water molecules within the peaks of a Fo-Fc plot with a profile of 3.0 σ. This was then refined using REFMAC5, and all water samples were checked using COOT's validation tools. The criteria for a list of suspected water samples were: a B factor greater than 80 Ų, a 2Fo-Fc plot less than 1.2 σ, and a distance from the nearest contact point less than 2.3 Å or greater than 3.5 Å. Data within the resolution range of 73.5–2.24 Å were included in the final refinement cycle, with Rcryst and RfreeR factors of 22.3% and 26.8%, respectively. The final model's Ramachandran plot showed all residues in the most favorable region (89.8%), the additionally allowed region (8.8%), and the generously allowed region (0.7%). For a summary overview, see Table 12. Table 12. Detailed Statistics of Fab24F8 / TIGIT Wrong Combination Hu24F8.2 Fab / Human TIGIT ECD Resolution [Å] 73.52-2.24 Number of reflections (operational / testing) 50688 / 2467 R cryst [%] 22.3 R free [%] 1 26.8 Total number of atoms: protein 12244 water 427 ligands - sulfates 30 Deviation from ideal geometry: 2 Bond length [Å] 0.010 Key angle [°] 1.47 Bond of B' [Å 2 ] 3 1.7 Ramachandran Curve: 4 89.8 Most favorable area [%] Additional allowed area [%] 8.8 Generous allowance for certain areas [%] 0.7 Areas not allowed [%] 0.7 1 The test set contains a 4.6-meter measurement of reflectance. 2 Root mean square difference with geometric target value 3 Calculated using MOLEMAN 4 Calculated using PROCHECK

[0244] The structure of Fab24F8 bound to human TIGIT consists of three independent molecules of a complex within a crystalline asymmetric unit, with their atomic coordinates overlapping in pairs, exhibiting a root-mean-square deviation of 0.53–1.13 Å for all non-hydrogen atoms. The final model includes the following residues: Gln1 to Ser223 of the Fab heavy chain, Glu1 to Cys214 of the Fab light chain, and Met22 to Ser129 of TIGIT. Some short-ring regions are not adequately defined by electron density and are not included in the final model.

[0245] The Fab heavy chain HC-CDR2 and HC-CDR3, as well as all three LC-CDRs of the light chain, form extensive interactions with the large β-sheet structure of TIGIT, namely the β-strands C, C', C'' and F, and the rings C'C'' and C''D composed of the polypeptide chains 55TQVNWEQQDQLLAICNADLGWHISPSFK82 and 109IYH111 of SEQ ID NO: 80 (Figures 8 and 9). This binding interaction results in a protein-protein interface surface area of ​​790 ± 10 Å2 (n=3) between the Fab fragment and TIGIT (PISA, EMBL-EBI). The molecular properties of this interaction are both hydrophilic and hydrophobic. TIGIT residues Thr55, Gln56, Asn58, Glu60, Asp72, Ser80, and Lys82 (shown as a bar diagram in Figure 9) form direct or water-mediated hydrogen bond interactions with residues of the Fab heavy and light chain CDRs (donor / acceptor atomic distance not exceeding 3.1 Å). Additionally, TIGIT residue Glu60 forms a salt bridge with Arg30 on the Fab light chain (Table 13). TIGIT hydrophobic residues Leu65, Ile68, Leu73, Pro79, and Ile109 (shown as spheres in Figure 9) form van der Waals contacts with residues of the Fab heavy and light chains. Table 13. Antigenic determinant residues of TIGIT that form hydrogen bonds and salt bridges with complementary residues of the Fab 24F8 heavy and light chains. TIGIT antigenic determinant Fab24F8 complementary bit (Kabat number) Thr 55 LC-CDR3 Thr 94 Gln 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 bonds

[0246] Figure 10A shows a schematic diagram of the complex structure of the N-terminal Ig-like domain (SEQ ID NO: 91) of human CD155 bound to human TIGIT (represented as the molecular surface), as reported by Stengel et al. in 2012. Figure 10B shows a schematic diagram of CD155 superimposed on a crystal structure complex of Fab24F8 bound to TIGIT (represented as the surface of each free molecule) in the same orientation as in Figure 10A. It can be clearly demonstrated that Hu24F8.2 or other antibodies derived from mouse antibody 24F8 binding to the extracellular domain of TIGIT will block the binding of CD155 to TIGIT.

[0247] Example 7. Anti-TIGIT antibodies can enhance T cell responses alone or in combination with anti-PD-1 antibodies. This example demonstrates that Hu24F8.2-IgG1, alone or in combination with anti-PD-1 antibodies (such as AB122), enhances primary T cell responses in healthy or cancerous individuals. Compared to isotype controls, PBMCs from healthy and cancerous individuals treated with 0.1, 1, or 10 µg / mL Hu24F8.2-IgG1 showed significantly increased IL-2 concentrations. In PBMCs from healthy individuals, treatment with a combination of 10 µg / mL Hu24F8.2-IgG1 and 1 µg / mL anti-human PD-1 antibody (AB122, cepallimab) resulted in significantly higher IL-2 levels compared to treatment with AB122 alone.

[0248] PBMCs from healthy individuals were isolated from chambers using the leukoreduction system (LRS), and PBMCs from cancer individuals were isolated from CPT tubes. These PBMCs were then cultured in vitro at 1 ng / mL SEA using 0.1, 1, or 10 µg / mL Hu24F8.2, 1 µg / mL AB122, or a combination of 10 µg / mL Hu24F8.2-IgG1 and 1 µg / mL AB122. After four days, the IL-2 concentration in the supernatant was measured using a cytological bead array (CBA). IgG1 isotype was included as a negative control. Methods

[0249] PBMCs from healthy individuals were isolated from the LRS chamber, while PBMCs from cancerous individuals were isolated from CPT tubes. PBMCs were resuspended at a concentration of 2 × 10⁶ cells / mL, and 100 µL / well was aliquoted into 96-well round-bottomed dishes. 4× concentrated antibody, resuspended in CTS Optimizer medium, was added at 50 µL / well to appropriate wells: Hu24F8.2-IgG1 or human IgG1 isotype control was added to obtain final concentrations of 0.1, 1, and 10 µg / mL; AB122 or human IgG4 isotype control was added to obtain a final concentration of 1 µg / mL. The analytical dishes were incubated at 37°C and 5% CO₂ for 1 hour. 50 µL / well of 4× concentrated staphylococcal enterotoxin A (SEA) was resuspended in CTS Optimizer medium and added to appropriate wells to obtain a final concentration of 1 ng / mL. The analytical trays with a final well volume of 200 µL were incubated at 37°C and 5% CO2 for 4 days, and the supernatant was collected for subsequent quantification of secreted IL-2. The supernatant was diluted 1:2 with analytical diluent from the human soluble protein master buffer kit. Analysis, data acquisition, and quantification were performed using the human IL-2 flexure kit and the human soluble protein master buffer kit according to the manufacturer's instructions. Results

[0250] Verify that PBMCs from all donors used in the study respond to SEA stimulation of the CD14+ monocyte population and TIGIT (the ligand of TIGIT) in a non-T regulatory CD4+ T cell population. PBMCs isolated from 10 healthy individuals and 7 cancer individuals were cultured in the presence of 1 ng / mL SEA with 0.1, 1, or 10 µg / mL Hu24F8.2-IgG1 alone, 1 µg / mL AB122, or a combination of 10 µg / mL Hu24F8.2-IgG1 and 1 µg / mL AB122. After 4 days, IL-2 concentrations were measured in the supernatant. IL-2 levels from the Hu24F8.2 treatment group were compared to IL-2 levels from individual IgG1 isotype control treatment groups, and IL-2 levels from the AB122 and Hu24F8.2-IgG1 combination treatment group were compared to IL-2 levels from the AB122 monotherapy group (Tables 14 and 15). Figure 11 shows an example of IL-2 levels in a healthy individual (#566), applicable to all concentrations of Hu24F8.2-IgG1. For both individuals, IL-2 secretion was statistically significantly increased at all concentrations of Hu24F8.2-IgG1 compared to individual IgG1 isotype controls. For healthy individual #566, IL-2 secretion was also statistically significantly increased with AB122 and Hu24F8.2-IgG1 combination treatment compared to AB122 monotherapy. At the cohort level, in both healthy and cancer-affected PBMCs, treatment with 10 µg / mL Hu24F8.2-IgG1 resulted in a statistically significant increase in IL-2 secretion compared to IgG1 isotype controls (Figure 12A). Furthermore, in healthy PBMCs, treatment with a combination of AB122 and Hu24F8.2-IgG1 resulted in a statistically significant increase in IL-2 secretion compared to AB122 treatment alone (Figure 12B).In summary, 0.1 µg / mL Hu24F8.2-IgG1 significantly increased IL-2 concentrations (compared to isotypes) in 6 / 10 healthy individuals' PBMC samples (1.1-3.4-fold) and 2 / 5 cancer individuals' PBMC samples (1.6-1.7-fold); 1 µg / mL Hu24F8.2-IgG1 significantly increased IL-2 concentrations (compared to isotypes) in 7 / 10 healthy individuals' PBMC samples (1.4-4.0-fold) and 4 / 7 cancer individuals' PBMC samples (1.6-2.0-fold); 10 µg / mL Hu24F8.2-IgG1 significantly increased IL-2 concentrations (compared to isotypes) in 7 / 10 healthy individuals' PBMC samples (1.2-4.0-fold) and 4 / 7 cancer individuals' PBMC samples (1.3-2.0-fold); and 10 µg / mL Hu24F8.2-IgG1+ AB122 increased IL-2 concentrations in 6 / 10 healthy individual PBMC samples (1.9–8.3-fold increase compared to AB122 alone). Table 14. Mean aIL-2 levels (pg / mL) in human healthy individual PBMCs. individual 223 225 226 229 272 273 566 967 568 969 0.1 µg / mL Same type 452.8 3282 1552 2629 nt nt 717.9 643.5 509.6 2625 Hu24F8.2-IgG1 377.8 3735 2353 3837 nt nt 1763 998.8 1725 6706 Significance b NS * * * - - ** NS *** ** Multiple change 0.83 1.13 1.51 1.45 - - 2.45 1.55 3.38 2.55 1.0 µg / mL Same type 385.2 3276 1920 2517 1035 321.4 735.9 565.1 510.6 2442 Hu24F8.2-IgG1 394.3 3671 2361 3588 1944 1296 2268 1270 1919 8256 Significance b NS NS NS * *** **** *** * **** **** Multiple change 1.02 1.12 1.22 1.42 1.88 4.03 3.08 2.24 3.75 3.38 10.0 µg / mL Same type 380.1 3269 2017 3448 1385 673.1 804.4 854.9 519.9 2847 Hu24F8.2-IgG1 294.5 3775 2329 4175 1779 1006 2515 1649 1949 8510 Significance b NS * NS * NS * **** * **** **** Multiple change 0.77 1.15 1.15 1.21 1.87 4.03 3.12 1.92 3.74 2.98 Same type 430.2 3421 2070 3025 1154 785 928.1 733.3 593.4 nt AB122 1080 5628 3455 4841 1728 1453 2650 2074 1593 nt 1 µg / ml AB122+ 10 µg / ml Hu24F8.2-IgG1 1222 5762 3672 7077 2240 2067 7665 3614 4510 nt AB122 and AB122 + Hu24F8.2-IgG1 Significance b NS NS NS * * *** **** **** **** - Multiple change 2.84 1.68 1.77 2.33 1.94 2.63 8.25 4.92 7.6 - One-way ANOVA of repeated bSidak multiple comparison tests using an=3 technique (isotype with Hu24F8.2 or AB122 + Hu24F8.2 with AB122); NS, not significant; nt, not tested; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Table 15. Mean aIL-2 levels (pg / mL) in PBMCs of human cancer individuals. individual 3 29 60 33 27 6 12 0.1 µg / mL Same type nt 84.68 nt 47.44 1097 3438 1349 Hu24F8.2-IgG1 nt 98.07 nt 87.85 1874 5447 1871 Significance b - NS - NS *** **** NS Multiple change - 1.16 - 1.85 1.71 1.58 1.38 1.0 µg / mL Same type 1583 95.57 792.3 170 1068 3396 1295 Hu24F8.2-IgG1 2074 91.4 1557 119.8 1746 5992 2221 Significance b NS NS * NS ** **** ** Multiple change 1.31 0.95 1.96 0.7 1.63 1.76 1.71 10.0 µg / mL Same type 1600 95.34 674.7 129.7 1153 3885 1765 Hu24F8.2-IgG1 1897 53.54 1363 94.35 1934 5073 3089 Significance b NS NS * NS ** ** **** Multiple change 1.18 0.56 2.02 0.72 1.67 1.3 1.75 One-way ANOVA with repeatable bSidak multiple comparison test (isotype and Hu24F8.2) using an=3 technique; NS, not significant; nt, not tested; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

[0251] These data confirm that Hu24F8.2-IgG1, alone or in combination with anti-PD-1 antibodies (such as AB122), enhances the primary T-cell response in healthy or cancerous individuals.

[0252] Example 8. Antibody Characterization by In Vitro Complement-Dependent Cytotoxicity (CDC) Assay. CDC is an immune response in which the complement system participates in antibody-dependent cell killing via the binding of complement component 1q (C1q) to the crystallizable (Fc) region of an antibody fragment. This triggers a complement cascade reaction, leading to the formation of membrane attack complexes that damage the cell membrane of target cells that express 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).

[0253] Cell lysis of CDC was determined using the Cell Titer-Glo® Assay Kit (Promega), which quantifies the number of viable cells in the culture based on ATP. In short, GS-J1 / TIGIT cells (5000 cells / well) were treated with 10 μg / mL Hu24F8.2-IgG1 or human IgG1 (Abcam) negative controls at 37°C and 5% CO2 in the presence of 5%, 10%, or 20% NHSC for 4 hours (%NHSC optimized analysis), or GS-J1 / TIGIT cells (5000 cells / well) were treated with serially diluted Hu24F8.2-IgG1 (10 μg / mL) or human IgG1 (10 μg / mL) at 37°C and 5% CO2 in the presence of 5% NHSC for 4 hours (CDC concentration effect study). As a positive control, Raji cells (ATCC CCL-86, (5000 cells / well)) were treated with serially diluted rituximab (10 μg / mL) for 4 hours at 37°C and 5% CO2 in the presence of 5% NHSC. After incubation with the test or control antibody, cell titer-Glo® reagent was added, and the samples were incubated at room temperature for 10–30 minutes, with luminescence read on a PHERAstar FSX (BMG LabTech). Cell lysis attributable to CDC was calculated using the following formula: Cell lysis % = 100% × (1 - (RLU sample - RLUNHSC) / (RLU cells + NHSC - RLUNHSC)).

[0254] The results of the systemic control (rituximab against Raji cells) met quality control standards in both tests. However, no concentration-dependent CDC activity was observed in the %NHSC optimization analysis or the CDC concentration-responsiveness study for either the test sample (Hu24F8.2-IgG1) or the negative control (human IgG1). The results are summarized in Tables 16 and 17, respectively. FACS analysis of Hu24F8.2-IgG1 bound to GS-J1 / TIGIT cells confirmed that the lack of CDC was not due to a lack of target cell binding (data not shown). Table 16. Results of %NHSC optimization analysis sample Concentration (μg / mL) target cells % NHSC Average percentage of target cell lysis EC 50 (µg / mL) Rituximab 10 Raji 5% 93.9 0.414 Hu24F8.2-IgG1 10 GS-J1 / TIGIT 5% 5.77 N / A 10% 17.5 20% -15.5 Human IgG1 10 GS-J1 / TIGIT 5% 5.39 N / A 10% 8.44 20% -12.3 Table 17. Results of the CDC concentration response study sample Maximum concentration (μg / mL) target cells % NHSC EC 50 (µg / mL) Rituximab 10 Raji 5% 0.275 Hu24F8.2-IgG1 10 GS-J1 / TIGIT 5% N / A Human IgG1 10 GS-J1 / TIGIT 5% N / A

[0255] Example 9. Antibody Characterization by SPR Using a BioRad ProteOn XPR36 instrument, antibodies were captured on six different densities of anti-human IgG coated wafers or protein A coated wafers to analyze Hu24F8.2-IgG1. The analyte was soluble hTIGIT-His (stock solution prepared in 33.3 μM), diluted to 33 nM as the highest concentration, and tested in triplicate serially on the surface of Hu24F8.2-IgG1 at three-fold dilutions. The run 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. Local Rmax was used to globally fit the data from all six surface densities into a 1:1 interaction model. The results are shown in Table 18. Table 18. Kinetic Binding Constants of Hu24F8.2-IgG1 to Human TIGIT Capture Method k a (M -1 s -1 ) k d (s -1 ) K D (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: The numbers in parentheses represent the standard error in the final report of the global fit from 6 surfaces of different densities. [Simplified Explanation of the Diagram]

[0256] Figure 1A shows the amino acid sequences of mature VH (SEQ ID NO: 1) and mature VL (SEQ ID NO: 2) of 21F8. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 36), HC-CDR2 (SEQ ID NO: 37) and HC-CDR3 (SEQ ID NO: 38), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 39), LC-CDR2 (SEQ ID NO: 40) and LC-CDR3 (SEQ ID NO: 41), respectively.

[0257] Figure 1B shows the amino acid sequences of mature VH (SEQ ID NO: 3) and mature VL (SEQ ID NO: 4) of 30M18. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 42), HC-CDR2 (SEQ ID NO: 43) and HC-CDR3 (SEQ ID NO: 44), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 45), LC-CDR2 (SEQ ID NO: 46) and LC-CDR3 (SEQ ID NO: 47), respectively.

[0258] Figure 1C shows the amino acid sequences of mature VH (SEQ ID NO: 5) and mature VL (SEQ ID NO: 6) of 24F8. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49) and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52) and LC-CDR3 (SEQ ID NO: 53), respectively.

[0259] Figure 1D shows the amino acid sequences of mature VH (SEQ ID NO: 7) and mature VL (SEQ ID NO: 8) of 5J24. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 54), HC-CDR2 (SEQ ID NO: 55) and HC-CDR3 (SEQ ID NO: 56), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 57), LC-CDR2 (SEQ ID NO: 58) and LC-CDR3 (SEQ ID NO: 59), respectively.

[0260] Figure 1E shows the amino acid sequences of mature VH (SEQ ID NO: 9) and mature VL (SEQ ID NO: 10) of 21B9. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 60), HC-CDR2 (SEQ ID NO: 61) and HC-CDR3 (SEQ ID NO: 62), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 64) and LC-CDR3 (SEQ ID NO 65), respectively.

[0261] Figure 1F shows the amino acid sequences of mature VH (SEQ ID NO: 11) and mature VL (SEQ ID NO: 12) of 22B22. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 60), HC-CDR2 (SEQ ID NO: 66) and HC-CDR3 (SEQ ID NO: 67), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68) and LC-CDR3 (SEQ ID NO 65), respectively.

[0262] Figure 1G shows the amino acid sequences of mature VH (SEQ ID NO: 13) and mature VL (SEQ ID NO: 14) of 28P24. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 69), HC-CDR2 (SEQ ID NO: 55) and HC-CDR3 (SEQ ID NO: 70), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 71), LC-CDR2 (SEQ ID NO: 68) and LC-CDR3 (SEQ ID NO 65), respectively.

[0263] Figure 1H shows the amino acid sequences of mature VH (SEQ ID NO: 15) and mature VL (SEQ ID NO: 16) of 21B16. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 72), HC-CDR2 (SEQ ID NO: 73) and HC-CDR3 (SEQ ID NO: 67), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68) and LC-CDR3 (SEQ ID NO 65), respectively.

[0264] Figure 1I shows the amino acid sequences of mature VH (SEQ ID NO: 17) and mature VL (SEQ ID NO: 12) of 28O12. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 74), HC-CDR2 (SEQ ID NO: 75) and HC-CDR3 (SEQ ID NO: 67), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 63), LC-CDR2 (SEQ ID NO: 68) and LC-CDR3 (SEQ ID NO 65), respectively.

[0265] Figure 1J shows the amino acid sequences of mature VH (SEQ ID NO: 76) and mature VL (SEQ ID NO: 77) of Hu24F8.1. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49) and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52) and LC-CDR3 (SEQ ID NO: 53), respectively.

[0266] Figure 1K shows the amino acid sequences of mature VH (SEQ ID NO: 78) and mature VL (SEQ ID NO: 77) of Hu24F8.2. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49) and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52) and LC-CDR3 (SEQ ID NO: 53), respectively.

[0267] Figure 1L shows the amino acid sequences of mature VH (SEQ ID NO: 78) and mature VL (SEQ ID NO: 79) of Hu24F8.3. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49) and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52) and LC-CDR3 (SEQ ID NO: 53), respectively.

[0268] Figure 1M shows the amino acid sequences of mature VH (SEQ ID NO: 76) and mature VL (SEQ ID NO: 79) of Hu24F8.4. The CDR1, CDR2 and CDR3 amino acid sequences of VH are underlined and identified as HC-CDR1 (SEQ ID NO: 48), HC-CDR2 (SEQ ID NO: 49) and HC-CDR3 (SEQ ID NO: 50), respectively. The CDR1, CDR2 and CDR3 amino acid sequences of VL are underlined and identified as LC-CDR1 (SEQ ID NO: 51), LC-CDR2 (SEQ ID NO: 52) and LC-CDR3 (SEQ ID NO: 53), respectively.

[0269] Figure 2 shows that Ch24F8, Ch28O12, and Ch22B22 can bind to TIGIT expressed on CD4+ and CD8+ cells of cynomolgus monkeys. The geometric mean of fluorescence intensity (gMFI) was obtained, and the data are presented as the fold increase of gMFI relative to the isotype control.

[0270] Figure 3A shows the binding of humanized anti-TIGIT antibody to CHO-K1 cells that overexpress human TIGIT.

[0271] Figure 3B shows the binding of humanized anti-TIGIT antibody to CHO-K1 cells of cynomolgus monkeys that overexpress TIGIT.

[0272] Figure 4A shows the binding of humanized anti-TIGIT to CHO-K1 cells overexpressing mouse TIGIT.

[0273] Figure 4B shows the binding of humanized anti-TIGIT to CHO-K1 cells overexpressing rat TIGIT.

[0274] Figure 5A shows the binding of humanized anti-TIGIT antibody to human unactivated CD8+ T cells.

[0275] Figure 5B shows the binding of humanized anti-TIGIT antibody to human activated CD8+ T cells.

[0276] Figure 6 shows the inhibition of human CD155 binding to CHO-K1 cells that overexpress human TIGIT by humanized anti-TIGIT antibody.

[0277] Figure 7 shows the inhibition of human CD155 binding to human TIGIT by humanized anti-TIGIT antibody in the Jurkat dual reporter somatic cell line blocking analysis.

[0278] Figure 8 shows the combination of Fab24F8 and TIGIT.

[0279] Figure 9 shows the TIGIT residues that have hydrogen bonds, salt bridges and van der Waals interactions with Fab24F8.

[0280] Figures 10A and 10B illustrate the binding of CD155 to TIGIT blocked by Fab24F8. Figure 10A shows a schematic diagram of the complex structure of human CD155 (represented as bands) bound to human TIGIT (represented as molecular surfaces). Figure 10B shows a schematic diagram of CD155 superimposed on a crystal structure complex of Fab24F8 bound to TIGIT (represented as the surface of each free molecule) in the same orientation as in Figure 10A.

[0281] Figure 11 shows the IL-2 response of a single individual to SEA in the presence of Hu24F8.2-IgG1, AB122, AB122, and Hu24F8.2-IgG1 or isotype control. Bar plots and errors depict mean ± standard error mean. ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA of Sidak multiple comparison test (Hu24F8.2-IgG1 and IgG1 at each concentration and AB122 + Hu24F8.2-IgG1 and AB122 alone or Hu24F8.2-IgG1 alone).

[0282] Figure 12A shows the IL-2 response of PBMCs in healthy or cancerous individuals to SEA in the presence of Hu24F8.2-IgG1 or isotype controls. Each symbol represents an individual. *p<0.05, paired t-test.

[0283] Figure 12B shows the IL-2 response of healthy individuals' PBMCs to SEA in the presence of AB122 compared to AB122 and Hu24F8.2-IgG1. Each symbol represents an individual. *p<0.05, paired t-test. [Sequence List]

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Claims

1. An anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, comprising: a heavy chain variable region comprising HC-CDR1 having the amino acid sequence of SEQ ID NO: 48, HC-CDR2 having the amino acid sequence of SEQ ID NO: 49, and HC-CDR3 having the amino acid sequence of SEQ ID NO: 50; and a light chain variable region comprising LC-CDR1 having the amino acid sequence of SEQ ID NO: 51, LC-CDR2 having the amino acid sequence of SEQ ID NO: 52, and LC-CDR3 having the amino acid sequence of SEQ ID NO:

53.

2. The anti-TIGIT antibody or its antigen-binding fragment as claimed in claim 1, comprising (a) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 5; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 6; (b) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 76; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 77; (c) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 78; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 77; (d) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 76; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 79; or (e) a heavy chain variable region having at least 80% sequence identity with SEQ ID NO: 78; and a light chain variable region having at least 80% sequence identity with SEQ ID NO: 79; 79 Light chain variable regions with at least 80% sequence identity.

3. The anti-TIGIT antibody or its antigen-binding fragment as claimed in claim 1, wherein the anti-TIGIT antibody or its antigen-binding fragment is a monoclonal antibody.

4. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a chimeric, humanized, or face-modified antibody.

5. The anti-TIGIT antibody or its antigen-binding fragment as claimed in claim 4, wherein the chimeric antibody contains a human IgG1 / κ Fab constant domain.

6. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof is a human antibody.

7. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof inhibits the binding of TIGIT to CD155.

8. The anti-TIGIT antibody or its antigen-binding fragment as claimed in claim 1, wherein the antibody further comprises a variable heavy chain constant region selected from variant human IgG1, variant human IgG2, variant human IgG3 or variant human IgG4, and a human light chain constant region.

9. The anti-TIGIT antibody or its antigen-binding fragment as claimed in claim 8, wherein the variant heavy chain constant region has enhanced or reduced effector function relative to the wild-type heavy chain constant region.

10. The anti-TIGIT antibody or its antigen-binding fragment as claimed in claim 9, wherein the constant region of the human IgG heavy chain of the variant contains SEQ ID NO: 97, SEQ ID NO: 99 or SEQ ID NO:

101.

11. The anti-TIGIT antibody or its antigen-binding fragment as claimed in claim 1, wherein the antibody further comprises a constant region of the wild-type human IgG heavy chain and a constant region of the human light chain.

12. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 11, wherein the constant region of the wild-type human IgG heavy chain contains SEQ ID NO:

94.

13. The anti-TIGIT antibody or antigen-binding fragment thereof of claim 11, comprising a human light chain κ constant region, wherein the human light chain constant region comprises SEQ ID NO:

95.

14. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 1, 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.

15. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or its binding fragment (a) has an equilibrium binding constant (KD) of 0.01 × 10⁻¹¹ M to 100 × 10⁻¹¹ M, 0.1 × 10⁻¹¹ M to 100 × 10⁻¹¹ M, 0.1 × 10⁻¹¹ M to 10 × 10⁻¹¹ M, 1 × 10⁻¹¹ M to 100 × 10⁻¹¹ M or 1 × 10⁻¹¹ M to 10 × 10⁻¹¹ M as measured by surface plasma resonance; and (b) blocks the binding of soluble human CD155 ligand to human TIGIT on the cell surface at a half-maximum inhibitory concentration (IC50) of 0.2 nM to 2 nM, 0.2 nM to 0.8 nM, 0.6 nM to 0.8 nM or 0.6 nM to 0.8 nM. (c) Conjugated to an antigenic determinant comprising 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; (iii) D72 and K82 of SEQ ID NO: 80; (iv) E60, D72 and K82 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).

16. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 15, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof as claimed in claim 1 for binding to TIGIT.

17. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 15, wherein, as measured in a competitive binding assay, an excess of the antibody or antigen-binding fragment thereof competitively binds to TIGIT at least 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%, 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.

18. An anti-TIGIT antibody or antigen-binding fragment thereof that specifically binds to human TIGIT, comprising 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.

19. An in vitro method for inhibiting the binding of TIGIT to CD155, comprising contacting TIGIT with an anti-TIGIT antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 18.

20. Use of an anti-TIGIT antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 18, for the preparation of a medicament for treating cancer, wherein the cancer can be treated by inhibiting the binding of TIGIT to CD155.

21. As claimed in claim 20, wherein the cancer is a hematologic malignancy, Merkel cell carcinoma, urothelial carcinoma, 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, ganglion cancer, cancers of the central nervous system (CNS) and peripheral nervous system (PNS), as well as cancers of the hematopoietic system and the immune system.

22. As claimed in claim 20, wherein the individual is given tumor-infiltrating T cells activated by the antibody or its antigen-binding fragment.

23. As claimed in claim 20, wherein the individual is given a vaccine that induces an immune response against the cancer, the immune response being enhanced by the antibody or an antigen-binding fragment thereof.

24. As claimed in claim 23, wherein the vaccine contains an antigen or fragment thereof expressed on the surface of cancer cells.

25. As claimed in claim 20, wherein natural killer cells are administered to the individual, the cytotoxicity of these cells against the cancer being enhanced by the antibody or its antigen-binding fragment.

26. As claimed in claim 20, wherein the individual is further administered a second antibody against an antigen expressed on the surface of cancer cells, wherein the effector-mediated cytotoxicity of the second antibody against the cancer is enhanced by the antibody or its antigen-binding fragment.

27. As claimed in claim 20, wherein the individual is further administered a second antibody against an antigen expressed on the surface of an immune cell.

28. As used in claim 27, wherein the immune cell line is a T cell or a natural killer cell.

29. As claimed in claim 26, wherein the antigen is CTLA-4, PD-1, or PD-L1.

30. As claimed in claim 20, wherein the individual is further given one or more therapies selected from the group consisting of chemotherapy, radiation, cell-based therapies and surgery.

31. As claimed in claim 20, wherein the individual is further administered an inhibitor of one or more immune checkpoint receptors or ligands.

32. As claimed in claim 31, 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, A2aR, A2bR, A2a / A2bR, arginase, CD39, CD73, IDO, and TDO.

33. As claimed in claim 31, wherein the inhibitor is selected from the group consisting of: ipilimumab, tremelimumab, nivolumab, pembrolizumab, lambrolizumab, cemiplimab, tislelizumab, zimberelimab, durvalumab, and atezolizumab.

34. A pharmaceutical composition comprising an anti-TIGIT antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

35. An anti-TIGIT antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof binds to an antigenic determinant of human TIGIT, the antigenic determinant comprising at least one amino acid residue of SEQ ID NO 80: T55, Q56, N58, E60, D72, S80 and K82.

36. The anti-TIGIT antibody or antigen-binding fragment thereof as claimed in claim 15, wherein the antibody or its binding fragment is bound to an antigenic determinant comprising at least one of the following TIGIT residues: (i) E60 and D72 of SEQ ID NO: 80 and at least one of T55, Q56, N58, S80 and K82 of SEQ ID NO: 80; (ii) D72 and K82 of SEQ ID NO: 80 and at least one of T55, Q56, N58, E60 and S80 of SEQ ID NO: 80; or (iii) E60, D72 and K82 of SEQ ID NO: 80 and at least one of T55, Q56, N58 and S80 of SEQ ID NO: 80.

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