EA041301B1 - ANTIBODIES TO TIGIT - Google Patents

Description

Cross-reference to related applications

This application claims priority to U.S. Provisional Application No. 62/304045, filed March 4, 2016, and U.S. Provisional Application No. 62/413025, filed October 26, 2016, the disclosures of which are hereby incorporated by reference in their entirety. .

The field of technology to which the invention belongs

The present application provides, inter alia, monoclonal antibodies that specifically bind to immune checkpoint molecules, thus leading to significant activation of immune cells, as well as the use of such antibodies for the treatment of cancer and infectious disease, among others.

State of the art

The antigen-specific immune response is a complex biological process that is controlled by several levels of positive and negative regulators. T cells are initially stimulated through the T cell receptor (TCR) by recognition of their cognate peptide antigen presented by major histocompatibility complex (MHC) molecules on antigen presenting cells. Optimal T cell activation requires a second signal provided by co-stimulatory molecules such as CD28. The immune response is then upregulated by costimulatory molecules such as OX40, GITR and 4-1BB, which belong to the TNF receptor superfamily, and downregulated by checkpoint molecules such as PD-1 and CTLA-4. The function of checkpoint molecules is to prevent unwanted overreaction of the immune system in the body; however, they also limit the ability of the immune system to effectively fight cancer and infection. Blocking PD-1 or CTLA-4 function with an antagonistic monoclonal IgG antibody has been reported to be effective for human cancer immunotherapy (reviewed by Pardoll, Nat. Rev. Cancer, 12:252-264, 2012; Mahoney et al., Nat. Rev. Drug Discov. 14:561-584, 2015; Shin et al., Curr. Opin. Immunol. 33:23-35, 2015; Marquez-Rodas et al. Ann. Transl. Med. 3:267, 2015).

Other checkpoint molecules such as TIM-3, LAG-3, TIGIT, BTLA and VISTA have been reported (Mercier et al., Front. Immunol. 6:418, 2015). TIGIT (T-cell immunoreceptor with Ig and ITIM domains), a member of the immunoglobulin superfamily with a tyrosine-based immunoreceptor inhibitory motif (ITIM) in the cytoplasmic terminal segment, is expressed on subgroups of activated T cells and natural killer (NK) cells (Yu et al. , Nat Immunol 10:48-57, 2009). TIGIT is known to interact with CD155 (also called PVR and necl-5), CD112 (also called PVRL2 and nectin-2), and possibly CD113 (also called PVRL3 and nectin-3) (Mercier et al., supra; Martinet et al. ., Nat Rev Immunol 15:243-254, 2015). Binding of TIGIT to high-affinity CD155 ligands, which are expressed on antigen-presenting cells, has been reported to suppress T cell and NK cell function (Mercier et al., supra; Joller et al., J. Immunol. 186: 1338-1342, 2011; Stanietsky et al., Eur. J. Immunol. 43:2138-2150, 2013; Li et al., J. Biol. Chem. 289:17647-17657, 2014; Zhang et al. Cancer Immunol. Immunother. Epub on Feb. 3, 2016). TIGIT has also been reported to inhibit T cells indirectly by modulating dendritic cell cytokine production (Yu et al., supra).

Tumors create a highly suppressive microenvironment where infiltrating T cells are depleted and NK cells are downregulated by checkpoint molecules such as PD-1 and TIGIT to evade immune responses (Johnston et al., Cancer Cell. 26:926-937, 2014; Chauvin et al., J. Clin. Invest. 125:20462058, 2015; Inozume et al., J. Invest. Dermatol. Epub on Oct 12, 2015). High levels of TIGIT expression on CD8+ T cells have been reported to correlate with poor clinical outcome in AML patients (Kong et al., Clin. Cancer Res. Epub on Jan. 13, 2016). Functional defects in TIGIT+ depleted CD8+ T cells in AML patients have been reported to be reversible by miRNA-mediated knockdown of TIGIT expression (Kong et al., supra). It has also been reported that effector CD8+ T cells in HIV infection in the blood and HCV (swine influenza virus) infection in lymphoid tissue show higher levels of TIGIT (Chew et al., PLOS Pathogens, 12:e1005349, 2016). In addition, ex vivo blocking of TIGIT with an antibody has been reported to restore virus-specific CD8+ T cell effector responses.

Disclosure of the essence of the invention

The invention relates, inter alia, to an antibody that competes with any of TIG1, TIG2 or TIG3 for binding to human TIGIT. The TIG1 antibody is characterized by the mature light chain variable region of SEQ ID NO: 14 and the mature heavy chain variable region of SEQ ID NO: 10, the TIG2 antibody is characterized by the mature light chain variable region of SEQ ID NO: 22 and the mature heavy chain variable region of SEQ ID NO: 18, and the TIG3 antibody is characterized by the mature light chain variable region of SEQ ID NO: 30 and the mature heavy chain variable region of SEQ ID NO: 26, for specific binding to TIGIT. Some antibodies bind to the same epitope on human TIGIT as TIG1, TIG2, or TIG3. Some antibodies inhibit the binding of human TIGIT to CD155. Some antibodies include three light chain CDRs and three heavy chain CDRs, essentially from the corresponding three light chain CDRs and three heavy chain CDRs from TIG1. Some antibodies include three light chain CDRs and three heavy chain CDRs TIG1, TIG2, or TIG3. Some antibodies include three

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Heavy chain CDRs defined by Cabot nomenclature and three light chain CDRs defined by Cabot nomenclature, any of TIG1 (SEQ ID NO: 15-17 light chain and 11-13 heavy chain), TIG2 (SEQ ID NO: 23-25 light chain, 19-21 heavy chain) or TIG3 (SEQ ID NOs: 31-33 light chain and 27-29 heavy chain).

Some monoclonal antibodies bind to the human TIGIT epitope containing residues 35 and 37 of SEQ NO: 1 and/or residues 49 and 51 of SEQ ID NO: 1. Some monoclonal antibodies bind to the human TIGIT epitope containing residues 35, 37, 49 and 51 of SEQ ID NO: 1. Some monoclonal antibodies bind to a peptide consisting of residues 35-51 of SEQ ID NO: 1 and no more than five flanking amino acids from SEQ ID NO: 1 on both sides. Some monoclonal antibodies bind to a peptide consisting of residues 35-51 of SEQ ID NO: 1. Some such monoclonal antibodies bind to an epitope consisting of 3-20 contiguous residues of SEQ ID NO: 1.

Some antibodies are chimeric, humanized, veneered, or human. Some antibodies have the human IgG1 kappa isotype. Some antibodies have the human IgG4 kappa isotype. The antibody may be an intact antibody or a single chain antibody, Fab or F(ab')2 fragment.

The invention also relates to a pharmaceutical composition containing any of the above antibodies and a pharmaceutically acceptable carrier.

The invention also relates to methods for treating or preventing cancer in a patient, comprising administering to a patient suffering from cancer or having an increased risk of developing cancer, any of the above antibodies in an effective manner. In some methods, the patient is suffering from acute myeloid leukemia or adult T-cell leukemia.

In other embodiments, the implementation of the present invention relates to the use of antibodies described in this application, in combination with inhibitors of immune checkpoints. Blockade of immune checkpoints, which leads to the amplification of antigen-specific T-cell responses, has been shown to be a promising approach in the treatment of human cancer. Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated in various tumor cell types that are candidates for blockade, include PD-1 (programmed cell death protein 1); PD-L1 (programmed cell death ligand-1); BTLA (B and T lymphocyte attenuator); CTLA-4 (antigen 4 cytotoxic T-lymphocytes); TIM-3 (T cell membrane protein 3); LAG-3 (lymphocyte activation gene 3); V-domain immunoglobulin suppressor of T-cell activation (VISTA); CD96; A2aR (adenosine A2a receptor); A2bR (adenosine A2b receptor); CD73 (ecto-5'-nucleotidase); CD39 (ENTPD1, NTPDasel); Arginase; indolamine-pyrrole 2,3-dioxygenase (IDO); tryptophan 2,3 dioxygenase (TDO) and inhibitory killer receptors. Immune checkpoint inhibitors and combination therapy using such inhibitors are discussed in detail in this application.

The invention also provides methods for treating an individual infected with a pathogen, comprising administering to the individual any of the above antibodies in an effective manner. In some methods, the pathogen is HIV or HCV. In other methods, the pathogen is a viral, bacterial, fungal, or protozoan pathogen.

In additional aspects, provided herein is an anti-TIGIT antibody that binds to a TIGIT polypeptide at one or more amino acid residues including D51, wherein the TIGIT polypeptide has an amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments, the antibody is a monoclonal antibody . In some embodiments of any of the embodiments disclosed herein, the antibody is chimeric, humanized, or veneered. In some embodiments, the antibody is a human antibody. In some embodiments of any of the embodiments disclosed in this application, the antibody does not bind to one or more amino acid residues, including L44, 147 or H55. In some embodiments of any of the embodiments disclosed herein, the antibody comprises a mature light chain variable region of SEQ ID NO: 14 and a mature heavy chain variable region of SEQ ID NO: 10. In some embodiments of any of the embodiments disclosed herein application, the antibody binds to the same epitope as TIG1 at the amino acid sequence corresponding to SEQ ID NO: 1. In some embodiments of any of the embodiments disclosed herein, the antibody comprises three light chain CDRs comprising SEQ ID NO: : 15-17, and three heavy chain CDRs comprising SEQ ID NOs: 11-13. In some embodiments of any of the embodiments disclosed herein, the antibody comprises a mature heavy chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 35 and a mature light chain variable region that is at least 90 % is identical to the sequence of SEQ ID NO: 37. In some embodiments, the mature heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35, and the mature

- 2 041301 the light chain variable region comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments of any of the embodiments disclosed herein, the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 40, with the proviso that a C-terminal lysine may or may not be present and the mature light chain variable region is linked to a light chain constant region comprising SEQ ID NO: 41.

In the following aspects, the present application provides a monoclonal antibody that competes with any of TIG1, TIG2, or TIG3 for binding to human TIGIT, wherein the TIG1 antibody is characterized by the mature light chain variable region of SEQ ID NO: 14 and the mature heavy chain variable region of SEQ ID NO: 10, the TIG2 antibody is characterized by the mature light chain variable region of SEQ ID NO: 22 and the mature heavy chain variable region of SEQ ID NO: 18, and the TIG3 antibody is characterized by the mature light chain variable region of SEQ ID NO: 30 and the mature heavy chain variable region of SEQ ID NO: : 26, for specific binding to CD155. In some embodiments, the antibody binds to the same epitope on human TIGIT as any of TIG1, TIG2, or TIG3. In some embodiments of any of the embodiments disclosed herein, the antibody inhibits the binding of CD155 to human TIGIT. In some embodiments, the antibody comprises three light chain CDRs and three heavy chain CDRs corresponding to three light chain CDRs and three heavy chain CDRs of any one of TIG1, TIG2, or TIG3. In some embodiments, the antibody comprises three heavy chain CDRs and three light chain CDRs of any of TIG1, TIG2, or TIG3. In some embodiments, the antibody comprises three heavy chain CDRs defined by Cabot nomenclature and three light chain CDRs defined by Cabot nomenclature, any of TIG1 (SEQ ID NOS: 15-17 light chain and 11-13 heavy chain), TIG2 ( SEQ ID NOs 23-25 light chain, 19-21 heavy chain) or TIG3 (SEQ ID NOs: 31-33 light chain and 27-29 heavy chain). In some embodiments of any of the embodiments disclosed herein, the antibody is chimeric, humanized, or veneered. In some embodiments, the antibody is a human antibody. In some embodiments of any of the embodiments disclosed herein, the antibody is of the human IgG1 kappa isotype. In some embodiments of any of the embodiments disclosed herein, the antibody is an intact antibody. In some embodiments of any of the embodiments disclosed herein, the antibody is a single chain antibody, Fab, or F(ab') ₂ fragment. In some embodiments of any of the embodiments disclosed herein, the antibody comprises a mature heavy chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 35 and a mature light chain variable region that is at least 90 % identical to the sequence of SEQ ID NO: 37. In some embodiments, the mature heavy chain variable region is at least 95 or 99% identical to the sequence of SEQ ID NO: 35, and the mature light chain variable region is at least 95 or 99% identical to the sequence of SEQ ID NO: 37. In some embodiments, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 35, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 37. In some embodiments, the mature heavy chain variable region is linked to a constant heavy chain region comprising SEQ ID NO: 40, with y provided that a C-terminal lysine may or may not be present and the mature light chain variable region is linked to a light chain constant region comprising SEQ ID NO: 41. In some embodiments, the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 60, with the proviso that a C-terminal lysine may or may not be present and the mature light chain variable region is linked to a light chain constant region comprising SEQ ID NO: 64. In some embodiments, the mature heavy chain variable region is linked with a heavy chain constant region comprising SEQ ID NO: 61, provided that the C-terminal lysine may or may not be present and the mature light chain variable region is linked to the light chain constant region comprising SEQ ID NO: 64. In some embodiments, implementation of any of the embodiments disclosed in this application, the antibody contains a mature variable a heavy chain region that is at least 90% identical to the sequence of SEQ ID NO: 43; and a mature light chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 45. In some embodiments, the mature heavy chain variable region is at least 95% or 99% identical to the sequence of SEQ ID NO: 43, and the mature light chain variable region is at least 95 or 99% identical to the sequence of SEQ ID NO: 45. In some embodiments, the mature heavy chain variable region has the amino acid sequence SEQ ID NO: 43 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 45. In some embodiments, the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 48, provided that C- terminal lysine may or may not be present, and the mature light chain variable region is linked to the constant light chain region, including

- 3 041301 SEQ ID NO: 49. In some embodiments, the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 62, with the proviso that the C-terminal lysine may or may not be present, and the mature light chain variable region chain is linked to a light chain constant region comprising SEQ ID NO: 65. In some embodiments, a mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 63, provided that the C-terminal lysine may or may not be present. present, and the mature light chain variable region is linked to the light chain constant region comprising SEQ ID NO: 65. In some other aspects, the present application provides a monoclonal antibody that binds to an epitope comprising residues 35 and 37 of SEQ NO: 1 and/or residues 49 and 51 of SEQ ID NO: 1. In some embodiments, the antibody binds to an epitope including residues 35, 37, 49 and 5 1 SEQ ID NO: 1. In some embodiments, the antibody binds to a peptide consisting of residues 35-51 of SEQ ID NO: 1 and no more than five flanking amino acids from SEQ ID NO: 1 on either side. In some embodiments, the antibody binds to a peptide consisting of residues 35-51 of SEQ ID NO: 1. In some embodiments, the epitope consists of 3-20 contiguous residues of SEQ ID NO: 1. In some embodiments of any of the embodiments disclosed in of the present application, the antibody has one or more of the following properties: (a) inhibits TIGIT binding to CD155, optionally with an IC50 of 15-100 ng/mL, (b) increases natural T-cell activation in the presence of antigen-presenting cells expressing CD155, as measured in IL-2 production, optionally 1.5-3-fold, (c) increases antigen-specific T cell activation, as measured by IL-12 production, optionally 1.5-3-fold, (d) increases natural killer cell activation, as measured by production of any of IL-2, IL-6, TNFa or IFNy, optionally 1.5-3-fold, (e) increases T-cell production of at least one pro-inflammatory cytokine, optionally 1.5-3-fold, and (f) decrease aet production of at least one anti-inflammatory cytokine by T cells, optionally 1.5-3-fold.

In another aspect, this application provides pharmaceutical compositions comprising any of the antibodies described in this application, and a pharmaceutically acceptable carrier.

In other aspects, provided herein are methods for treating or preventing cancer comprising administering to an individual having cancer or an increased risk of developing cancer an effective regimen or a therapeutically effective amount of any of the antibodies disclosed herein. In some embodiments, the cancer is acute myeloid leukemia or adult T-cell leukemia. In some embodiments of any of the embodiments disclosed herein, the individual is administered tumor-infiltrating T cells that are activated by an antibody. In some embodiments of any of the embodiments disclosed herein, an individual is administered a vaccine that induces an immune response against cancer that is enhanced by an antibody. In some embodiments, the implementation of the vaccine includes an antigen or fragment thereof, expressed on the surface of cancer cells. In some embodiments of any of the embodiments disclosed herein, the individual is administered natural killer cells whose cytotoxicity against cancer is enhanced by an antibody. In some embodiments of any of the embodiments disclosed herein, the individual is also administered a second antibody against an antigen expressed on the surface of cancer cells, wherein the effector-mediated cytotoxicity of the second anti-cancer antibody is enhanced by the antibody. In some embodiments of any of the embodiments disclosed herein, the individual is also administered a second antibody against an antigen expressed on the surface of an immune cell. In some embodiments, the immune cell is a T cell or natural killer cell. In some embodiments of any of the embodiments disclosed herein, the antigen is CTLA-4, PD-1, or PD-L1. In some embodiments of any of the embodiments disclosed herein, the individual is also administered one or more treatments selected from the group consisting of chemotherapy, radiation, cell therapy, and surgery. In some embodiments of any of the embodiments disclosed herein, an inhibitor of one or more immune checkpoint receptors or ligands is also administered to the individual. In some embodiments, the inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab (lambrolizumab), and atezolizumab.

In additional aspects, the present application provides methods for treating an individual infected with a pathogen, comprising administering to the individual an effective regimen or a therapeutically effective amount of any of the antibodies disclosed in this application. In some embodiments, the implementation of the pathogen is a viral, bacterial, fungal or protozoan pathogen. In some embodiments, the pathogen is HIV, HCV, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, virus

- 4 041301 rubella virus, parvovirus, vaccinia virus, HTLV virus (human T lymphotropic virus), dengue virus, papillomavirus, mollusk virus, poliovirus, rabies virus, JC virus, arbovirus encephalitis virus, chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci , pneumococci, meningococci, gonococci, klebsiella, proteus, serration, pseudomonas, legionella, diphtheria bacillus, salmonella bacillus, bacteria that cause cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease. In some embodiments of any of the embodiments disclosed herein, the individual is treated with a vaccine that induces an immune response against a pathogen that is enhanced by an antibody. In some embodiments, the implementation of the vaccine includes a protein of the pathogen or a fragment thereof. In some embodiments of any of the embodiments disclosed herein, the individual is also administered a second antibody against the pathogen, wherein the effector-mediated cytotoxicity of the second antibody against the pathogen is enhanced by the antibody. In some embodiments of any of the embodiments disclosed herein, the individual is also administered one or more of an antiviral agent, an antiparasitic agent, an antibacterial agent, or an antifungal agent.

In another aspect, provided herein are methods to aid in the treatment of cancer comprising administering to an individual having cancer a therapeutically effective amount of any of the antibodies disclosed herein. In some embodiments, the cancer is acute myeloid leukemia or adult T-cell leukemia. In some embodiments of any of the embodiments disclosed herein, the individual is administered tumor-infiltrating T cells that are activated by an antibody. In some embodiments of any of the embodiments disclosed herein, an individual is administered a vaccine that induces an immune response against cancer that is enhanced by an antibody. In some embodiments, the implementation of the vaccine includes an antigen expressed on the surface of cancer cells, or a fragment thereof. In some embodiments of any of the embodiments disclosed herein, the individual is administered natural killer cells whose cytotoxicity against cancer is enhanced by an antibody. In some embodiments of any of the embodiments disclosed herein, a second antibody against an antigen expressed on the surface of cancer cells is administered to the individual, wherein the effector-mediated cytotoxicity of the second anti-cancer antibody is enhanced by the antibody. In some embodiments of any of the embodiments disclosed herein, the individual is also administered a second antibody against an antigen expressed on the surface of an immune cell. In some embodiments, the immune cell is a T cell or natural killer cell. In some embodiments of any of the embodiments disclosed herein, the antigen is CTLA-4, PD-1, or PD-L1. In some embodiments of any of the embodiments disclosed herein, the individual is also administered one or more treatments selected from the group consisting of chemotherapy, radiation, cell therapy, and surgery. In some embodiments of any of the embodiments disclosed herein, an inhibitor of one or more immune checkpoint receptors or ligands is also administered to the individual. In some embodiments, one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, and PD-L1. In some embodiments, the inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab (lambrolizumab), and atezolizumab.

Each of the aspects and embodiments described herein may be used together unless it is expressly or specifically excluded from the context of the embodiment or aspect.

Brief description of the drawings

Fig. 1 is a schematic representation of the structure of expression vectors for recombinant TIGIT and CD155 proteins.

Fig. 2 represents inhibition of TIGIT-CD155 interaction by anti-TIGIT antibodies.

Fig. 3 represents the amino acid sequence of TIG1 VH.

Fig. 4 represents the amino acid sequence of TIG1 VL.

Fig. 5 represents the amino acid sequence of TIG2 VH.

Fig. 6 represents the amino acid sequence of TIG2 VL.

Fig. 7 represents the amino acid sequence of TIG3 VH.

Fig. 8 represents the amino acid sequence of TIG3 VL.

Fig. 9A shows the nucleotide sequence of the HuTIG1 VH gene and the encoded amino acid sequence, while FIG. 9B shows the nucleotide sequence of the HuTIG1 VL gene and the encoded amino acid sequence.

Fig. 10 is a schematic representation of the structure of the pHuTIG1.AA expression vector.

Fig. 11 shows the results of an ELISA analysis of the binding of HuTIG1-IgG1.AA to human TIGIT.

Fig. 12 - FACS analysis of the binding of HuTIG1-IgG1.AA and HuTIG3-IgG1.AA to human TIGIT.

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Fig. 13 is a graph showing the blocking of interaction between human TIGIT and human CD155 by HuTIG1-IgG1.AA and HuTIG3-IgG1.AA.

Fig. 14A shows the nucleotide sequence of the HuTIG3 VH gene and the encoded amino acid sequence, while FIG. 14B shows the nucleotide sequence of the HuTIG3 VL gene and the encoded amino acid sequence.

Fig. 15 is an ELISA assay of the binding of HuTIG3-IgG1.AA to human TIGIT.

Fig. 16 shows increased production of IL-2 induced by TIG1 upon stimulation of an antigen-specific immune response.

Fig. 17 represents increased CD4+ and CD8+ T-cell proliferation caused by TIG1 upon stimulation of an antigen-specific immune response.

Fig. 18 shows CD155 expression on K562 cells (top) and TIGIT on NK cells (bottom).

Fig. 19 represents the increased NK cell mediated cytotoxicity induced by TIG1 on K562 target cells.

Fig. 20 shows that HuTIG1-IgG1.AA potentiates cytokine effector responses.

Fig. 21 shows the expression of human TIGIT by the Jurkat-TIGIT cell line on the cell surface.

Fig. 22 shows that HuTIG1-IgG1.AA and HuTIG3-IgG1.AA do not induce CDC activity.

Fig. 23 shows that HuTIG1-IgG1.AA and HuTIG3-IgG1.AA increased natural T cell activation in an in vitro T cell antagonist activity assay for anti-human TIGIT antibody.

Fig. 24 shows CD112, CD155 and PD-L1 expressed by GM-CSF/IL-4 monocyte-derived differentiated dendritic cells (moDCs).

Fig. 25 represents increased secretion of IFNy when added with HuTIG1-IgG1.AA, anti-PD-L1 antibody and HuTIG1-IgG1.AA in combination with anti-PD-L1 antibody.

Fig. 26A shows the expression of TIGIT and CD96 on human lymphoid and myeloid cells.

Fig. 26B is representative histograms of anti-TIGIT staining at different expression levels.

Fig. 27 shows HuTIG1-IgG1.AA epitope mapping by flow cytometry.

Fig. 28 is a three-dimensional ribbon representation of the extracellular IgV domain of human TIGIT with side chain numbered epitope amino acid residues.

Fig. 29A and 29B show the results of analysis of the CD4+ T cell subset to determine TIGIT expression.

Fig. 30A and 30B show the results of an analysis of the CD8+ T cell subset to determine TIGIT expression.

Fig. 31 shows increased NK cell-mediated cytotoxicity induced by HuTIG1-IgG1.AA and HuTIG3-IgG1.AA on K562 target cells.

Fig. 32A shows TIGIT expression on tumor-infiltrating lymphocytes from dissociated tumor samples.

Fig. 32B is representative histograms of anti-TIGIT staining at different expression levels.

Detailed description

The invention provides, inter alia, monoclonal antibodies that specifically bind to the extracellular domain of TIGIT, which is a member of the immunoglobulin superfamily with an immunoreceptor tyrosine inhibitory motif (ITIM) in the cytoplasmic terminal segment. Monoclonal antibodies inhibit TIGIT binding to CD155 and thus can activate T cells and/or NK cells. Monoclonal antibodies can be used to treat cancer and infectious disease, among other uses.

I. Definitions

Monoclonal antibodies or other biological compounds, such as a TIGIT fragment, are typically provided in isolated form. This means that the antibody or other biological compound is typically at least 50% wt./wt. free from interfering proteins and other impurities arising from its preparation or purification, but it is possible that the monoclonal antibody is combined with an excess amount of pharmaceutically acceptable carrier(s) or other excipient intended to facilitate its use. Sometimes monoclonal antibodies at least 60, 70, 80, 90, 95 or 99% wt./wt. free of interfering proteins and impurities from the manufacturing or purification process. Often, the isolated monoclonal antibody or other biological compound is the predominant macromolecular particle remaining after purification.

Specific binding of a monoclonal antibody to its target antigen means an affinity (association constant or Ka) of at least 106, 10 ⁷ , 10 ⁸ , 109 or 10 ¹⁰ M' ¹ as determined, for example, in the assay of Example 15. Specific binding is markedly higher in magnitude and different from

- 6 041301 specific binding occurring with at least one unrelated target. Specific binding may result from the formation of bonds between specific functional groups or a specific spatially relevant structure (eg, like a lock and key), while non-specific binding is usually the result of van der Waals forces. Specific binding, however, does not necessarily imply that the monoclonal antibody binds to one and only one target.

The basic structural unit of an antibody is the tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, with each pair having one light (about 25 kDa) and one heavy chain (about 50-70 kDa). The amino terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed associated with a cleaved signal peptide. The variable region without the signal peptide is sometimes referred to as the mature variable region. Thus, for example, a mature light chain variable region means a light chain variable region without a light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region predominantly responsible for effector function.

Light chains are classified as either kappa or lambda chains. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon chains and define the isotype of the antibody, such as IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a J region of about 12 or more amino acids, while the heavy chain also includes a D'' region of about 10 or more amino acids. (See, for a general description, Fundamental Immunology (Paul W., ed., 2nd ed. Raven Press, N.Y., 1989, Ch. 7, the entire contents of which are incorporated by reference in their entirety for all purposes).

The mature variable regions of each light/heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. With the exception of bifunctional or bispecific antibodies, these two binding sites are the same. All chains show the same overall structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions, or CDRs. The CDRs from the two strands of each pair are linked via framework regions, allowing binding to a specific epitope. From the N-terminus to the C-terminus, the light and heavy chains include the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The assignment of amino acids to each domain is as defined by Cabot, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). In addition, Cabat provides a widely used numbering system (Cabot numbering) in which the corresponding residues between different heavy chains or between different light chains are assigned the same number.

The term antibody includes intact antibodies and their binding fragments. Thus, any reference to an antibody should be understood as referring to the antibody in intact form or binding fragment, unless the context dictates otherwise. Typically, fragments compete with the intact antibody from which they are derived for specific target binding, including individual heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and scFv. , diabody, scFv-Fc, minibody, IgNARs, V-NAR, hcIgG, bis-scFv, triabody, and tetrabody. Fragments can be obtained by recombinant DNA methods or by enzymatic or chemical cleavage of intact immunoglobulins. The term antibody also refers to a bispecific antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites (see, for example, Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol 148: 1547-53 (1992)).

The term epitope refers to the site of an antigen to which an antibody binds. An epitope can be formed from contiguous or non-contiguous amino acids that are superimposed on each other when stacked in the tertiary structure of one or more proteins. Epitopes derived from contiguous amino acids (also known as linear epitopes) are typically retained upon exposure to denaturing solvents, while epitopes derived from tertiary structure formation (also known as conformational epitopes) are typically lost upon exposure to denaturing solvents. An epitope typically includes at least 3 or more, usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Protocols for Epitope Mapping in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can be identified using a conventional immunoassay that detects the ability of one antibody to compete for binding to a target antigen with another antibody. The epitope of an antibody can also be determined by X-ray crystallography of an antibody bound to its antigen for identification.

- 7 041301 tification of contact residues. Alternatively, two antibodies share the same epitope 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. Two antibodies have overlapping epitopes if some amino acid mutation that reduces or eliminates the binding of one antibody reduces or eliminates the binding of another antibody.

Antibody competition is determined by an assay in which the antibody, under test conditions, inhibits the specific binding of a control antibody to a common antigen (see, for example, Junghans et al., Cancer Res. 50: 1495, 1990). A test antibody competes with a control antibody if an excess of test antibody (e.g., at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x , 60x, 70x, 80x, 90x, 100x or more, including values between the indicated values) inhibits control antibody binding by at least 50%, but preferably by 75%, 90% or 99%. In other embodiments, a test antibody competes with a control antibody if an excess of test antibody inhibits binding of the control antibody by any amount of at least about 55%, 60%, 65%, 70%, or preferably at least about 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 competitive analysis binding. Antibodies identified by competitive binding methods (competing antibodies) include antibodies that bind to the same epitope as the control antibody and antibodies that bind to an adjacent epitope proximal to the epitope to which the control antibody binds to form spatial difficulty. Preferably, competition is evaluated as described in Example 14.

The term individual includes humans and other mammalian individuals. In some cases, the methods of the invention can be used in experimental animals, in veterinary medicine, and in developing animal models for disease, including, but not limited to, rodents, including mice, rats, hamsters, as well as primates such as monkeys. In some embodiments, individuals are receiving, or are candidates for, either prophylactic or therapeutic treatment.

In the context of the present application, an amino acid residue from an amino acid sequence of interest that corresponds to or is corresponding to or in accordance with an amino acid residue of a reference amino acid sequence indicates that the amino acid residue from the sequence of interest is in a position homologous or equivalent to the specified residue in the reference amino acid sequence. One skilled in the art will be able to determine if the position of a particular amino acid residue in a polypeptide, such as the TIGIT polypeptide, corresponds to the position of that residue in a homologous reference sequence. For example, the TIGIT polypeptide sequence can be aligned to a reference sequence using known methods (eg, basic local alignment finder (BLAST), ClustalW2, structure-based sequence alignment programs (STRAP), etc.). In addition, the crystal structure coordinates of a reference sequence can be used as an aid in determining the three-dimensional structure of a homologous polypeptide residue (Stengel et al., Proc. Natl. Acad. Sci., USA, 109: 5399-5404, 2012. In another aspect, equivalent residues can be identified by determining homology at the tertiary structure level.Using such methods, the amino acid residues of a TIGIT polypeptide variant can be numbered according to the amino acid position numbering of a reference sequence.For example, the amino acid sequence of SEQ ID NO: 1 can be used to determine the amino acid position numbering residues for each amino acid residue of the TIGIT variant or epitope of interest.In some embodiments, one amino acid sequence corresponds to another amino acid sequence if they share at least 3 sequence identities. here about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

For the purposes of classifying amino acid substitutions as conservative or non-conservative, the 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 (major side chains): Asn, Gln, His, Lys, Arg; Group V (residues affecting chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions include substitutions between amino acids of the same class. Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

Percent sequence identity is determined by maximal alignment of antibody sequences using the Cabot numbering system. After alignment, if an antibody region of interest (e.g., the entire mature heavy or light chain variable region) is compared to the same region of a reference antibody, then the percent sequence identity between the regions of the comparison and the reference antibody is the number of positions occupied by the same amino acid as in the region compared and reference antibodies, de

- 8 041301 divided by the total number of aligned positions of the two areas, with gaps not taken into account, multiplied by 100 to convert to a percentage.

Compositions or methods comprising one or more of the enumerated elements may include other elements not specifically listed. For example, a composition that includes an antibody may contain the antibody alone, or the antibody in combination with other ingredients.

Unless otherwise evident from the context, a range reference includes all integers within that range and all subranges defined by such integers.

II. target molecules

Unless otherwise noted, TIGIT means human TIGIT. An exemplary human sequence has been assigned Swiss-Prot accession number Q495A1. The complete human TIGIT sequence contains 244 amino acids, of which amino acids 1-21 are the signal peptide and amino acids 22-244 form the mature protein (SEQ ID NO: 1). Approximately residues 22-141 form the extracellular domain (SEQ ID NO: 3). Approximately residues 142-162 form the transmembrane domain, and approximately residues 163-244 form the cytoplasmic domain.

Unless otherwise noted, CD155 refers to the human form of this protein. An exemplary human sequence for human CD155 is designated Swiss-Prot P15151, which is a 417 amino acid protein, of which approximately residues 1-20 are the signal peptide, 21-343 are the extracellular domain (SEQ ID NO: 6), 344- 367 are the transmembrane domain and 368-417 are the cytoplasmic domain.

Unless clearly apparent from the context, reference to one of the above proteins means at least the extracellular domain of the protein, and usually the complete protein with the exception of the cleaved signal peptide.

III. Antibodies of the invention

A. Binding specificity and functional properties

The invention provides monoclonal antibodies that bind to epitopes in the extracellular domain of the TIGIT protein. The antibodies designated TIG1, TIG2, and TIG3 are three such exemplary murine antibodies. The sequences of the mature heavy and light chain variable regions of these antibodies are designated as SEQ ID NOs: 10 and 14, 18 and 22, and 26 and 30, respectively. TIG1, TIG2 and TIG3 specifically bind to the extracellular domain of human TIGIT.

Some antibodies of the invention bind to the same or overlapping epitope as an antibody designated TIG1, TIG2, or TIG3. Other antibodies with such binding specificity can be obtained by immunizing mice with TIGIT or a portion thereof, including the target epitope, and screening the resulting antibodies for binding to the extracellular domain of TIGIT, optionally in competition with TIG1, TIG2 or TIG3. Antibodies can also be screened against mutagenic forms of the TIGIT antigen to identify an antibody showing the same or similar binding profile to a collection of mutations as TIG1, TIG2, or TIG3. Mutations can be a systemic substitution of alanine (or serine if alanine is already present) for one residue at a time, or at wider intervals, throughout the extracellular domain of the TIGIT antibody, or the portion of it known to contain the epitope.

Example 16 maps residues 35, 37, 49, and 51 of SEQ ID NO: 1 as being residues that form the epitope of a TIG1 antibody. Substitution of alanine at any of these residues essentially eliminates antibody binding. The invention thus includes other antibodies that bind to a human TIGIT epitope comprising residues 35 and 37 of SEQ NO: 1 and/or residues 49 and 51 of SEQ ID NO: 1, and preferably an epitope comprising all of these residues. An epitope may be linear (eg, 3-20, 3-17, or 5-10 contiguous residues) or conformational. Some such antibodies bind to a peptide consisting of residues 35-51 of SEQ ID NO: 1 and no more than 1, 2, 3, 4, or 5 flanking amino acids from SEQ ID NO: 1 on either side. Some of these antibodies bind to a peptide consisting of residues 35-51 of SEQ ID NO: 1. Some of these antibodies can be generated by immunization with such peptides. Example 19 also shows that residue 90 of SEQ ID NO: 1 is critical for the binding of a humanized (Hu)TIG1 antibody to TIGIT, in addition to residues 35, 37, 49, and 51 of SEQ ID NO: 1. In other embodiments, the antibody (such as a HuTIG1 antibody) binds to an epitope comprising one or more of the residues corresponding to amino acid positions 35, 37, 49, 51, and/or 90 of SEQ ID NO: 1. In other embodiments, an antibody (such as a HuTIG1 antibody) does not bind to one or several residues corresponding to amino acid positions 34, 39, 44, 47, 52, 55, 86, 88, 92 and/or 96 of SEQ ID NO: 1.

Antibodies having the binding specificity of the selected mouse antibody (eg, TIG1, TIG2, or TIG3) can also be generated using a variation of the phage display method. See Winter, WO 92/20791. This method is particularly suitable for the production of human antibodies. In this method, either the heavy or light chain variable region of a selected mouse antibody is used as starting material. If, for example, a light chain variable region is chosen as the starting material, a phage library is constructed in which members exhibit the same light chain variable region (i.e., mouse starting material) and different

- 9 041301 heavy chain regions. Heavy chain variable regions, for example, can be obtained from a library of rearranged human heavy chain variable regions. Choose phage showing strong specific binding for TIGIT (for example, at least 10 ⁸ and preferably at least 10 ⁹ M ^-1 ). The heavy chain variable region from this phage then serves as starting material for the construction of an additional phage library. In this library, each of the phages displays the same heavy chain variable region (ie, the region identified from the first phage display library) and different light chain variable regions. Light chain variable regions can be obtained, for example, from a library of rearranged human light chain variable regions. Again, a phage showing strong specific binding for TIGIT is selected. The resulting antibodies typically have the same or similar epitope specificity as the mouse starting material.

Some antibodies have a mature heavy chain variable region, including CDRs H1, H2, and H3, and a mature light chain region, including CDRs L1, L2, and L3, wholly or substantially from TIG1. Some antibodies have a mature heavy chain variable region, including CDRs H1, H2, and H3, and a mature light chain variable region, including CDRs L1, L2, and L3, wholly or substantially from TIG2. Some antibodies have a mature heavy chain variable region, including CDRs H1, H2, and H3, and a mature light chain variable region, including CDRs L1, L2, and L3, wholly or substantially from TIG3. CDRs can be defined according to any accepted definition, including Cabot, Chothia, Cabot and Chothia, AbM, or Contact as shown in the table below: _______________________________

a loop Cabot ALM Chothia Contact L1 L-24--L34 L24--34 L24--L34 L30--L36 L2 L50--L56 L50--156 L50---L56 L46--L55 L3 L89--L97 L89--97 L89--L97 L89--L96 H1 H31—H35V (Numbering by Cabot) H26--H35b H26--H32..34 H30--H35V H1 H31-H35 (Numbering according to Chothia) H26--H35 H26--H32 H30--H35 H2 H50--H65 H50--H58 H52--H56 H47--H58 NZ H95--H102 H95--H102 H95--H102 H93--H10

Other antibodies can be generated by mutagenesis of cDNA encoding the heavy and light chains of an exemplary antibody, such as TIG1, TIG2, or TIG3. Monoclonal antibodies that are at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 % are identical to TIG1, TIG2 or TIG3 in the amino acid sequence of mature heavy and/or light chain variable regions and retain their functional properties, and/or which differ from the corresponding antibody by a small number of functionally non-essential amino acid substitutions (for example, conservative substitutions), deletions or insertions, also included in the invention. Amino acids in the variable region frameworks that may be important for binding can be identified as described in the humanization sections below. Monoclonal antibodies containing at least one and preferably all six CDR(s) defined by Cabot's nomenclature, which are any of the following values about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the corresponding TIG1, TIG2, or TIG3 CDRs are also included.

Antibodies preferably have one or more of the following characteristics (i) inhibition of binding of human TIGIT to human CD155, (ii) inhibition of binding of TIGIT to other ligands such as CD112 and CD113, (iii) enhancement of antigen-specific T cell responses, (iv) activation natural killer cells, (v) stimulation of natural T cell activations, and (vi) stimulation of production of one or more immunostimulatory cytokines and/or reduction of production of one or more immunosuppressive cytokines by T cells and other cells of the immune system. Illustrative assays for measuring these properties are provided in the examples.

Preferred antibodies completely or partially inhibit the binding of TIGIT to CD155. Some antibodies can inhibit this interaction with any of the following IC values of about 25-300 ng/mL, 25-75 ng/mL, 25-50 ng/mL, 40-75 ng/mL, 50-75 ng/mL, 50- 90 ng/ml, 50-100 ng/ml, 75-100 ng/ml, 50-150, 75-175 ng/ml, 100-200 ng/ml, 125-225 ng/ml, 100-250 ng/ml , 150-300 ng/ml, 175-250 ng/ml, 200-300 ng/ml, 25-275 ng/ml, 250-300 ng/ml, 49+/-10% ng/ml, 65+/- 10% ng/ml or 76+/-10% ng/ml measured as described in the examples. In other embodiments, the antibodies can completely or partially inhibit the binding of TIGIT to CD155 with any of the following IC ₅₀ values of about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 ng/mL, or more, including concentrations between these values. Some antibodies can enhance antigen-specific T-cell responses

- 10 041301 you 1.5-3 times, for example, with any of the following response gain values of about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2, 2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 times or more as measured in the examples. Some antibodies can increase production of 1, 2, 3, or all of IL-2, IL-6, TNFa, and IFNy by NK cells 1.5-3-fold, for example, with any of the following increase values of about 1.5, 1.6 , 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 times or more as measured in the examples. Some antibodies can increase natural T cell activation 1.5-3-fold, for example, with any of the following activation increase values of about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2 .1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 times or more as measured in the examples. Some antibodies can inhibit cancer or infectious disease as shown in animal models or clinical trials. Animal models of cancer in which human cancer cells are injected into an immunodeficient laboratory animal such as a mouse or rat are widely available.

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

B. Non-human antibodies

Obtaining other non-human monoclonal antibodies, for example, murine, guinea pigs, primates, rabbits, chickens or rats, against TIGIT can be done, for example, by immunizing the animal with TIGIT or its fragment or cells bearing TIGIT. See Harlow & Lane, Antobodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such an immunogen can be obtained from a natural source, by peptide synthesis or by recombinant expression. Optionally, the immunogen can be administered in a fusion or otherwise complexed with a carrier protein. Optionally, the immunogen may be administered with an adjuvant. Several types of adjuvants can be used, as described below. For immunization of laboratory animals Freund's complete adjuvant is preferred followed by incomplete adjuvant. Rabbits or guinea pigs are usually used to obtain polyclonal antibodies. Mice are commonly used to obtain monoclonal antibodies. Antibodies are screened for specific binding to TIGIT. Optionally, the antibodies are further screened for binding to a specific TIGIT region. Such screening can be done by determining the binding of the antibody to a collection of TIGIT deletion mutants and determining which deletion mutants bind to the antibody. Binding can be assessed, for example, by Western blotting, FACS or ELISA.

C. Humanized antibodies

Reducing or eliminating the response of HAMA (human anti-mouse (also applicable to human anti-rat or human anti-rabbit or human anti-hamster, etc.) antibody) is an important aspect of 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 in this application, the invention provides antibodies that are humanized so that the HAMA response is reduced or eliminated. Variants of these antibodies can then be obtained using routine methods known in the art, some of which are described below.

A humanized antibody is a genetically engineered antibody in which CDRs from a non-human donor antibody are grafted into human acceptor antibody sequences (see, for example, Queen, US 5530101 and 5585089; Winter, US 5225539; Carter, US 6407213; , Foote, US 6881557). Acceptor antibody sequences can be, for example, a mature human antibody sequence, a mixture of such sequences, a consensus sequence from human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody containing some or all of the CDRs wholly or substantially from the donor antibody, and variable region framework sequences and constant regions, if present, wholly or substantially from human antibody sequences. Similarly, a humanized heavy chain contains at least one, two, and usually all three CDRs wholly or substantially from the donor antibody's heavy chain, and a heavy chain variable region framework sequence and a heavy chain constant region, if present, substantially from human framework sequences. the variable region of the heavy chain; and the constant region. Similarly, a humanized light chain contains at least one, two, and usually all three CDRs wholly or substantially from the light chain of the donor antibody and a light chain variable region framework sequence and a light chain constant region, if present, substantially from human framework sequences. the variable region of the light chain; and the constant region. Unlike nanobodies and dAbs, a humanized antibody includes

- 11 041301 humanized heavy chain and humanized light chain. Here and throughout this application, the CDR in the subject antibody is essentially 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 respective residues (as determined by Cabot) are identical between the respective CDRs; however, the H2 CDR, as defined by Cabot's nomenclature, in the antibody in question is derived substantially from the corresponding CDR in the reference antibody when at least about 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 The % of the respective residues (as determined by Cabot) are identical between the respective CDRs. An antibody variable region framework or an antibody chain constant region is derived substantially from a human variable region framework or human constant region, respectively, 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 Cabot residues are identical.

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

In some antibodies, only a portion of the CDR, namely the set of CDR residues required for binding, called SDRs, are required to maintain binding in a humanized antibody. CDR residues not in contact with antigen and not in SDR can be identified on the basis of previous research (for example, H60-H65 residues in CDR H2 are often not required) from Cabot CDR regions located outside Chothia's hypervariable loops (Chothia, J Mol. Biol. 196:901, 1987), by molecular modeling and/or empirically or as described in Gonzales et al., Mol. Immunol. 41: 863, 2004. In such humanized antibodies, at positions where one or more of the donor CDR residues is missing, or where the entire donor CDR is missing, the amino acid occupying that position may be the amino acid occupying the corresponding position (as numbered Cabot) in the sequence of the acceptor antibody. The number of such substitutions to include acceptor amino acids instead of donor amino acids in CDRs reflects a balance of competitive considerations. Such substitutions are potentially beneficial in reducing the number of murine amino acids in a humanized antibody and, as a result, reducing potential immunogenicity. However, substitutions can also cause changes in affinity, and it is preferable to avoid a significant decrease in affinity. Positions for substitution within the CDR and amino acids for substitution can also be chosen empirically.

Although the acceptor may be sequence identical to the selected human framework sequence, whether derived from human immunoglobulin or human consensus framework sequence, the present invention contemplates that the acceptor sequence may include pre-existing amino acid substitutions relative to the human immunoglobulin sequence or human consensus framework sequence. These pre-existing substitutions are preferably minimal; typically only four, three, two, or one amino acid difference relative to the human immunoglobulin sequence or consensus framework sequence.

The human acceptor antibody sequences may optionally be selected from a variety of known human antibody sequences to provide a high degree of sequence identity (e.g., 65-85% identity) between the human acceptor sequence variable region frameworks and the corresponding donor antibody chain variable region frameworks.

Some amino acids from the framework residues of the human variable region may be selected for substitution based on their possible effect on CDR conformation and/or antigen binding. Modeling, characterization of amino acids at specific positions, or empirical observation of the effects of substitutions or mutagenesis of specific amino acids is used to explore such possible influences.

For example, in the event of amino acid differences between a non-human variable region framework residue and a selected human variable region framework residue, the human framework amino acid may be substituted with an equivalent framework amino acid from a non-human antibody when the amino acid is expected to:

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

Other candidates for substitution are amino acids of the acceptor human scaffold

- 12 041301 areas that are unusual for human immunoglobulin at this position. These amino acids can be replaced with amino acids from the equivalent position of the non-human donor antibody or from the equivalent positions of more typical human immunoglobulins. Other candidates for substitution are human acceptor framework amino acids, which are unusual for human immunoglobulin at this position.

A preferred humanized antibody contains a mature heavy chain variable region of 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: 35 and a mature light chain variable region of 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: 37. Preferably, any change can take place at variable region framework residues except those identified as possibly important for binding (see paragraph [0073]). Preferably, any change is a conservative amino acid substitution. A preferred antibody comprises a mature heavy chain variable region of SEQ ID NO: 35 and a mature light chain variable region of SEQ ID NO: 37. For full-length antibody expression, a mature heavy chain variable region is preferably linked to a heavy chain constant region consisting of, or comprising SEQ ID NO: 40, with the proviso that a C-terminal lysine may or may not be present, and the mature light chain variable region is preferably linked to a light chain constant region consisting of or including SEQ ID NO: 41.

Another preferred humanized antibody contains a mature heavy chain variable region of 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: 43 and a mature light chain variable region of at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 9θ, 91, 92, 93, 94, 95, 96, 97, 98, 99, or less than 100% identical to SEQ ID NO: 45. Preferably, any change occurs in the variable region framework residues, except for those identified as possibly important for binding (See paragraph [0073] and examples). Preferably, any change is a conservative amino acid substitution. A preferred antibody comprises a mature heavy chain variable region having the sequence of SEQ ID NO: 43 and a mature light chain variable region having the sequence of SEQ ID NO: 45. For full-length antibody expression, the mature heavy chain variable region is preferably linked to a heavy chain constant region consisting of of or including SEQ ID NO: 48, with the proviso that a C-terminal lysine may or may not be present, and the mature light chain variable region is preferably linked to a light chain constant region consisting of, or including, SEQ ID NO: 49.

D. Chimeric and veneered antibodies

The invention also provides chimeric and veneered forms of non-human antibodies, in particular the TIG1, TIG2, and TIG3 antibodies of the examples.

A chimeric antibody is an antibody in which the mature light and heavy chain variable regions of a non-human (eg, murine) antibody are present in combination with human light and heavy chain constant regions. Such antibodies essentially or completely retain the binding specificity of the non-human antibody and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains some, and usually all, of the CDRs and some of the non-human variable region framework residues of the non-human antibody, but with the replacement of other variable region framework residues that may facilitate interaction with B- or T-cell epitopes, e.g. , exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues from the corresponding positions in the human antibody sequence. The result is an antibody in which the CDRs are wholly or essentially from the non-human antibody, and the non-human antibody variable region frameworks are made more human-like by substitutions. Veneered forms of TIG1, TIG2 or TIG3 antibodies are included in the invention.

E. Human antibodies

Human anti-TIGIT antibodies are provided using the various methods described below. Some human antibodies are selected by performing competitive binding experiments, by the Winter phage display method described above, or by having the same epitope specificity as a particular mouse antibody, such as one of the mouse monoclonal antibodies described in the examples. Human antibodies can also be screened for specificity for a particular epitope using only the TIGIT fragment as the target antigen and/or by screening the antibody against a collection of TIGIT deletion mutants.

Methods for making human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, US Patent No. 4634664; and Engleman et al., US Pat. No. 4,634,666, the use of transgenic mice comprising human immunoglobulin genes (see, for example, Lonberg et al., WO93/12227 (1993); US 5877397, US 5874299, US 5814318, US 5789650, US US 5770429

- 13 041301

5661016, US 5633425, US 5625126, US 5569825, US 5545806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991) and phage display methods (see for example, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, US 5877218, US 5871907, US 5858657, US 5837242, US 5733743 and US 5565332).

F. Constant Region Selection

The heavy and light chain variable regions of chimeric, humanized (including veneered), or human antibodies can be linked to at least a portion of a human constant region. The choice of constant region depends in part on whether antibody-dependent complement and/or cell-mediated cytotoxicity is desired. For example, the human IgG1 and IgG3 isotypes have complement-mediated cytotoxicity, while the human IgG2 and IgG4 isotypes do not. Light chain constant regions can be lambda or kappa. For immunotherapy against cancer or a non-TIGIT-expressing pathogen, human IgG2 or IgG4 or an attenuated form of human IgG1 with reduced effector function is often preferred. For human IgG4, it is often preferable to include an S228P (Eu numbering) genetically engineered mutation on the heavy chain to prevent the exchange of Fab fragments. However, for elimination of TIGIT-expressing cancer cells (eg, from T-cell or NK-cell tumors) or for immunosuppression, human IgG1 or IgG3 is often preferred.

Human constant regions show allotypic variation and isoallotype variation between individuals, ie constant regions may differ from individual to individual at one or more polymorphic positions. Isoallotypes differ from allotypes in that the isoallotype-recognizing serum proteins bind to a non-polymorphic region of one or more other isotypes. Reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying polymorphic positions in natural allotypes.

One or more amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or may be converted to some or all of these molecules. Substitutions can be in constant regions to reduce or increase effector function, such as complement-mediated cytotoxicity or ADCC [see, for example, Winter et al., US Pat. No. 5,624,821; Tso et al., US Pat. No. 5,834,597 and Lazar et al., Proc. Natl. Acad. sci. USA, 103:4005, 2006) or to extend the half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004). Illustrative substitutions include Gln at position 250 and/or Leu at position 428 (Eu numbering) to increase the half-life of the antibody.

Some antibodies of the invention are engineered by introducing mutation(s) in the constant region to produce reduced effector functions such as CDC and ADCC or ADCP compared to the same antibody without the mutation(s). Preferably, each or all of these effector functions are reduced by at least 50%, 75%, 90% or 95% compared to non-mutated antibodies. The examples presented show how CDC can be measured. Other assays are described in Shields et al, 2001 J. Biol. Chem., Vol. 276, p 6591-6604; Chappel et al, 1993 J. Biol. Chem., Vol 268, p 2512425131; Lazar et al, 2006 PNAS, 103; 4005-4010.

Substitution of any or all positions 234, 235, 236 and/or 237 reduces the affinity for Fcy receptors, in particular the FcyRI receptor (see, for example, US 6624821). Alanine is the preferred replacement residue, and L234A/L235A is the preferred double mutation for reduced effector function. Other mutation combinations with reduced effector functions include L234A/L235A/G237A, E233P/L234V/L235A/G236, A327G/A330S/P331S, K322A, L234A and L235A, L234F/L235E/P331S. Optionally, positions 234, 236 and/or 237 in human IgG2 are replaced by alanine and position 235 by glutamine (see, for example, US 5624821). Two amino acid substitutions at the C1q complement binding site at positions 330 and 331 (EU index) decrease complement fixation (see Tao et al., J. Exp. Med. 178:661 (1993) and Canfield and Morrison, J. Exp. Med 173:1483 (1991)). The substitution in human IgG1 or IgG2 residues at positions 233-236 and in IgG4 residues at positions 327, 330 and 331 greatly reduces ADCC and CDC (see, for example, Armor 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 numbering) mutations reduce glycosylation and thus effector functions.

G. Expression of recombinant antibodies

Chimeric, humanized (including veneered), and human antibodies are generally produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of the antibody chains, including naturally associated or heterologous promoter regions. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been introduced into a suitable host, the host is maintained under conditions suitable for a high level of expression of the nucleotide sequences.

- 14 041301 values, and collect and purify recombinant antibodies.

Mammalian cells are the preferred host for the expression of nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Various suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody producing myeloma including Sp2/0 and NS0. Preferably the cells are non-human. Expression vectors for these cells may include expression control sequences such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary information processing sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

Once the antibodies have been expressed, they can be purified according to standard procedures known in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. (See generally Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

IV. Therapeutic Applications

Antibodies can be used to enhance immune responses in the treatment of cancer and infectious diseases. The disorders treatable with the antibodies of the invention include, without limitation, cancer, including hematologic malignancies and solid tumors. Such cancers may or may not express TIGIT or CD155. Anti-TIGIT antibodies are effective against non-TIGIT-expressing cancers because inhibition of TIGIT interaction with CD155 stimulates an immune response against such cancers. Examples of hematological malignancies include leukemias, lymphomas, and myelomas, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute monocytic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. lymphoma and multiple myeloma. Examples of solid tumors include, without limitation, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcomas, and brain tumors (eg, gliomas such as glioblastomas)

The methods of the invention may be practiced under adjuvant therapy. Adjuvant therapy refers to a clinical situation where an individual has a history of a proliferative disease, especially malignancy, and has typically (but not necessarily) responded to treatment, which includes, but is not limited to, surgery, radiation therapy, and/or chemotherapy. However, due to a history of proliferative disease, these individuals are considered at risk for developing this disease. Treatment or administration under adjuvant therapy refers to the subsequent method of treatment. In some embodiments, provided herein is a method for treating or preventing cancer, comprising administering to an individual having cancer or an increased risk of developing cancer a therapeutically effective amount of any of the antibodies disclosed herein under adjuvant therapy.

The methods presented in this application can also be carried out in non-adjuvant conditions, that is, the method can be carried out before primary/radical therapy. In some aspects, the individual has previously received treatment. In other aspects, the individual has not previously received treatment. In some aspects, the treatment is a first line therapy. In some embodiments, provided herein is a method for treating or preventing cancer, comprising administering to an individual having cancer or an increased risk of developing cancer a therapeutically effective amount of any of the antibodies disclosed herein under non-adjuvant conditions.

Other disorders that can be treated with the antibodies of the invention include infectious diseases caused by viruses, bacteria, fungi, protozoa, and other pathogens (e.g. hepatitis (A, B or C), herpes virus (e.g. VZV, HSV-1, HAV -6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, HIV, HCV and arbovirus encephalitis virus, chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and gonococci, klebsiella, proteus, serration, pseudomonas, legionella, diphtheria bacillus, bacillus

- 15 041301 salmonella, bacteria that cause cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease.

A. Administration of antibodies

The antibodies described herein are administered in an effective regimen, meaning a dosage, route of administration, and frequency of administration, that delays the onset of development, reduces the severity, inhibits further deterioration, and/or improves at least one sign or symptom of the disorder. If the individual is already suffering from the disorder, the regimen may be referred to as a therapeutically effective regimen. If an individual has an increased risk of disease compared to the general population, but is not yet experiencing symptoms, the regimen may be termed a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy may be observed in an individual subject relative to historical controls or past experience in the same individual. In other cases, therapeutic or prophylactic efficacy may be demonstrated in preclinical or clinical trials in a population of treated individuals compared to a control population of untreated individuals.

In some aspects, any of the methods described in this application includes the introduction of a therapeutically effective amount of one or more antibodies described in this application, individuals in need of it. Used in this application, the term therapeutically effective amount or therapeutically effective dose of anticancer therapy (such as any of the antibodies against TIGIT described in this application) is an amount sufficient to obtain useful or desired results. For therapeutic use, useful or desirable results include clinical results such as a reduction in one or more of the symptoms caused by cancer, an improvement in the quality of life of individuals suffering from cancer, a reduction in the dose of other drugs needed to treat cancer, an increase in the effect of another drug eg by targeting, delaying disease progression and/or prolonging survival. An effective dose may be administered in one or more administrations. For the purposes of the present invention, an effective dosage of an anticancer therapy is an amount sufficient to effect a therapeutic or prophylactic treatment, either directly or indirectly. As understood in the clinical context, a therapeutically effective dose of an anticancer therapy may or may not be achieved in combination with another anticancer therapy.

Illustrative doses for any of the antibodies described in this application are about 0.1-20 mg/kg or 0.5-5 mg/kg body weight (for example, about 0.5, 1, 2, 3, 4, 5 g, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg/kg) or 10-1500 mg (for example, any dose of less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or 1500 mg or more, including values between these figures), as a fixed dose. The dose depends on the condition of the individual and the response to prior treatment, if any, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic, among other things.

Administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intratumoral, topical, intranasal, or intramuscular. Preferred is the introduction into the systemic circulation by intravenous or subcutaneous administration. Intravenous administration can be accomplished, for example, by infusion over a period such as 30-90 minutes.

The frequency of administration depends on the circulating half-life of the antibody, the condition of the individual, and the route of administration, among other things. The frequency may be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the individual's condition or progression of the disorder being treated. By way of example, the frequency for intravenous administration is from once a week to once a quarter during a continuous treatment period, although more or less frequent administration is also possible. For subcutaneous administration, an administration frequency of once a day to once a month can be exemplified, although more or less frequent administration is possible.

The number of doses administered depends on whether the disease is acute or chronic and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, 1 to 10 doses are often sufficient. Sometimes a single bolus dose, optionally in divided doses, is sufficient for an acute disorder or an acute exacerbation of a chronic disorder. Treatment may be repeated if the acute disorder or acute exacerbation recurs. In chronic diseases, the antibody can be administered at regular intervals, for example, weekly, biweekly, monthly, quarterly, every six months for at least 1, 5, or 10 years, or for the lifetime of the individual.

Anti-TIGIT antibody treatment can alleviate disease by increasing median progression-free survival or overall survival time in patients with cancer by at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47,

- 16 041301

48, 49%, but preferably at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94%, 95%, 96%, 97%, 98%, 99% or even 100% compared to control individuals, or increase any of these times by 2 weeks, 1, 2 or 3 months, or preferably by 4 or 6 months, or even by 9 months or a year. Additionally or alternatively, treatment comprising an anti-TIGIT antibody can increase the percentage of patients with complete response, partial response, or objective response (complete + partial) by at least about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49%, but preferably at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or even 100% compared to control individuals. Control individuals receive the same treatment as those receiving the anti-TIGIT antibody, with the exception of the anti-TIGIT antibody. Thus, control individuals may receive a placebo alone or a combination of a placebo and some chemotherapeutic agent other than an anti-TIGIT antibody, if such agent is also taken by individuals taking an anti-TIGIT antibody.

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

Generally, in clinical trials (e.g., phase II, phase II/III, or phase III), the increase in median progression-free survival and/or response rate of individuals treated with anti-TIGIT antibody compared to controls of individuals is statistically significant, e.g. , at p=0.05 or 0.01 or even 0.001. The percentages of complete and partial response patients are determined by objective criteria commonly used in clinical trials for the treatment of cancer, such as those listed or accepted by the National Cancer Institute and/or the Food and Drug Administration, and may include, for example, tumor sizing, number of tumors, metastases, survival time and quality of life, among others.

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP (Good Manufacturing Practices) conditions. Pharmaceutical compositions may be presented in unit dosage form (ie, single administration dose). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients or excipients. The dosage form depends on the chosen route of administration. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). The solution may contain formulation agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibodies may be in lyophilized form for reconstitution with a suitable vehicle, such as sterile, pyrogen-free water, prior to use. The concentration of the antibody in the liquid compositions can vary from about 10-150 mg/ml. In some compositions, the concentration is about 25-50 mg/ml.

B. Combination therapies

The present invention provides for the use of anti-TIGIT antibodies in combination with one or more active therapeutic agents (eg, chemotherapeutic agents) or other prophylactic or therapeutic modalities (eg, radiation). In such combination therapy, the different active agents often have different complementary mechanisms of action. Such a combination therapy may be particularly advantageous, allowing the dose of one or more agents to be reduced, thereby reducing or eliminating the side effects associated with one or more agents. In addition, such combination therapy may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder or condition.

As used herein, the term combination means therapeutic agents that can be administered separately, for example, formulated separately for separate administration (for example, as may be provided in a kit), and therapeutic agents that can be administered together in one composition (i.e., joint composition).

In some embodiments, any of the anti-TIGIT antibodies disclosed herein is administered or used sequentially, such as when one agent is administered before one or more other agents. In other embodiments, the antibodies are administered simultaneously, for example, when two or more agents are administered at about the same time; two or more agents may be present in two or more separate compositions or combined into one composition (then

- 17 041301 there is a joint composition). Whether two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of the present invention.

The antibodies of the present invention may be used in combination with at least one other (active) agent in any manner appropriate to the circumstances. In one embodiment, treatment with at least one active agent and at least one anti-TIGIT antibody of the present invention is maintained for a certain period of time. In another embodiment, treatment with at least one active agent is reduced or discontinued (eg, when the individual is stable) while treatment with an 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 reduced or discontinued (eg, when the individual is stable), while treatment with an anti-TIGIT antibody of the present invention is reduced (eg, lower dose, less frequent administration, or shorter treatment regimen). In yet another embodiment, treatment with at least one active agent is reduced or discontinued (eg, when the individual is stable) and treatment with an anti-TIGIT antibody of the present invention is increased (eg, higher dose, more frequent administration, or longer treatment regimen). In yet another embodiment, treatment with at least one active agent is maintained and treatment with the anti-TIGIT antibody of the present invention is reduced or discontinued (eg, lower dose, less frequent administration, or shorter treatment regimen). In yet another embodiment, treatment with at least one active agent and treatment with anti-TIGIT antibodies of the present invention is reduced or discontinued (eg, lower dose, less frequent administration, or shorter treatment regimen).

Treatment with antibodies of the invention may be combined with other treatments effective against the disorder in question. When used in the treatment of a proliferative condition, malignancy, tumor or precancerous disease, disorder or condition, the antibodies of the invention may be combined with chemotherapy, radiation (e.g., localized radiation therapy or total body radiation therapy), stem cell therapy, surgery, or treatment with other biological agents. .

In some embodiments, the present invention relates to methods for suppressing tumor or tumor growth, comprising administering anti-TIGIT antibodies described herein in combination with a signal transduction inhibitor (STI) to achieve additive or synergistic suppression of tumor growth. As used herein, the term signal transduction inhibitor refers to an agent that selectively inhibits one or more steps in a signaling pathway. The signal transduction inhibitors (STIs) of the present invention include: (i) BCR-Abl kinase inhibitors (eg, imatinib mesylate (GLEEVEC)); (ii) epidermal growth factor (EGF) receptor inhibitors, including kinase inhibitors and antibodies; (iii) HER2/nei receptor inhibitors (eg, HERCEPTIN); (iv) Akt family kinase or Akt pathway inhibitors (eg rapamycin); (v) cell cycle kinase inhibitors (eg, flavopiridol); and (vi) phosphatidylinositkinase inhibitors. Immunomodulatory agents can also be used in combination with the anti-TIGIT antibodies described herein to suppress tumor growth in cancer patients.

Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, imsulfan and pyrosulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelalamide; nitrogen mustards such as chiorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednimustine, trofosfamide, uraciliprite; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carbabicin, kaminomycin, carcinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-Norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, β-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, fluxuridine, 5-FU; androgens such as calastone, dromonostanol propionate, epithiotonol, mepitiostane, testolactone; adrenal depressants such as aminoglutethimide, mitotane, trilostane; a folic acid substitute such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elformitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxan

- 18 041301 throne; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; podophyllic acid; 2ethylhydrazide; procarbazine; razoxane; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Apa-C); cyclophosphamide; thiotepa; toxoids (eg taxanes), eg paclitaxel, taxol and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbin; novantron; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; camptothecins, difluoromethylornithine (DMFO); retinoic acid; esperamycins; capecitabine; antimetabolites (eg, azathioprine); anthracyclines; plant alkaloids (including, for example, vinca alkaloids); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

Chemotherapeutic agents also include antihormonal agents that act to regulate or inhibit hormonal effects on tumors, such as antiestrogen including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4hydroxytamoxifen, trioxifene, keoxifene, and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In some embodiments, the combination therapy comprises the administration of a hormone or an appropriate hormonal agent.

Additional therapies that can be used in combination with the anti-TIGIT antibodies described herein include radiation therapy, anti-tumor antigen monoclonal antibody, monoclonal antibody-toxin complex, T-cell adjuvant, bone marrow transplantation, or use of antigen-presenting cells ( e.g. dendritic cell therapy) or cell therapies. As used herein, the term cell therapy refers to any therapy that involves the administration of cells in order to achieve a desired therapeutic effect, for example, to restore damaged or supersaturated adult cell populations or tissues, or to enhance the immune response against a pathogen or proliferating cell (such as a cancer cell). ). In other embodiments, the cell therapy comprises administering an undifferentiated cell (such as a stem cell or other multi- or totipotent cell) capable of differentiating into the particular cell type required to achieve the desired therapeutic effect, such as repairing damaged or supersaturated cell populations or tissues in adults or enhancing the immune response against a pathogen or proliferative cell (such as a cancer cell). In some embodiments, antibodies of the invention can also be administered with tumor-infiltrating T cells or natural killer cells, which have optionally been expanded or selected for ex vivo homing to the tumor. The antibody of the invention promotes the activation of these cells.

A preferred combination is an antibody of the invention with a second antibody directed to a surface antigen preferentially expressed on cancer cells compared to control normal tissue. Some examples of antibodies that can be administered in combination therapy with the antibodies of the invention for the treatment of cancer include Herceptin® (trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) against VEGF, or antibodies to the EGF receptor such as (Erbitux®, cetuximab ) and Vectibix® (panitumumab). Other agents that may be administered include antibodies or other inhibitors of any of PD-1, PDL1, CTLA-4, 4-1BB, BTLA, VISTA, TIM-3, and LAG-3; or other signaling pathway inhibitors, eg mTOR and GSK3e inhibitors; and cytokines such as interferon-γ, IL-2 and IL-15. Some specific examples of additional agents include: ipilimumab, pazopanib, sunitinib, dasatinib, pembrolizumab, INCR024360, dabrafenib, trametinib, atezolizumab (MPDL3280A), rarceva, cobimetinib, and nivolumab. The choice of the second antibody or other agent for combination therapy depends on the treatment of the malignancy. Optionally, the cancer is tested for expression or preferential expression of the antigen to guide selection of the appropriate antibody. The isotype of the second antibody is preferably human IgG1 to promote effector functions such as ADCC, CDC and phagocytosis.

The antibodies of the invention can also be administered with vaccines that elicit an immune response against cancer. Such an immune response is enhanced by an antibody of the invention. The vaccine may include an antigen expressed on the surface of the cancer, or a fragment thereof effective to induce an immune response, optionally associated with a carrier molecule.

The present invention contemplates the use of the anti-TIGIT antibodies described herein in combination with immune checkpoint inhibitors.

A huge number of genetic and epigenetic changes, characteristic of all types of malignant neoplasm, provide a diverse set of antigens that the immune system

- 19 041301 topic can be used to distinguish tumor cells from their normal counterparts. In the case of T cells, the maximum amplitude (eg, levels of cytokine production or proliferation) and quality (eg, type of immune response generated, such as a pattern of cytokine production) of the response that is initiated by antigen recognition by the T cell receptor (TCR) is governed by a balance between costimulatory and inhibitory signals (immune checkpoints). Under normal physiological conditions, immune checkpoints are critical for preventing autoimmunity (i.e., maintaining self-resistance) as well as protecting tissues from damage when the immune system responds to a pathogenic infection. Expression of immune checkpoint proteins may be dysregulated by tumors as an important mechanism of immune resistance.

Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated in various tumor cell types that are candidates for blockade, include PD-1 (programmed cell death protein 1); PD-L1 (programmed cell death ligand-1); BTLA (B and T lymphocyte attenuator); CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4); TIM-3 (T-cell membrane protein 3); LAG-3 (lymphocyte activation gene 3); V-domain immunoglobulin suppressor of T-cell activation (VISTA); CD96; A2aR (adenosine A2a receptor); A2bR (adenosine A2b receptor); CD73 (ecto-5'nucleotidase); CD39 (ENTPD1, NTPDase1); Arginase; indolamine-pyrrole 2,3-dioxygenase (IDO); tryptophan 2,3-dioxygenase (TDO); and inhibitory killer receptors, which can be divided into two classes based on their structural features: i) killer immunoglobulin-like receptors (KIRs), and ii) C-type lectin receptors (members of the type II transmembrane receptor family). Other immune checkpoints that have not been as well defined have been described in the literature, including both receptors (eg, the 2B4 (also known as CD244) receptor) and ligands (eg, some inhibitory ligands of the B7 family 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 12:25264.

The present invention contemplates the use of any of the anti-TIGIT antibodies described herein in combination with inhibitors of the aforementioned immune checkpoint receptors and ligands, as well as as yet undescribed immune checkpoint receptors and ligands. Some immune checkpoint modulators are currently available, while others are in the last stages of development. To illustrate, when it was approved for the treatment of melanoma in 2011, the fully humanized CTLA-4 monoclonal antibody ipilimumab (YERVOY, Bristol-Myers Squibb) became the first immune checkpoint inhibitor to receive official approval in the US. Fusion proteins containing CTLA-4 and an antibody (CTLA-4-Ig, abatacept (ORENCIA, Bristol-Myers Squibb)) have been used to treat rheumatoid arthritis, and other fusion proteins have been shown to be effective in kidney transplant patients who are sensitized to the Epstein Barr virus. Anti-PD-1 antibodies are under development (eg, nivolumab (Bristol-Myers Squibb) and pembrolizumab (lambrolizumab) (Merck)), as well as anti-PD-L1 antibodies (eg, MPDL3280A (Roche)). Nivolumab and pembrolizumab have shown potential in patients with melanoma, lung and kidney cancer.

Such combination therapy can be used to treat or prevent infectious diseases such as viral, bacterial, fungal and parasitic diseases, disorders and conditions, and related disorders. For example, an antibody of the invention can be combined with an antibody directed against a pathogen, or with a vaccine against the pathogen, such as palivizumab against Rous sarcoma virus. The vaccine may be a pathogen protein or fragment thereof effective to induce an immune response. An antibody of the invention enhances the immune response of an antibody or vaccine directed against a pathogen. The antibody of the invention can also be administered with ex vivo expanded T cells or natural killer cells.

Such combination therapy includes antiviral agents that target different stages of the viral life cycle and have different mechanisms of action, including, but not limited to, the following: inhibitors of viral decapsidation (eg, amantadine and rimantidine); reverse transcriptase inhibitors (eg, acyclovir, zidovudine, and lamivudine); means aimed at integrase; agents that block the attachment of transcription factors to viral DNA; agents (eg, antisense molecules) that affect translation (eg, fomivirsen); agents that modulate the function of translation/ribozymes; protease inhibitors; virus assembly modulators (eg, rifampicin); antiretroviral agents such as, for example, nucleoside reverse transcriptase inhibitors (eg, azidothymidine (AZT), ddl, ddC, 3TC, d4T); non-nucleoside reverse transcriptase inhibitors (eg, efavirenz, nevirapine); nucleotide reverse transcriptase inhibitors; and agents that prevent the release of viral particles (eg zanamivir and oseltamivir). For the treatment and/or prevention of certain viral infections (eg HIV) often requires a group (cocktail) of antiviral agents.

- 20 041301

Other antiviral agents for use in combination with any of the anti-TIGIT antibodies described herein include, but are not limited to, the following: abacavir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevirertet, cidofovir, combrivir, darunavir, delavirdine, didanosine, docosanol, edoxydin, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, phosphonet, ganciclovir, ibacitabine, iminovir, idoxuridine, imiquimod, indinavir, inosine, various interferons (eg, peginterferon alfa-2a) , lopinavir, lorizide, maraviroc, moroxidine, metizazon, nelfinavir, nexavir, penciclovir, perimivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, ritonavir, pyramidin, saquinavir, stavudine, telopravir, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir , valganciclovir, vicriviroc, vidarabine, viramidine, and zalcitabine.

The present invention contemplates the use of any of the anti-TIGIT antibodies disclosed herein in combination with antiparasitic agents. Such agents include, but are not limited to, thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide, bithionol, oxamnikin, metriconate, ivermectin, albendazole, eflornithine, melarsoprol, pentamidine, bennidazole, nifurtimox, and nitroimidazole. Those skilled in the art are aware of other agents that may be useful in the treatment of parasitic disorders.

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

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

The present invention encompasses pharmaceutically acceptable salts, acids or derivatives of the agents (and representatives of the classes of agents) listed above.

Anti-TIGIT antibodies can be used in combination with any of the second antibodies or agents described for use in co-treatment as components of a pharmaceutical composition. In a pharmaceutical composition, the agents may be combined with one or more pharmaceutically acceptable carriers as described.

V. Other applications

Anti-TIGIT antibodies can be used to detect TIGIT in the context of clinical diagnosis or treatment, or in research. For example, antibodies can be used to detect the presence of TIGIT on T cells, natural killer cells, and cancer cells as an indication that an individual is suffering from a treatable cancer or infectious disease. Expression of TIGIT on T cells, natural killer cells and/or cancer cells of an individual suffering from a malignant neoplasm or infectious disease also indicates that the cancer or infectious disease is amenable to treatment with the antibodies of the present invention. Antibodies can also be sold as research reagents for laboratory testing to detect T-cells, natural killer cells and malignant cells and their response to various stimuli. In such applications, monoclonal antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided as a kit with all necessary reagents to perform a TIGIT assay. Antibodies can also be used to purify TIGIT, for example using affinity chromatography.

VI. Sets

Anti-TIGIT antibodies can be combined with any of the second antibodies or agents described for use in co-treatment as kit components. The invention disclosed in this application provides one or more kits containing one or more of the described

- 21 041301 antibodies, as well as one or more pharmaceutically acceptable excipients or carriers (such as, without limitation, phosphate-buffered saline solutions, water, sterile water, polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil, emulsions such as oil/water or water/oil emulsions, microemulsions, nanocarriers and various types of wetting agents). Additives such as alcohols, oils, glycols, preservatives, flavors, colorants, suspending agents, and the like may also be included in the kits of the present invention along with a carrier, diluent, or excipient. In one embodiment, a pharmaceutically acceptable carrier suitable for use in the antibody compositions described herein is sterile, pathogen-free and/or otherwise safe for administration to an individual without the risk of associated infection and other undesirable side effects. In a kit, the respective substances may be presented in separate vials with instructions for combination and subsequent administration, or with instructions for single administration. The kit may also include written instructions for the correct handling and storage of any anti-TIGIT antibodies described herein.

It is intended that each maximum numerical limit given in the present description, includes each lower numerical limit, as if such lower numerical limits were explicitly stated in this application. Each minimum numeric limit provided herein will include all higher numeric limits as if such higher numeric limits were explicitly stated herein. Each numerical range specified in the present description will include all narrower numerical ranges that fall within such a broader numerical range, as if such narrower numerical ranges were explicitly stated in this application.

All patent applications, websites, other publications, access numbers, etc. referenced above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were specifically and is individually identified for inclusion by reference. When different sequence versions are associated with an access number at different times, the version associated with the access number at the current filing date of this application is meant. Valid filing date means earlier than the actual filing date or the filing date of the priority application referring to the access number, if applicable. Similarly, if different versions of a publication, website, or the like are published at different times, meaning the version that was most recently published on the current filing date, unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention may be used in conjunction with any other, unless specifically noted otherwise.

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

Examples

The following examples discuss the production, description, and humanization of anti-human TIGIT monoclonal antibodies, and provide illustrative methods by which the binding characteristics of the antibodies described in this application can be determined.

Example 1 Expression of Recombinant Human TIGIT Proteins

Gene cloning, mutagenesis, and plasmid construction in this work were performed using standard molecular biology techniques such as those described in Sambrook and Russel (Molecular Cloning, A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), Kostelny et al. (Int. J. Cancer 93:556-565, 2001) and Cole et al. (J. Immunol. 159:3613-3621, 1997).

The mammalian expression vector pFCm404 (FIG. 1) for the production of the human TIGIT extracellular region fused to the Fc region of the human immunoglobulin γ1 chain (hTIGIT-Fc; SEQ ID NO: 59) contains the following genetic components. Clockwise from the SalI site of pFCm404 in FIG. 1, the plasmid contains a transcription unit for hTIGIT-Fc, starting with a major immediate early promoter and enhancer (CMV-P in FIG. 1) from human cytomegalovirus (CMV). The CMV promoter is followed by the signal peptide coding regions (sp; SEQ ID NO: 2), human TIGIT extracellular region (hTIGIT; SEQ ID NO: 3), polypeptide linker (SEQ ID NO: 4), and human γ1 Fc region (SEQ ID NO: : 5), followed by the polyadenylation site of the human heavy chain γ1 gene. The hTIGIT-Fc gene is followed by the SV40 early promoter (SV40-P), the puromycin N-acetyltransferase (puro) gene for puromycin resistance, and the segment containing the SV40 polyadenylation site (SV40-A). Finally, pFCm404 contains a portion of the pUC19 plasmid including the bacterial origin of replication (pUC ori) and the β-lactamase (β-lactamase) gene. The arrows in the figure show the orientation of the transcription. The corresponding sites for restriction enzymes are shown in the drawings.

The human TIGIT extracellular coding region in pFCm404 was replaced with a DNA fragment encoding the human CD155 extracellular region (hCD155; SEQ ID NO: 6), resulting in a new expression vector pFCm406 (FIG. 1). The extracellular region of the human

- 22 041301

CD155 fused to the human γ1 Fc region (hCD155-Fc; SEQ ID NO: 7) was expressed from pFCm406.

The coding region of the γ1 Fc region between the AgeI and EagI sites in pFCm404 was replaced with a DNA fragment encoding the FLAG peptide (FLAG; SEQ ID: 8) followed by the human CD55 glycosylphosphatidylinositol binding signal (GPI; SEQ ID: 9), resulting in a new vector expression of pFCm407 (Fig. 1). The extracellular region of human TIGIT fused to FLAG and GPI (hTIGIT-GPI)-encoded pFCm406 was expressed on the cell surface.

To obtain cell lines stably producing hTIGIT-Fc and hCD155-Fc in culture supernatants, expression vectors pFCm404 and pFCm406, respectively, were introduced into the chromosome of the mouse myeloma cell line NS0 (European Collection of Animal Cell Cultures, Salisbury, Wiltshire, UK). NS0 cells were grown in DME medium containing 10% fetal bovine serum (FBS) at 37°C in a 7.5% CO2 incubator. Stable transfection into NS0 was performed by electroporation as described in Bebbington et al. (Bio/Technology 10: 169-175, 1992). Prior to transfection, the expression vectors were linearized with FspI. Approximately 10 ⁷ cells were transfected with 10 μg of the linearized plasmid, suspended in DME medium containing 10% FBS, and plated in several 96-well plates. After 24 hours, selective medium (DME medium containing 10% FBS and 3 μg/ml puromycin) was applied. Approximately 10 days after the start of selection, culture supernatants were analyzed for production of Fc fusion proteins by ELISA using goat anti-human gamma chain antibody for coating and HRP-conjugated goat anti-human gamma chain antibody for detection of Fc fusion proteins.

NS0 stable transfectants producing high levels of Fc fusion proteins were adapted to and expanded in Hybridoma-SFM medium (Thermo Fisher Scientific, Waltham, MA) and grown to exhaustion in roller flasks. After centrifugation and filtration, the culture supernatant was loaded onto a protein-A-sepharose column (GE Healthcare, Piscataway, NJ). The column was washed with phosphate buffered saline (PBS), then the Fc fusion proteins were eluted using 0.1 M glycine-HCl (pH 3.0). After neutralization with 1 M Tris-HCl (pH 8), the Fc-eluting fusion protein buffer was exchanged for PBS by dialysis.

To obtain cell lines expressing hTIGIT-GPI on the surface, the pFCm407 expression vector was stably transfected into NS0 cells. Expression of hTIGIT-GPI on the cell surface was monitored by flow cytometry using a rat anti-FLAG monoclonal antibody and a PE-labeled goat anti-rat IgG, Fc-specific antibody. NS0 cell lines expressing high levels of hTIGIT-GPI on the cell surface (NS0-hTIGIT) were used to screen for anti-TIGIT antibodies.

Example 2 Generation and Characterization of Anti-TIGIT Monoclonal Antibodies

Mouse hybridomas producing anti-human TIGIT monoclonal antibodies were generated at JN Biosciences (Mountain View, CA) using standard hybridoma technology such as GenomONE CF EX Cell Fusion Reagent (Cosmo Bio, Carlsbad, CA). Human TIGIT-Fc fusion proteins and NS0-hTIGIT cells were used as immunogens.

Mouse antibodies secreted in hybridoma cell culture supernatants were subjected to a series of screens to identify antibodies with the following properties: (1) binding to purified hTIGIT-Fc fusion protein, (2) binding to NS0-hTIGIT cells, (3) not binding to NS0 cells , (4) binding to CD3+ T cells derived from phytohemagglutinin-treated human peripheral blood mononuclear cells, and (5) blocking the binding of the hCD155-Fc fusion protein to NS0-hTIGIT cells. The first binding property to hTIGIT-Fc was tested by ELISA using HRP-conjugated goat anti-mouse kappa and lambda light chain antibodies (SouthernBiotech, Birmingham, AL) to identify mouse anti-TIGIT antibodies. The second, third and fourth properties were examined by flow cytometry using a PE-labeled goat anti-mouse gamma chain antibody (SouthernBiotech) to detect cell-bound mouse antibodies. The fifth property was analyzed by flow cytometry as described below. Three mouse monoclonal antibodies (TIG1, TIG2 and TIG3) were found to have all of these five properties. TIG1, TIG2 and TIG3 monoclonal antibodies were purified from the hybridoma cell culture supernatant by protein A column chromatography as described above. The isotypes TIG1, TIG2 and TIG3 are mouse IgG2b/kappa, IgG2a/kappa and IgG2a/kappa, respectively.

The activity of TIG1, TIG2 and TIG3 to block the interaction between TIGIT and CD155 was analyzed by flow cytometry. NS0-hTIGIT cells were incubated with a subsaturating concentration of hCD155-Fc in the presence (or absence) of various concentrations of anti-TIGIT monoclonal antibody as a competitor. Detection of hCD155-Fc associated with NS0-hTIGIT cells was performed using a DyLight488-labeled goat anti-human IgG, Fc-specific antibody (Jackson ImmunoResearch, West Grove, PA). As shown in FIG. 2, TIG1, TIG2, and TIG3 inhibited hCD155-Fc binding to NS0-hTIGIT cells in a dose-dependent manner. Half-maximal inhibitory concentration (IC ₅₀ ) to block the interaction between hCD155-Fc and

- 23 041301

NSO-hTIGIT cells was 49 ng/ml for TIG1, 197 ng/ml for TIG2 and 460 ng/ml for TIG3. The antibody concentration required to block more than 95% of CD155 binding to TIGIT was 0.63 μg/ml for TIG1, 1.25 μg/ml for TIG2, and 2.5 μg/ml for TIG3.

Example 3 V Gene Sequencing of Anti-TIGIT Monoclonal Antibodies

Sequencing of the heavy and light chain variable regions (VH and VL, respectively) of mouse anti-TIGIT antibodies was performed following the procedures described in Tsurushita et al (Methods 36:69-83, 2005). Total RNA was extracted from about 10 ⁷ cells using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the supplier's protocol. Oligo-dT-primed 5'-RACE cDNA was synthesized using the SMARTer RACE cDNA amplification kit (Clontech, Mountain View, CA) following the vendor's protocol. VH and VL cDNA were amplified by polymerase chain reaction (PCR) using Phusion DNA polymerase (Thermo Fisher Scientific, Waltham, MA) using 3' primers that hybridize specifically to mouse heavy or light chain constant regions (Tsurushita et al. , supra) respectively, and the 5' primer provided in the SMARTer RACE set for cDNA amplification. The amplified VH and VL genes were cloned using the CloneJet PCR cloning kit (Thermo Fisher Scientific) and sequenced with the primers provided in the CloneJet PCR cloning kit. Several VH and VL clones were sequenced and identified unique sequences homologous to typical murine heavy and light chain variable regions.

The amino acid sequence of mature VH TIG1 (SEQ ID NO: 10) is shown in FIG. 3. The amino acid sequences of CDR1, 2 and 3 of the TIG1 VH regions are NFGMH (SEQ ID NO: 11), FISSGSSSIYYADTVKG (SEQ ID NO: 12) and MRLDYYAMDY (SEQ ID NO: 13), respectively.

The amino acid sequence of mature VL TIG1 (SEQ ID NO: 14) is shown in FIG. 4. The amino acid sequences of CDR1, 2 and 3 of the TIG1 VL regions are RASKSISKYLA (SEQ ID NO: 15), SGSTLQS (SEQ ID NO: 16) and QQHNEYPWT (SEQ ID NO: 17), respectively.

The amino acid sequence of mature VH TIG2 (SEQ ID NO: 18) is shown in FIG. 5. The amino acid sequences of CDR1, 2 and 3 of the TIG2 VH regions are EYTMH (SEQ ID NO: 19), GINPNNGGTSYNQKFKG (SEQ ID NO: 20) and PGWYNYAMDY (SEQ ID NO: 21), respectively.

The amino acid sequence of mature VL TIG2 (SEQ ID NO: 22) is shown in FIG. 6. The amino acid sequences of CDR1, 2 and 3 of the TIG2 VL regions are KASQGVSTAVA (SEQ ID NO: 23), SASYRYT (SEQ ID NO: 24) and QQHYITPWT (SEQ ID NO: 25), respectively.

The amino acid sequence of mature VH TIG3 (SEQ ID NO: 26) is shown in FIG. 7. The amino acid sequences of CDR1, 2 and 3 of the TIG3 VH regions are DYDMS (SEQ ID NO: 27), YISDGGYNTYYPDTVKG (SEQ ID NO: 28) and QILLRYYFDY (SEQ ID NO: 29), respectively.

The amino acid sequence of mature VL TIG3 (SEQ ID NO: 30) is shown in FIG. 8. The amino acid sequences of CDR1, 2 and 3 of the TIG3 VL regions are KSSQSLLYSSNQKNYLA (SEQ ID NO: 31), WASTRES (SEQ ID NO: 32) and QQYHSYPWT (SEQ ID NO: 33), respectively.

Assignment of CDR sequences and numbering of amino acid positions in FIG. 3-8 are presented according to Kabat et al. (1991). The sequences CDR1, CDR2 and CDR3 are underlined in the drawings.

Example 4 Construction and expression of a humanized TIG1 antibody

Humanization of VH and VL TIG1 was performed as described in Queen et al. (Proc. Natl. Acad. Sci. USA. 86:10029-10033, 1989) following the procedures of Tsurushita et al. (Methods 36:69-83, 2005). Briefly, a three-dimensional molecular model of TIG1 variable regions was first constructed using an algorithm developed by JN Biosciences. Then identified framework amino acid residues important for the formation of the structure of complementarity determining regions (CDR) TIG1 using a molecular model. In parallel, cDNA-derived human VH and VL amino acid sequences with high homology to TIG1 VH and VL, respectively, were selected. Finally, CDR sequences were grafted along with framework amino acid residues important for maintaining the CDR structure from TIG1 VH and VL into the appropriate selected human framework sequences.

The amino acid sequence of the engineered humanized TIG1 VH (HuTIG1 VH) is

MDSRLNLVFLVLILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGL

EWVAFISSGSSSIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYW GQGTMVTVSS (SEQ ID NO:34).

Amino acid sequence of mature HuTIG1 VH

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIY

YADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVSS (SEQ ID NO:35)

- 24 041301 starts at position 20 in SEQ ID NO: 34.

The amino acid sequence of the engineered humanized TIG1 VL (HuTIG1 VL) is

MRFQVQVLGLLLLWISGAQCDIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAP

KLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKVEIK (SEQ ID NO: 36) .

Amino acid sequence of mature HuTIG1 VL

DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSG

SGSGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKVEIK (SEQ ID NO:37), starts at position 21 in SEQ ID NO:36.

The gene encoding HuTIG1 VH was synthesized as an exon including a splicing donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment. The nucleotide sequence of the synthetic HuTIG1 VH gene flanked by SpeI and HindIII sites (SEQ ID NO: 38) is shown along with the predicted amino acid sequence (SEQ ID NO: 34) in FIG. 9A.

The gene encoding HuTIG1 VL was synthesized as an exon including a splicing donor signal at the 3' end of the coding region, a NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment. The nucleotide sequence of the synthetic HuTIG1 VL gene flanked by NheI and EcoRI sites (SEQ ID NO: 39) is shown along with the predicted amino acid sequence (SEQ ID NO: 36) in FIG. 9b.

The mammalian expression vector pHuTIG1.AA (FIG. 10) for the production of a humanized IgG1/kappα form of the mouse monoclonal anti-human anti-TIGIT antibody, TIG1 (HuTIG1IgG1.AA) was constructed to contain the following genetic components. Clockwise from the SalI site in FIG. 10, the vector contains a heavy chain transcription unit, starting with a major immediate early promoter and enhancer from human cytomegalovirus (CMV) (CMV-P in FIG. ) to initiate transcription of the antibody heavy chain gene. The CMV promoter is followed by an exon encoding the heavy chain variable region of a humanized form of TIG1 flanked by SpeI and HindIII sites (HuTIG1 VH), a genomic sequence containing the human heavy chain γ1 constant regions, including exons CH1, hinge, CH2 and CH3 with intervening introns, and a polyadenylation site for the human γ1 heavy chain gene. The CH2 region encoded in pHuTIG1.AA contains amino acid substitutions for alanine residues at positions 234 and 235 (Eu numbering) (L234A/L235A) to eliminate effector functions (Hezareh et al., J. Virol. 75:12161-12168, 2001 ). After the heavy chain gene sequence, the light chain transcriptional unit begins with a CMV promoter and enhancer (CMV-P), followed by an exon encoding the light chain variable region of a humanized form of TIG1 flanked by NheI and EcoRI sites (HuTIG1 VL), a genomic sequence containing the human exon constant region of the kappa chain (CK) with the part of the intron preceding it, and the polyadenylation site of the human kappa chain gene after the CK exon. The light chain gene is then followed by the SV40 early promoter (SV40-P), the puromycin N-acetyl transferase (puro) gene for puromycin resistance, and the segment containing the SV40 polyadenylation site (SV40-A). Finally, pHuTIG1.AA contains a portion of the pUC19 plasmid including the bacterial origin of replication (pUC ori) and the β-lactamase (β-lactamase) gene. The arrows in Fig. show the orientation of transcription.

The amino acid sequence of the mature gamma heavy chain HuTIG1-IgG1.AA encoded in pHuTIG1.AA is

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIYYADTV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG К (SEQ ID NO:40).

The C-terminal lysine may or may not be present.

The amino acid sequence of the mature HuTIG1-IgG1.AA kappa light chain encoded in pHuTIG1.AA is

DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSG

SGSGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:41).

The expression vector pHuTIG1.AA was introduced into the chromosomes in the CHO-K1 cell line of the ovaries.

- 25 041301 Thai hamster (ATCC, Manassas, VA) to obtain cell lines that stably produce HuTIG1-IgG1.AA. CHO-K1 cells were grown in SFM4CHO medium (GE Healthcare Life Sciences, Logan, UT) at 37° C. in a 7.5% CO2 incubator. Stable transfection into CHO-K1 was performed by electroporation. Prior to transfection, pHuTIG1.AA was linearized with FspI. In a typical experiment, approximately 10 ⁷ cells were transfected with 20 μg of the linearized plasmid, suspended in SFM4CHO medium, and plated at 100 μl/well in several 96-well plates after appropriate cell dilutions. After 48 hours, SFM4CHO medium containing 20 μg/ml puromycin was added at 100 μl/well to isolate stable transfectants. Approximately ten days after the start of selection, the culture supernatants of the transfectants were analyzed for antibody production.

Expression of HuTIG1-IgG1.AA was measured by sandwich ELISA. In a typical ELISA experiment, the plate was coated with a goat anti-human IgG Fc-specific polyclonal antibody, washed with wash buffer (PBS containing 0.05% Tween 20) and blocked with ELISA buffer (PBS containing 2% skimmed milk and 0.05% Tween 20). ). After washing with wash buffer, the test samples, appropriately diluted in ELISA buffer, were applied to the ELISA plate. The corresponding humanized IgG1/k antibody was used as a standard. After incubating the ELISA plate for 1 hour at room temperature and washing with wash buffer, bound antibodies were detected using an HRP-conjugated goat polyclonal anti-human kappa chain antibody. After incubating the plate for 0.5 h at room temperature and washing with wash buffer, color development was initiated by adding 100 µl/well of ABTS substrate (Sigma-Aldrich) and stopped with 100 µl/well of 2% oxalic acid. Absorbance was read at 405 nm. CHO-K1 stable transfectants producing high levels of HuTIG1-IgG1.AA were expanded in SFM4CHO until cell viability was less than 50%. After centrifugation and filtration, culture supernatants were loaded onto a Protein A column (HiTrap MABSelect SuRe, GE Healthcare, Piscataway, NJ). The column was washed with PBS, then the antibody was eluted using 0.1 M glycine-HCl (pH 3.0). The eluting antibody buffer was neutralized with 1 M Tris-HCl (pH 8) and then exchanged with PBS by dialysis. The antibody concentration was determined by measuring the absorbance at 280 nm (1 mg/ml=1.4 OD).

Example 5 Characterization of HuTIG1-IgG1.AA

The binding of HuTIG1-IgG1.AA to human TIGIT was examined by ELISA. ELISA plate was coated with 5 μg/ml hTIGIT-Fc in PBS (100 μl/well) at 4°C overnight, washed with wash buffer (PBS containing 0.05% Tween 20) and blocked with 200 μl/well SuperBlock blocking buffer (Thermo Fisher Scientific). After washing the wells with wash buffer, HuTIG1-IgG1.AA was added starting at 2.5 μg/ml and serial 2-fold dilutions in SuperBlock blocking buffer to bind to plate-bound human TIGIT. After incubating the ELISA plate for 1 hour at room temperature and washing with wash buffer, bound antibodies were detected using 100 μl/well HRP-conjugated goat anti-human kappa chain polyclonal antibody (Southern Biotech), 1/2000-diluted in ELISA Buffer. After incubation for 30 minutes at room temperature and washing with wash buffer, color development was initiated using 100 μl/well of ABTS substrate and stopped with 100 μl/well of 2% oxalic acid. Absorbance was read at 405 nm. As shown in FIG. 11, HuTIG1-IgG1.AA associated with human TIGIT in a dose-dependent manner. The half maximum effective concentration (EC ₅₀ ) of HuTIG1-IgG1.AA for TIGIT binding was 65 ng/ml. This shows that HuTIG1-IgG1.AA is a good binder for human TIGIT.

Binding of HuTIG1-IgG1.AA to human TIGIT was also examined by flow cytometry. NS0-hTIGIT cells (8x105 cells) were incubated in 160 µl FACS Buffer (PBS containing 0.5% bovine serum albumin and 0.05% sodium azide) in the presence (or absence) of various concentrations of HuTIG1-IgG1.AA starting from 10 μg/ml and serial 2-fold dilutions, for 20 min at 4°C. After washing with FACS Buffer, HuTIG1-IgG1.AA binding on NS0-hTIGIT cells was determined using PE-labeled goat anti-human IgG antibody in FACS buffer. After incubation for 20 minutes and washing with FACS Buffer, cells were subjected to flow cytometry using a FACScan flow cytometer (BD Biosciences, San Jose, CA) to obtain mid-channel fluorescence (MCF) at each antibody concentration. Fig. 12 shows a plot of MCF (vertical axis) at each antibody concentration (horizontal axis). The EC ₅₀ value was 60 ng/ml. This shows that HuTIG1-IgG1.AA is a good binder for human TIGIT expressed on the cell surface.

The activity of HuTIG1-IgG1.AA to block the interaction between human TIGIT and human CD155 was analyzed by flow cytometry using NS0-hTIGIT cells. NS0-hTIGIT cells (106 cells) were incubated with a sub-saturating concentration (125 ng/ml) of FITC-labeled hCD155-Fc fusion protein (hCD155-Fc-FITC) in 200 µl of FACS Buffer in the presence (or absence) of various concentrations of HuTIG1-IgG1 .AA starting at 10 µg/mL and serial 2x times

- 26 041301 trials, for 20 min at 4°C. After washing with FACS Buffer NS0-hTIGIT, cells were subjected to flow cytometry using a FACScan flow cytometer (BD Biosciences, San Jose, CA). To study the binding of hCD155-Fc-FITC on cells received MCF at each concentration of antibodies. As shown in FIG. 13, HuTIG1-IgG1.AA blocked interactions between hCD155-Fc-FITC and TIGIT on the cell surface in a dose-dependent manner. The half-maximal inhibitory concentration (IC ₅₀ ) of HuTIG1-IgG1.AA to block the TIGIT-CD155 interaction was 67 ng/ml. This shows that HuTIG1-IgG1.AA is an effective blocker of the interaction between CD155 and TIGIT.

Example 6 Construction and Expression of a Humanized TIG3 Antibody

Humanization of VH and VL TIG3 was performed following the general procedure described in Example 4. The amino acid sequence of the engineered humanized VH TIG3 (HuTIG3 VH) is

MNFGLRLIFLVLTLKGVNCEVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGL

EWVAYISDGGYNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYW GQGTTVTVSS (SEQ ID NO:42).

Amino acid sequence of mature VH HuTIG3

EVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTYYPDTV

KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSS (SEQ ID NO:43) starts at position 20 in SEQ ID NO:42.

The amino acid sequence of the engineered humanized TIG3 VL (HuTIG3 VL) is

MDSQAQVLMLLLLWVSGTCGDIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQ

KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGT KVEIK (SEQ ID NO: 44).

Amino acid sequence of mature VL HuTIG3

DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGV

PSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGTKVEIK (SEQ ID

NO :4 5) starts at position 21 in SEQ ID NO: 44.

The gene encoding HuTIG3 VH was synthesized as an exon including a splicing donor signal at the 3' end of the coding region, a SpeI site at the 5' end of the fragment, and a HindIII site at the 3' end of the fragment. The nucleotide sequence of the synthetic HuTIG3 VH gene (SEQ ID NO: 46) is shown along with the predicted amino acid sequence (SEQ ID NO: 42) in FIG. 14A.

The gene encoding humanized TIG3 VL (HuTIG3 VL) was synthesized as an exon including a splicing donor signal at the 3' end of the coding region, a NheI site at the 5' end of the fragment, and an EcoRI site at the 3' end of the fragment. The nucleotide sequence of the synthetic HuTIG3 VL gene (SEQ ID NO: 47) is shown along with the predicted amino acid sequence (SEQ ID NO: 44) in FIG. 14b.

The pHuTIG3.AA mammalian expression vector for the production of a humanized IgG1/kappa form of the TIG3 anti-human TIGIT monoclonal antibody (HuTIG3-IgG1.AA) was constructed by modifying pHuTIG1.AA as follows: (1) The HuTIG1 VH gene was replaced with the HuTIG3 VH gene between the SpeI and HindIII sites , and (2) the HuTIG1 VL gene was replaced with the HuTIG3 VL gene between the NheI and EcoRI sites. The CH2 region encoded in pHuTIG3.AA contains amino acid substitutions for alanine residues at positions 234 and 235 (Eu numbering) (L234A/L235A) to eliminate effector functions.

The amino acid sequence of the mature gamma heavy chain HuTIG3-IgG1.AA encoded in pHuTIG3.AA is

EVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTYYPDTV

KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSSASTKGPSVF

PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS

SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE

YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE

SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO:48)

The C-terminal lysine may or may not be present.

The amino acid sequence of the mature HuTIG3-IgG1.AA kappa light chain encoded in pHuTIG3.AA is

DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGV

PSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGTKVEIKRTVAAPSVFIFPPSD

EQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:49).

Transient transfection of pHuTIG3.AA into 100 ml exponentially growing Expi293 cells

- 27 041301 (Thermo Fisher Scientific) was performed according to the supplier's protocol. CHO-K1 stable transfectants producing high levels of HuTIG3-IgG1.AA were also prepared by pHuTIG3.AA electroporation as described above and expanded in SFM4CHO until cell viability was less than 50%. HuTIG3-IgG1.AA was purified from culture supernatants of each transiently transfected Expi293 cells and stably transfected CHO-K1 cells on a protein A affinity column as described above.

Example 7 Characterization of HuTIG3-IgG1.AA

Binding of HuTIG3-IgG1.AA to human TIGIT was examined by ELISA as described in Example 5. As shown in FIG. 15, HuTIG3-IgG1.AA binds to human TIGIT in a dose dependent manner. The half maximum effective concentration (EC ₅₀ ) of HuTIG3-IgG1.AA for TIGIT binding was 85 ng/ml. This shows that HuTIG3-IgG1.AA is a good binder for human TIGIT.

Binding of HuTIG3-IgG1.AA to human TIGIT was also examined by flow cytometry as described in Example 5. As shown in FIG. 12, HuTIG3-IgG1.AA binds to human TIGIT in a dose dependent manner. The half maximum effective concentration (EC ₅₀ ) of HuTIG3-IgG1.AA for binding to TIGIT was 370 ng/ml. This shows that HuTIG3-IgG1.AA is a good binder for human TIGIT.

The activity of HuTIG3-IgG1.AA to block TIGIT interaction with CD155 was analyzed by flow cytometry using NS0-hTIGIT cells and FITC-labeled hCD155-Fc as described in Example 5. As shown in FIG. 13, HuTIG3-IgG1.AA blocks the interaction between TIGIT and CD155 in a dose-dependent manner. The half-maximal inhibitory concentration (IC ₅₀ ) of HuTIG3IgG1.AA to block the TIGIT-CD155 interaction was 279 ng/mL. This shows that HuTIG3-IgG1.AA is an effective blocker of the interaction between CD155 and TIGIT.

Example 8 IL-2 Cytokine Response to Tetanus Toxoid in Human T Cells Enhanced by Blockade of TIGIT

The ability of mouse anti-TIGIT antibodies to enhance antigen-specific T-cell responses was investigated using an in vitro antigen-specific response to tetanus toxoid assay (see, e.g., Piersma et al., Vaccine. 2006 Apr 12;24(16):3076-83; Zaunders et al., J Immunol 2009 Aug 15;183(4):2827-36). Sufficient vaccine protection for tetanus toxoid is achieved with booster injections that allow the immune system to induce CD4+ and CD8+ memory T cell responses. As a surrogate measure of efficacy (see, e.g., Plotkin et al., Clin Vaccine Immunol. 2010 Jul; 17(7):1055-1065; Goulon et al. Nov. 1972 Presse Med. 1:3049-3050.), Serum levels of 0.1 IU/mL of tetanus toxoid indicate maintenance of the immune response.

Peripheral blood mononuclear cells (PBMCs) from human volunteers vaccinated with tetanus toxoid were obtained (iQ Biosciences) with confirmation of a tetanus titer in the blood of volunteers higher than 1.0 IU/ml by ELISA method (Genway). 800,000 PBMCs were cultured in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS) in 96-well round bottom plates (Nunc). 2 μg/ml tetanus toxoid (List Biological Laboratories) was added to wells containing PBMCs in the presence of mouse anti-PD-1 antibody (Biolegend, clone EH12.2H7) or anti-TIGIT antibody (TIG1) at 3.3 or 10 μg /ml for 4 days at 37°C in a 5% CO ₂ incubator. As a control, 2 μg/ml tetanus toxoid was added to PBMC without any antibodies. Supernatants from cultured wells were harvested and cytokines indicative of T cell activation, IL-2, were measured by multiplex microsphere analysis (BD Pharmingen, CBA Th1/Th2 Cytokine Kit) by flow cytometry (BD FACSCalibur). As shown in FIG. 16, IL-2 production upon stimulation with tetanus toxoid alone was 31 pg/mL. IL-2 production was further increased to 70 and 86 pg/ml in the presence of mouse anti-TIGIT antibody TIG1 at 3.3 μg/ml and 10 μg/ml, respectively. IL-2 production upon stimulation with tetanus toxoid was also increased to 39 pg/ml and 57 pg/ml in the presence of mouse anti-PD-1 antibody EH12.2H7 at 3.3 and 10 μg/ml, respectively. These data indicate that TIG1 has the functional ability to enhance antigen-specific responses of T-cell cytokines.

Example 9 Human T cell proliferative response to tetanus toxoid is enhanced by blockade of TIGIT

The ability of mouse anti-TIGIT antibodies to enhance antigen-specific T-cell responses was investigated using an in vitro antigen-specific response to tetanus toxoid assay (see, e.g., Piersma et al., Vaccine. 2006 Apr 12;24(16):3076-83; Zaunders et al., J Immunol 2009 Aug 15;183(4):2827-36). PBMCs from human volunteers vaccinated with tetanus toxoid were prepared (iQ Biosciences) as described in Example 8. PBMCs were labeled with carboxyfluorescein succinimidyl ether (CFSE) (ThermoFisher), which is a reagent used for in vitro and in vivo labeling of cells to track multiple generations with using dye dilution by flow cytometry. 250,000 CFSE-labeled PBMCs were cultured in AIM-V medium (Invitrogen) in 96 well round bottom plates (Nunc). 2 μg/ml tetanus toxoid (Astarte Biologies, LLC) was added to each well in the presence of mouse anti-PD-1 antibody (Biolegend clone EH12.2H7) or

- 28 041301

TIG1 at 2.5, 5, or 10 µg/mL in each well. Cells were incubated for 4 days at 37°C in a 5% CO ₂ incubator. On Day 4, IL-2 [50 units/mL] (Peprotech) was added to cultures, and on Day 6, PBMCs were harvested for proliferation measurements by flow cytometry (BD FACSCalibur) to determine CFSE dilution on CD4+ T cells or CD8+ T cells.

As shown in FIG. 17 (upper panels), tetanus toxoid induced proliferation of 4.5 and 6.5% of human CD4+ T cells derived from donors 1 and 2, respectively. The addition of TIG1 further increased CD4+ T cell proliferation at all antibody concentrations tested (2.5, 5 and 10 μg/ml), with similar proliferative effects observed with anti-PD-1 antibody at these dose ranges.

As shown in FIG. 17 (lower panels), tetanus toxoid induced proliferation of 15 and 15.5% of human CD8+ T cells derived from donors 1 and 2, respectively. The addition of TIG1 further increased CD8+ T cell proliferation at all antibody concentrations tested (2.5, 5 and 10 μg/mL), with similar proliferative effects observed with anti-PD-1 antibody EH12.2H7 at these dose ranges. These results indicate that TIG1 has the ability to enhance antigen-specific T-cell proliferative responses.

Example 10 Enhancement of NK Cell Mediated Cytotoxicity Using Human Primary Effector Cells and K562 Target Cells via TIGIT Blockade

Human NK cells are capable of inducing native cytotoxicity to target cells that do not contain MHC I, such as K562, a chronic myelogenous leukemia (CML) cell line (see, for example, Nagel et al., Cancer Res 1981;41:2284-2288; Andersson et al., Int J Cancer 1979 Feb;23(2):143-7.; Lozzio et al., Leuk Res. 1979;3(6):363-70). Expression of CD155 (PVR) on K562 cells (ATCC) was confirmed using a commercial antibody (Biolegend anti-CD 155 clone SKII.4). As shown in FIG. 18 (upper panel), 98% of K562 cells were found to express high levels of CD155 (black bar graph). Expression of TIGIT on human CD56 positive NK cells in PBMC (iQ Biosciences) using a commercial antibody (eBiosciences anti-TIGIT clone MBSA43 and Biolegend anti-CD56 clone HCD56) was also confirmed in 3 representative human donors (FIG. 18, bottom panels).

Lysis of K562 cells mediated by NK cells was measured with and without TIG1. PBMC were cultured overnight in AIM-V medium (Invitrogen) in the presence of interleukin-2 (IL-2) [200 units/ml] (Peprotech) at 37°C in a 5% CO2 incubator. The next day, K562 cells were labeled with CFSE and co-cultured 1:100 with PBMCs (Target: PBMCs; one K562 cell per hundred PBMCs that contained approximately 5% NK cells) for 4 h at 37°C in a 5% CO2 incubator in presence of 10 μg/ml TIG1. After incubation, cells were harvested and stained with 5 nM Sytox Red (ThermoFisher) to distinguish between dead target cells with (CFSE+ Sytox Red+). The percentages of K562 target cells lysed by NK cells were determined by flow cytometry (FACSCalibur; FlowJo Analysis). As shown in FIG. 19.2% of K562 cells were lysed without TIG1. When TIG1 was added, 4.4% of K562 cells were lysed. The addition of TIG1 increased the NK cell-mediated intrinsic cytotoxicity of K562 cells by a factor of two, demonstrating that TIG1 has the ability to increase the level of killing of target cells by a subpopulation of human effector cells.

Example 11 SEB-Induced Cytokine Production by Human T Cells Enhanced by Blockade of TIGIT

Superantigens such as SEB (Staphylococcus Enterotoxin B) activate T cells by binding class II MHC molecules on antigen presenting cells to the ve element of the TCR, resulting in the production of cytokines including interleukin-2 (IL-2), interleukin-6 (IL- 6), tumor necrosis factor alpha (TNFa) and interferon gamma (IFNy) (see, for example, Krakauer et al., Toxins (Basel). 2010 Aug; 2(8):19631983). Compared to a typical antigen-induced T cell response, where 0.1-0.001% of T cells can be activated, SEB is able to activate up to 10-20% of T cells in human blood, depending on the fraction of T cells containing ve3, ve12, ve14 and ve17 found in the blood of each specific donor. Therefore, SEB can be used for T-cell cytokine secretion assay to determine the level of target modulation through TIGIT blockade using human whole blood cells (WBC).

Freshly isolated WBC samples (Stanford Blood Center) were obtained using sodium heparin no later than 4 hours after blood sampling with no visual signs of hemolysis. 250 µl were aliquoted into wells in 96-well round bottom plates (Nunc) and stimulated with SEB (Toxin Technology) at 1 µg/ml final concentration in the presence of 10 µg/ml humanized anti-PD-1 monoclonal antibody or 10 µg/ml HuTIG1-IgG1.AA for 24 hours at 37°C in a 5% CO2 incubator. After 24 hours, the 96-well plates were rapidly centrifuged to separate the plasma layer for collection. After plasma sampling, expression levels of IL-2, IL-6, TNFa and IFNy were measured by cytokine microsphere multiplex assay (Biolegend, LEGENDplex™ Human CD8/NK Panel) by flow cytometry (BD FACSCalibur) and analyzed using Biolegend programs for quantitative determination.

- 29 041301

To determine the change in expression level of each of IL-2, IL-6, TNFa, and IFNy, the cytokine level in the presence of test antibody (plus SEB) was divided by the cytokine level in the absence of antibody for each donor. A 2-fold change (eg, determined in the presence of anti-TIGIT) thus means that the absolute concentration of cytokines measured in the experiment is twice that found in SEB-stimulated control conditions. As shown in FIG. 20, SEB-stimulated production of IL-2, IL-6, TNFa, or IFNy by healthy donor blood cells was increased in the presence of 10 μg/ml humanized anti-PD-1 or 10 μg/ml HuTIG1-IgG1.AA. Under these conditions, SEB-induced cytokine production by WBC cells and its modulation by HuTIG1IgG1.AA is a recordable indicator of potentiation of cytokine effector responses. HuTIG1-IgG1.AA is able to potentiate immune responses as demonstrated by stimulating the production of IL-2, IL-6, TNFa and IFNy in the assay shown.

Example 12 Characterization of HuTIG1-IgG1.AA and HuTIG3-IgG1.AA-Mediated Complement-Dependent Cytotoxicity

Complement dependent cytotoxicity (CDC) refers to the lysis of a cell that expresses target molecules in the presence of complement (see, for example, Gazzano-Santoro et al., J. Immunol. Methods, 202:163). Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are bound to their recognized antigen on the cell surface. To assess complement activation, a Jurkat human T cell double reporter parental cell line (Dual Jurkat; Invivogen) was engineered to stably express human TIGIT (Jurkat-TIGIT) on the surface. As shown in FIG. 21, TIGIT expression on the surface of Jurkat-TIGIT cells was confirmed by flow cytometry using a commercial PE-labeled anti-TIGIT antibody (eBiosciences clone MSBA43). Jurkat-TIGIT has therefore been used as target cells that constitutively express membrane-bound TIGIT and thus can undergo antibody-bound CDC activity in the presence of complement.

CDC analysis was performed using Jurkat-TIGIT cells and human complement in the presence of HuTIG1-IgG1.AA, HuTIG3-IgG1.AA or rabbit antithymocyte globulin (ATG) (Fresenius Biotech GmbH) at increasing concentrations up to 50 μg/ml. ATG was used as a positive control because of its reactivity with Jurkat cells and documented complement-dependent cytotoxic activity with Jurkat cells (see, e.g., Eiermann et al., J Hematother Stem Cell Res. 2001 Jun; 10(3):385- 90.; Ayuk et al., AntiCancer Research 29: 1355-1360 (2009) 50,000 Jurkat-TIGIT cells in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS) were plated in 96-well round bottom plates ( Nunc) _. incubation, human complement was added and left to incubate for an additional 3 h at 37° C. in a 5% CO2 incubator After completion of this 3 h incubation, 5 μg/ml propidium iodide (ThermoFisher) was added and samples were analyzed by flow cytometry (BD F ACSCalibur) to determine the percentage of propidium iodide positive as an indicator of cell death.

To determine normalized changes, the percentage of viable cells was measured by flow cytometry and the percentage of propidium iodide-positive cells was evaluated as dead cells. Values for untreated samples were normalized to 100%. Percent viability of samples in the presence of antibody was divided by normalized untreated to obtain a fold change in viability. As shown in FIG. 22, neither HuTIG1-IgG1.AA nor HuTIG3-IgG1.AA up to 50 μg/ml induced CDC activity. These data show that binding of both HuTIG1IgG1.AA and HuTIG3-IgG1.AA to TIGIT does not lead to complement-mediated cell death of T cells.

Example 13 Characterization of HuTIG1-IgG1.AA and HuTIG3-IgG1.AA Using the Jurkat Dual Reporter Cell Line

The functional consequence of blocking the human TIGIT receptor was analyzed using a Jurkat-TIGIT reporter cell line that carries a secreted luciferase reporter gene in the chromosome, which is driven by an IFNe minimal promoter fused to five copies of the NF-κB transcriptional response consensus element and three copies of the c-Rel binding site. . Jurkat-TIGIT cells also express a secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of the ISG54 minimal promoter along with five IFN-stimulated response elements.

The assays include two cell lines representing T effector cells (Jurkat-TIGIT) and antigen presenting cells. T effector cells stably express the luciferase reporter, which is activated downstream of the T cell receptor (TCR). Antigen-presenting cells (artificial antigen-presenting cells or aAPCs) stably express the activator protein

- 30 041301

T cell that binds to and activates a T effector cell such as Jurkat-TIGIT in an antigen-independent manner (Promega). aAPC cells are also engineered to express CD155. When a T effector cell is co-cultured with its corresponding aAPC cell, the interaction of TIGIT with CD155 inhibits T effector cell activation and reduces luciferase expression. The addition of an anti-TIGIT blocking antibody releases an inhibitory signal allowing the expression of luciferase activity.

In this assay, 16,000 aAPCs that express human CD155 and T cell activator protein (TCR CD155 Activator CHO-K1) (Promega) were plated in a 96-well half-well plate and incubated at 37°C in a 5% CO2 incubator for 24 h. The next day, 50,000 human Jurkat-TIGIT cells were co-cultured with TCR CD155 Activator CHO-K1 in the absence or presence of HuTIG1-IgG1.AA or HuTIG3-IgG1.AA, starting at 10 μg/mL with 2-fold serial dilution, for another 24 hours Supernatants were harvested and secreted luciferase was measured using QUANTI-Luc (Invivogen) and a multi-mode plate reader (Perkin Elmer EnSpire) as relative light units. To determine the fold change in relative light units, the relative light units of Jurkat-TIGIT co-cultured with TCR Activator CD155 CHO-K1 in the presence of various concentrations of anti-TIGIT antibodies were divided by relative light units in the absence of antibodies. Therefore, by way of example, a 2-fold increase (e.g., determined in the presence of anti-TIGIT) thus means that the relative light units measured in the experiment are two times greater than the amount determined in control conditions stimulated with TCR alone. activator.

As shown in FIG. 23, and HuTIG1-IgG1.AA and HuTIG3-IgG1.AA could increase natural T cell activating signaling, as determined by the increase in relative light units induced in the presence of each TIGIT blocking antibody, from 0.6 μg/mL to 10 μg/mL. approximately 1.5-2.5x magnification. These data show that anti-TIGIT antibodies have antagonistic activity, blocking TIGIT function, and increase natural T-cell activity.

Example 14 Competitive Binding with TIGIT

Flow cytometry is used to identify an antibody that competes with TIG1 for binding to human TIGIT. TIG1 is labeled with a FITC fluorescent dye using a Pierce FITC antibody labeling kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Binding of the resulting FITC-labeled TIG1 to human TIGIT was examined by flow cytometry using NS0-hTIGIT cells. One hundred thousand NS0-hTIGIT cells are incubated with various concentrations of FITC-labeled TIG1, starting at 10 µg/ml and 2-fold serial dilutions, in 200 µl FACS Buffer (PBS containing 0.5% bovine serum albumin and 0.05% sodium azide) at 4°C for 30 min. After washing with 2 ml of FACS Buffer NS0-hTIGIT, cells were suspended in 0.2 ml of FACS Buffer and subjected to flow cytometry analysis using a FACScan flow cytometer (BD Biosciences, San Jose, CA) and mean channel fluorescence (MCF) was obtained at each antibody concentration . The subsaturating concentration where 90% of the maximum fluorescence level is reached is determined for FITC-labeled TIG1.

A subsaturating concentration of FITC-labeled TIG1 is incubated with one hundred thousand NS0-hTIGIT cells in the presence (or absence) of 200-fold higher concentration of test antibody in 200 µl of FACS Buffer at 4° C. for 30 minutes. For example, when 0.1 μg/ml of FITC-labeled TIG1 is used to bind to NS0-hTIGIT cells, 20 μg/ml of test antibody is added to the cell suspension. As a background control, one hundred thousand NS0-hTIGIT cells are incubated without any antibodies. After washing with 2 ml FACS Buffer, NS0-hTIGIT cells are suspended in 0.2 ml FACS Buffer and analyzed by flow cytometry.

The MCF value for NS0-hTIGIT cells incubated with FITC-labeled TIG1 [MCF A] is normalized to 100%, and the MCF value for NS0-hTIGIT cells incubated without any antibodies [MCF B] is normalized to 0%. The MCF value for NS0-hTIGIT cells incubated with FITC-labeled TIG1 and test antibody [MCF C] is normalized using the following formula: 100 x ([MCF C] - [MCF B])/([MCF A] - [MCF B]). When the normalized MCF value for NS0-hTIGIT incubated with FITC-labeled TIG1 and test antibody is less than 20%, the test antibody is determined to compete with TIG1 for binding to human TIGIT.

The same assay can be used to identify antibodies that compete with TIG2 or TIG3.

Example 15 Affinity Measurement

The antigen-binding affinity of monoclonal antibodies can be measured using markerless surface plasmon resonance (SPR) optical biosensors such as Biacore T100 (GE Healthcare Life Sciences, Marlborough, MA), ProteOn XPR36 (BioRad, Hercules, CA), Octet RED384 (ForteBio, Menlo Park, CA) and IBIS MX96 (Wasatch Microfluidics, Salt Lake City, UT) (Yang et al., Anal. Biochem. 508:78-96, 2016). Antigen binding affinity for each of TIG1, TIG2 and TIG3 was measured using Biacore T100 as described by Yang et al. (supra). Protein A/G (Thermo Fisher Scien

- 31 041301 tific) were immobilized on flow cells in a CM5 sensor chip (GE Healthcare Life Sciences) using a standard binding protocol. The test antibody (TIG1, TIG2, or TIG3) was captured by the A/G protein on the CM5 sensor chip. Various concentrations of recombinant human TIGIT proteins, such as Recombinant Human TIGIT, His-tagged (Creative BioMart, Shirley, NY), were used on-line to bind to the sensor chip test antibody. Association rate (ka) and dissociation rate (kd) values for the interaction between test antibody and human TIGIT were obtained using the BIAevaluation program (GE Healthcare Life Sciences). The association constant (Ka) was calculated by dividing the association rate (ka) by the dissociation rate (kd) for each of TIG1, TIG2 and TIG3. The dissociation constant (Kd) was calculated by dividing the dissociation rate (kd) by the association rate (ka).

Biosensor studies for HuTIG1-IgG1.AA were performed on a BioRad ProteOn XPR36 system in 10 mM HEPES, 150 mM NaCl, pH 7.4, 0.005% Tween-20, and 0.2 mg/mL BSA at 25°C. HuTIG1-IgG1.AA was diluted to 3.7, 1.2 and 0.4 nM and captured for 5 min on a GLM sensor chip coated with ˜10,000 RU of anti-human IgG antibody from GE. His-labeled soluble recombinant human TIGIT (Cat # TIT-H52H3; Acro Biosystems, Newark, DE) was tested at 100 nM at the highest concentration in 3-fold serial dilutions up to 1.2 nM. Data from three different antibody surface densities were globally fitted to a 1:1 interaction model using a locally set Rmax. The resulting association rate (ka) and dissociation rate (kd) were (4.42 ± 0.02) x 105 M's' ¹ and (4.09 ± 0.02) x 10 ^-4 s ^-1 , respectively. The calculated dissociation constant (Kd) was 925 ± 7 pM.

Example 16 Epitope Mapping

To localize the epitope recognized by each of TIG1, TIG2, and TIG3 in the TIGIT molecule, single amino acid substitution mutants were generated in the extracellular region of TIGIT (SEQ ID NO: 3) by site-directed mutagenesis using overlap-extension PCR (Higuchi, R. , 1989, in PCR Technology: Principles and Applications for DNA Amplification, Erlich H. A., ed., Stockton Press, New York, NY, pp 61-70). The following six mutants in the TIGIT extracellular region were used for analysis: Q35A (SEQ ID NO: 50), N37A (SEQ ID NO: 51), Q39A (SEQ ID NO: 52), N49A (SEQ ID NO: 53), D51A (SEQ ID NO: 54) and F86A (SEQ ID NO: 55). The letter on the left, the number in the middle, and the letter on the right in the name of each mutant denote the amino acid residue in wild-type TIGIT, the location counting from the N-terminal methionine residue in the extracellular region of TIGIT (SEQ ID NO: 3), and the amino acid in the mutant, respectively. Amino acid residues are indicated by a one-letter code. Each of these six mutants contains an amino acid substitution near the junction of the TIGITCD155 complex (Stengel, Proc. Natl. Acad. Sci. USA, 109:5399-5404, 2012).

The mammalian expression vector pFCm179 had the same structure as pFCm404 (FIG. 1), except that (i) the puromycin N-acetyltransferase gene was replaced by the E. coli xanthineguanine phosphoribosyltransferase gene, and (ii) the extracellular region of human TIGIT was replaced with human IL-15 . DNA fragments encoding six human TIGIT extracellular region alanine substitution mutants were individually inserted into pFCm179 to replace the IL-15 coding region. The resulting expression vectors, pFCm179-Q35A, pFCm179-N37A, pFCm179-Q39A, pFCm179-N49A, pFCm179-D51A, and pFCm179-F86A, contained Q35A, N37A, Q39A, N49A, D51A, and F86A mutants in the TIGIT coding region, respectively, and expressed the following the same sequence of TIGIT-Fc fusion proteins as encoded in pFCm4 04, except for one amino acid substitution specific to each mutant.

Wild-type and mutant TIGIT-Fc fusion proteins were transiently expressed in the human embryonic kidney cell line HEK293. HEK293 cells were grown in DMEM containing 10% FCS at 37° C. in a 7.5% CO ₂ incubator. Expression vectors for wild-type and mutant TIGIT-Fc fusion proteins (pFCm404, pFCm179-Q35A, pFCm179-N37A, pFCm179-Q39A, pFCm179-N49A, pFCm179-D51A and pFCm179-F86A) were individually transfected into HEK293 cells by the polyethyleneimine method (Durocher et al Nucl Acids Res 30: e9, 2002). Production levels of TIGIT-Fc fusion proteins in culture supernatants were measured by ELISA. Briefly, an ELISA plate was coated with 100 µl/well goat anti-human IgG Fc-specific polyclonal antibody (SigmaAldrich), 1/2000-diluted in PBS, at 4° C. overnight, washed with wash buffer and blocked with ELISA Buffer 200 µl/well for 20 min at room temperature. After washing with wash buffer, culture supernatants of transiently transfected HEK293 cells, appropriately diluted in ELISA Buffer (100 μl/well), were loaded onto an ELISA plate. Purified hTIGIT-Fc was used as a standard. After incubating the ELISA plate for 30 min at room temperature and washing with wash buffer, bound Fc fusion proteins were detected using 100 μl/well HRP-conjugated goat anti-human IgG polyclonal antibody (SouthernBiotech), 12,000-diluted in ELISA Buffer. After incubating the plate for 30 minutes at room temperature and washing with wash buffer, color development was initiated by adding 100 µl/well of ABTS substrate (Sigma-Aldrich) and stopped at

- 32 041301 Aid 100 µl/well 2% oxalic acid. Absorbance was read at 405 nm.

The binding of each of the mouse TIG1, TIG2 and TIG3 antibodies to the TIGIT mutants was examined by ELISA. The ELISA plate was coated with 100 µl/well goat anti-human IgG Fc-specific polyclonal antibody (Sigma-Aldrich) 1/2000 diluted in PBS at 4° C. overnight, washed with wash buffer and blocked with 200 µl/well ELISA Buffer in for 20 min at room temperature. After washing with wash buffer 0.5 μg/ml transiently expressed TIGIT-Fc fusion proteins containing either wild-type TIGIT or one of the six TIGIT mutants in ELISA Buffer (100 μl/well) were applied to the ELISA plate in two parallel experiments for incubation at room temperature for 30 min. Culture supernatants of untransfected HEK293 cells were used as a negative control. After washing with wash buffer, 100 μl/well of 200 ng/ml of test antibody (TIG1, TIG2 or TIG3) was applied in ELISA Buffer (100 μl/well). After incubating the ELISA plate for 30 minutes at room temperature and washing with wash buffer, bound antibodies were determined using 100 μl/well HRP-conjugated goat anti-mouse kappa chain polyclonal antibody (Bethyl Laboratories, Montgomery, TX), 1/2000 diluted in ELISA Buffer . After incubating the plate for 0.5 h at room temperature and washing with wash buffer, color development was initiated by adding 100 µl/well of ABTS substrate (Sigma-Aldrich) and stopped with 100 µl/well of 2% oxalic acid. Absorbance was read at 405 nm.

Table 1 Binding of TIG1, TIG2 and TIG3 to TIGIT mutants

Relative binding to TIGIT (%)

TIGIT mutant TIG1 TIG2 TIG3 Q35A (SEQ ID NO: 50) -0.1 29.1 57.8 N37A (SEQ ID NO:51) 0.8 75.1 83.5 E39A (SEQ ID NO:52) 71.1 69.1 94.2 N49A (SEQ ID NO:53) 0.5 42.6 37.2 D51A (SEQ ID NO:54) 1.8 61.0 41.1 F86A (SEQ ID NO:55) 49.5 40.5 43.2

For each of TIG1, TIG2 and TIG3, the relative binding to each TIGIT mutant was calculated using the following formula. The mean uptake from the wild-type TIGIT-Fc fusion proteins [Abs A] was normalized to 100%, and the average uptake from culture supernatants of untransfected HEK293 cells [Abs B] was normalized to 0%. Mean absorbance with the mutant TIGIT-Fc fusion protein [Abs C] was normalized using the following formula: 100 x ([Abs C] - [Abs B])/([Abs A] [Abs B]). The result is presented in table. 1. The relative binding of TIG1 was less than 5% when the amino acid residue was replaced with alanine at position 35, 37, 49, or 51, indicating that the amino acid residues at these four positions are critical for binding TIG1 to TIGIT. None of the TIGIT mutants used in this assay abolished TIG2 and TIG3 binding to TIGIT.

Example 17 Increased IFNy secretion by the addition of HuTIG1-IgG1.AA, anti-PD-L1 antibody and HuTIG1-IgG1.AA in combination with anti-PD-L1 antibody

Human PBMCs were first isolated from the buffy coat by layering blood on gradient media (Lymphoprep; STEMCELL; 07801) and harvesting the PBMCs after centrifugation from interphase. In the next step, human monocytes were isolated from PBMCs by CD14 positive selection (EasySep human CD14 positive selection kit; STEMCELL; Cat# 18058) according to the manufacturer's protocol and cultured for six days in RPMI medium supplemented with 5% FBS and 0.1 µg/ml GM-CSF (R&D;Cat# 215-GM-050/CF) and 0.1 µg/ml IL-4 (R&D;Cat# 204-IL050/CF). Differentiated monocytic dendritic cells (moDCs) were harvested and checked for CD155, CD112 and PD-L1 expression by flow cytometry (FIG. 24).

Human CD4+ cells from 4 different donors were isolated from buffy coats by negative depletion (RosetteSep cocktail enriched in human CD4 T cells; STEMCELL; #15062) and determined for purity by flow cytometry.

moDCs and CD4+ cells were pooled in a 1:4 ratio (25,000 moDCs; 100,000 CD4+ cells) and cultured for four days in serum-free medium (X-Vivo-20; LONZA; Cat# 04-448Q) in the presence of HuTIG1-IgG1.AA ( 5 µg/mL or 15 µg/mL) or anti-PD-L1 antibody (10 µg/mL; clone 243.55.S1; SEQ ID NOs: 56 & 57) or both HuTIG1-IgG1.AA (15 µg/mL ) and anti-PD-L1 antibody (10 μg/mL) for 4 days prior to quantification of IFNy using a Cytometric Microsphere Assay (CBA; Human IFNy Flex Set; BD; Cat# 560111). Human IgG1 Fc (25 μg/ml; BioXcell; Cat# BE0096) was used as a control.

As shown in FIG. 25, blockade of HuTIG1-IgG1.AA and anti-PD-L1 antibody independently increased IFNy production compared to the corresponding isotype control antibody. The combination of HuTIG1-IgG1.AA with an anti-PD-L1 antibody significantly and synergistically increased IFNy production compared to any monotherapy used alone.

Example 18 HuTIG1-IgG1.AA and HuTIG3-IgG1.AA recognize TIGIT expressed on human lymphoid and myeloid cells

Total human PBMCs isolated from ten donors were costained directly

- 33 041301 directly conjugated antibodies, distinguishing between T-cells, B-cells, NK-cells and subpopulations of the myeloid lineage. Dead cells were excluded using the Live/dead Fixable Aqua Dead Cell Stain kit. TIGIT expression was determined using HuTIG1-IgG1.AA and HuTIG3-IgG1.AA antibodies conjugated to Alexa47 dye. CD96 expression was determined using a commercially available anti-CD96 antibody (clone NK92.39). In FIG. 26A, a plus sign (+) means expression above background, and (+++) means the highest observed expression. A plus/minus sign (+/-) indicates that only a subpopulation of cells in the designated population expresses TIGIT or CD96 above background, and a minus sign (-) indicates no expression above background. In all cases, the background value was determined using a modified Fluorescence Minus One (FMO) method in which all fluorochromes used in the staining panel were added except for the target antibody (i.e. HuTIG1-IgG1.AA, HuTIG3-IgG1.AA, anti-CD96 antibody). Conversely, the target antibody was substituted for IgG with a specific isotype control antibody of the same configuration. Fig. 26B shows representative histograms of antiTIGIT antibody staining in PBMCs at different expression levels, which were used to determine CD96 expression levels in FIG. 26A. Light histograms represent modified FMO. Dark histograms represent HuTIG1-IgG1.AA or HuTIG3-IgG1.AA staining.

Dissociated tumor cells from melanoma, colorectal cancer, non-small cell lung carcinoma, and clear cell renal cell carcinoma were obtained from a commercial source and stained with directly conjugated antibodies, distinguishing between T cells and antigen presenting cell subpopulations. Leukocytes were identified using the pan isoform of the anti-CD45 antibody (clone HI30). Dead cells were excluded using the Live/dead Fixable Aqua Dead Cell Stain kit. TIGIT expression was determined using the HuTIG1-IgG1.AA antibody conjugated to the Alexa47 dye. In all cases, the background value was determined using a modified Fluorescence Minus One (FMO) method in which all fluorochromes used in the staining panel were added except for the target antibody (ie HuTIG1-IgG1.AA). Conversely, the target antibody was substituted for IgG with a specific isotype control antibody of the same configuration. Fig. 32B shows representative histograms of anti-TIGIT staining on tumor-infiltrating lymphocytes (TILs) at various expression levels shown in FIG. 32A. Light histograms represent modified FMO. Dark histograms represent HuTIG1-IgG1.AA staining.

Example 19 Epitope Mapping of the Anti-Human TIGIT Antibody HuTIG1-IgG1.AA by Flow Cytometry

Human TIGIT cDNA was cloned into pcDNA 3.1 (+) with a 9 residue hemagglutinin (HA) peptide tag followed by a 10 residue peptide linker inserted after the 21 residue natural signal peptide and before the N-terminus of the mature TIGIT protein (SEQ ID NO: 58). Amino acid residues of interest (T34, Q35, N37, E39, L44, I47, N49, D51, L52, H55, F86, I88, H90, Y92, and T96) in the extracellular region of human TIGIT were mutated to alanine, one amino acid each at once. Plasmids were purified and transiently transfected into CHO-K1 cells using Fugene 6 transfection reagent (Promega) according to the manufacturer's recommendations. Expression of the HA-labeled human TIGIT mutant was confirmed by detection with an anti-HA antibody.

For flow cytometry analysis of anti-hTIGIT antibody epitope binding, HA-labeled human TIGIT proteins with or without single amino acid mutation were transiently transfected into CHO-K1 cells. After 24 hours, transfected cells were suspended in HBSS buffer (Thermo Fisher Scientific, Cat# 14175095) and incubated with HuTIG1-IgG1.AA antibody at varying concentrations in a 50 μl volume. After incubation for 1 hour at 4° C., cells were washed and a second incubation was performed with 50 μl of Alexa488-conjugated anti-human IgG(H+L) secondary antibody (Thermo Fisher Scientific, Cat#A-11013) to detect cells bound by the antibody. Cells were incubated with anti-HA antibody (Sigma, Cat# H7411) as a positive control and untransfected CHO-K1 cells were used as a negative control. Cells were analyzed on an Attune NxT cytometer (Thermo Fisher Scientific) and data was processed using FlowJo software. For each mutant, the MFI (mean fluorescence intensity) for saturation antibody binding was normalized to 100% POC (percentage of control), and the MFI for the control with no primary antibody binding was normalized to 0% POC. The data was then used to generate a non-linear 4-parameter sigmoid curve using GraphPad Prism 7 curve fitting software using the following formula: Y=Lowpoint+(Highpoint-Lowpoint)/( 1 + 10((LogEC50x)*Hill Slope ))

In this assay, HuTIG1-IgG1.AA bound human wild-type TIGIT with an EC ₅₀ =1.28 nM. Five of the fifteen single point alanine TIGIT mutants bound to HuTIG1-IgG1.AA with over a 10-fold reduction in binding affinity. Human TIGIT (SEQ ID NO: 3) with N49A, D51A, H9OA, N37A, or Q35A mutations bound HuTIG1-IgG1.AA with activity decreasing in the following range of EC ₅₀ =17.7 to 176 nM, respectively (FIG. 27) . All of these remnants

- 34 041301 are localized to one discrete region on the anterior β-sheet structure of the IgV extracellular domain of TIGIT (Fig. 28).

Example 20 Ex vivo Immunophenotyping of T cell subpopulations from whole blood of non-human primates injected with HuTIG1-IgG1.AA

The pharmacokinetic study was carried out at Charles River Laboratories (Reno, NV). The aim of this study was to examine ex vivo whole blood samples from cynomolgus monkeys treated with a single dose of 10 mg/kg anti-TIGIT (HuTIG1-IgG1.AA) antibody to indirectly evaluate TIGIT receptor occupancy.

Three previously unexperimented female cynomolgus monkeys (#67, #68 &#69) were given a single intravenous dose of 10 mg/kg HuTIG1-IgG1.AA antibody. Whole blood samples in sodium heparin were obtained within 24 hours of the last sample collection at time points that corresponded to the following: pre-dose, Days 1, 2, 7, 14, 21 and 28 post-dose. For whole blood flow cytometric testing, BioLegend's fluorescently labeled anti-human TIGIT (clone MBSA43) was used to determine levels of TIGIT expression on cell subpopulations of cynomolgus monkey blood lymphocytes, as they have been shown to cross-react with non-human primates (PLoS Pathog. 2016 Jan; 12(1); BioLegend). Live cells were obtained using FACSCalibur and analyzed using the program FlowJo.

Anti-TIGIT (MBSA43) was able to bind to both CD4+ and CD8+ T cell subpopulations when staining pre-dose whole blood samples. Following a dose of anti-TIGIT (MBSA43), it was unable to detect TIGIT expression on these T-cell subpopulations until Day 14 or 21, depending on the different monkeys studied, with levels returning to pre-dose detection levels by Day 21 or 28, depending on the different animals studied (FIGS. 29B & 30B). This failure to detect TIGIT expression was not due to significant changes in the frequency of CD4+ or CD8+ T cells (FIGS. 29A & 30A, respectively), suggesting that this was due to occupancy of the TIGIT surface by the treated antibody (HuTIG1IgG1.AA) rather than due to the depletion of the T-cell subpopulation.

These data confirm that the anti-TIGIT antibody HuTIG1-IgG1.AA is cross-reactive in cynomolgus monkeys, and the use of BioLegend's anti-human TIGIT (clone MBSA43) provides an estimate of TIGIT receptor occupancy.

Example 21 Increased NK cell-mediated cytotoxicity induced by HuTIG1IgG1.AA and HuTIG3-IgG1.AA on K562 target cells

The NK cell mediated cytotoxicity assay described in Example 10 was repeated using purified NK cells instead of PBMCs. To obtain sufficient and suitable purified NK cells for these experiments, it is important to screen for donor PBMCs with high NK cell frequency, sufficient surface TIGIT expression, and sufficient CD226 expression. LeukoPak peripheral blood (consisting of 10 billion cells or more) was prepared and NK cell isolation was performed using the Miltenyi autoMACS Pro Separator according to the manufacturer's instructions (Miltenyi Biotec, catalog#130-092-657). Purified NK cells were resuspended in medium containing interleukin-2 (IL-2) [200 units/ml] in 6-well plates. The 6-well plates were placed in a 5% CO ₂ incubator at 37° C. for 24 hours before carrying out natural cytotoxicity studies with cells harvested from the wells.

K562 cell lysis mediated by purified NK cells was measured with and without anti-TIGIT antibody (TIG1). NK cells were cultured in RPMI 1640 medium (Invitrogen) in the presence of interleukin-2 (IL-2) [200 units/ml] (Peprotech) overnight at 37°C in a 5% CO2 incubator. The next day, K562 cells were labeled with CFSE and co-cultured 1:20 with NK cells (Target^^ one K562 cell per twenty NK cells) for 4 h at 37°C in a 5% CO2 incubator in the presence of 10 μg/ml Rituxan or humanized anti-TIGIT antibodies (HuTIG1-IgG1.AA or HuTIG3-IgG1.AA). Rituxan was used as a negative control because K562 cells do not express CD20. After incubation, cells were harvested and stained with 5 nM Sytox Red (ThermoFisher) to distinguish between dead target cells with (CFSE+ Sytox Red+). The percentages of K562 target cells lysed with NK cells were determined by flow cytometry (FACSCalibur; FlowJo Analysis).

As shown in FIG. 31, when using purified NK cells, the addition of anti-TIGIT antibodies increased the NK cell-mediated intrinsic cytotoxicity of K562 cells, demonstrating that anti-TIGIT antibodies (HuTIG1-IgG1.AA or HuTIG3-IgG1.AA) have the ability to increase cell killing- targets (by 28% or 19%, respectively, compared to normalized percentages Without Antibody or with Rituxan) by a subpopulation of human effector cells.

- 35 041301

Sequences

SEQ ID NO:1: Amino acid sequence of mature human TIGIT:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPS FKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLG AMAATLWICTAVIVWALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGTGSCVQAEAAPA GLCGEQRGEDCAELHDLCFNVLSYRS

SEQ ID NO:2: Amino acid sequence of the signal peptide upstream of the extracellular region of human TIGIT:

MGWSWIFFFLLSGTASVLS

SEQ ID NO:3: Amino acid sequence of human TIGIT extracellular region (hTIGIT):

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRV APGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPTG

SEQ ID NO:4: Amino acid sequence of polypeptide linker immediately downstream from hTIGIT: TGGG

SEQ ID NO:5: Amino acid sequence of the human γΐ Fc region:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYVMKTTFSVLDCSPGQHLSK

SEQ ID NO:6: Amino acid sequence of human CD155 extracellular region:

DWVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSY SESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAE VQKVQL Т GE PVPMARCVS Т GGRPPAQITWHS DLGGMPNT S QVPGFL S GTVTVT S LWILVP S S QV DGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYN WSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGMSR NA

SEQ ID NO:7: Amino acid sequence of human CD155 extracellular region fused to human γΐ chain Fc region (hCD155-Fc):

DWVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSY SESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAE VQKVQL Т GE PVPMARCVS Т GGRPPAQITWHS DLGGMPNT S QVPGFL S GTVTVT S LWILVP S S QV DGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYN WSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGMSR NATGGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:8: Amino acid sequence of FLAG peptide

- 36 041301 (FLAG): DYKDDDDK

SEQ ID NO:9: Amino acid sequence of human CD55 glycosylphosphatidylinositol (GPI) binding signal: PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT

SEQ ID NO:10: TIG1 VH amino acid sequence: DVQLVESGGGLVQPGGSRKLSCAASGFTFSNFGMHWVRQAPEKGLEWVAFISSGSSSIYYADTV KGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARMRLDYYAMDYWGQGTSVTVSS

SEQ ID NO:11: TIG1 VH CDR1 amino acid sequence: NFGMH

SEQ ID NO:12: Amino acid sequence of TIG1 VH

CDR2: FISSGSSSIYYADTVKG

SEQ ID NO:13: TIG1 VH amino acid sequence

CDR3: MRLDYYAMDY

SEQ ID NO:14: TIG1 VL amino acid sequence: DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSG SGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK

SEQ ID NO:15: TIG1 VL CDR1 amino acid sequence: RASKSISKYLA

SEQ ID NO:16: Amino acid sequence of TIG1 VL

CDR2: SGSTLQS

SEQ ID NO:17: Amino acid sequence of TIG1 VL

CDR3: QQHNEYPWT

SEQ ID NO:18: TIG2 VH amino acid sequence: EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKNLEWIGGINPNNGGTSYNQKF KGRATLTVDKS S S TAYMELRSLTSDDSAVYYCARPGWYNYAMDYWGQGT SVTVSS

SEQ ID NO:19: TIG2 VH CDR1 amino acid sequence: EYTMH

SEQ ID NO:20: Amino acid sequence of TIG2 VH

CDR2: GINPNNGGTSYNQKFKG

SEQ ID NO:21: Amino acid sequence of TIG2 VH

CDR3: PGWYNYAMDY

SEQ ID NO:22: TIG2 VL Amino Acid Sequence: DIVMTQSHKFMSTSVGDRVNITCKASQGVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTG SGSGTDFTFTISSVQAEDLAVYHCQQHYITPWTFGGGTKLEIK

SEQ ID NO:23: TIG2 VL CDR1 amino acid sequence: KASQGVSTAVA

- 37 041301

SEQ ID NO:24: Amino acid sequence of TIG2 VL

CDR2: SASYRYT

SEQ ID NO:25: Amino acid sequence of TIG2 VL

CDR3: QQHYITPWT

SEQ ID NO:26: TIG3 VH amino acid sequence: EVQLVESGGGLVKPGGSLKLSCAASGFAFSDYDMSWVRQTPEKRLEWVAYISDGGYNTYYPDTV KGRFTISRDNAKNTLYLQMSSLKSEDTAIYYCARQILLRYYFDYWGQGTTLTVSS

SEQ ID NO:27: TIG3 VH CDR1 amino acid sequence: DYDMS

SEQ ID NO:28: TIG3 VH amino acid sequence

CDR2: YISDGGYNTYYPDTVKG

SEQ ID NO:29: TIG3 VH amino acid sequence

CDR3: QILLRYYFDY

SEQ ID NO:30: TIG3 VL amino acid sequence: DIVMSQSPSSLAVSVGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGV PDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYHSYPWTFGGGTKLEIK

SEQ ID NO:31: TIG3 VL CDR1 amino acid sequence: KSSQSLLYSSNQKNYLA

SEQ ID NO:32: Amino acid sequence of TIG3 VL

CDR2: WASTERS

SEQ ID NO:33: Amino acid sequence of TIG3 VL

CDR3: QQYHSYPWT

SEQ ID NO:34: Humanized engineered TIG1 VH (HuTIGl VH) amino acid sequence: MDSRLNLVFLVLILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGL EWVAFISSGSSSIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYW GQGTMVTVSS

SEQ ID NO:35: Amino acid sequence of mature HuTIGl VH:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIY YADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVSS

SEQ ID NO:36: Humanized engineered TIG1 VL (HuTIGl VL) amino acid sequence: MRFQVQVLGLLLLWISGAQCDIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAP KLLIYSGSTLQSGVPSRFSGSGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKVEIK

SEQ ID NO:37: Amino acid sequence of mature

- 38 041301

HuTIGl VL:

DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLIYSGSTLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKVEIK

SEQ ID NO:38: Nucleotide sequence of the synthetic HuTIGl VH gene in pHuTIGl.AA:

ACTAGTACCACCATGGACTCCAGGCTCAATCTGGTTTTCCTTGTCCTTATTCTGAAAGG CGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTTTGGAATGCACTGGGTTCGACAGG CTCCAGGGAAGGGGCTGGAGTGGGTCGCATTCATTAGTAGTGGCAGTAGTTCCATCTACTATGC AGACACAGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACAGCCTGTACCTGCAA ATGAACAGTCTGAGGGCTGAGGACACTGCCGTGTATTACTGTGCAAGAATGAGACTGGATTACT ATGCTATGGACTACTGGGGTCAAGGAACCATGGTCACCGTCTCCTCAGGTAAGTATGGCCTCTA AGCTT

SEQ ID NO:39: Nucleotide sequence of the synthetic HuTIGl VL gene in pHuTIGl.AA:

GCTAGCACCACCATGAGGTTCCAGGTTCAGGTTCTGGGGCTCCTTCTGCTCTGGATCTC AGGAGCCCAGTGTGATATCCAGATGACCCAGTCTCCATCTTCTCTTTCTGCATCTGTTGGAGAT AGAGTCACTATTACTTGCAGGGCAAGTAAGAGCATTAGCAAATATCTGGCCTGGTATCAACAGA AACCTGGGAAAGCTCCTAAGCTGCTTATCTACTCTGGGTCCACTTTGCAATCTGGAGTTCCATC AAGATTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGCAGCCTGAA GATTTTGCAACCTATTACTGTCAACAGCATAATGAATACCCCTGGACCTTCGGCGGAGGCACCA AAG T C GAAAT СAAAC G TAAG TAGAAT С CAAAGAATT C

SEQ ID NO:40: Аминокислотная последовательность зрелой гамма тяжелой цепи HuTIGl-IgGl.АА, кодируемой в pHuTIGl.AA: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIYYADTV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG К

SEQ ID NO:41: Amino acid sequence of the mature HuTIGl-IgGl.AA light chain kappa encoded in pHuTIGl.AA:

- 39 041301

TASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:42: Humanized engineered TIG3 VH (HuTIG3 VH) amino acid sequence: MNFGLRLIFLVLTLKGVNCEVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGL EWVAYISDGGYNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDVSS

SEQ ID NO:43: Amino acid sequence of mature HuTIG3 VH:

EVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTY YPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSS

SEQ ID NO:44: Humanized engineered TIG3 VL (HuTIG3 VL) amino acid sequence: MDSQAQVLMLLLLWVSGTCGDIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQ KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGT KVEIK

SEQ ID NO:45: Amino acid sequence of mature HuTIG3 VL:

DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWAST RESGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGTKVEIK

SEQ ID NO:46: Nucleotide sequence of the synthetic HuTIG3 VH gene in pHuTIG3.AA:

ACTAGTACCACCATGAACTTTGGGCTCAGATTGATTTTCCTTGTCCTTACTCTGAAAGG CGTGAACTGTGAAGTCCAGCTCGTGGAGTCTGGGGGAGGCCTTGTGCAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTGACTATGACATGTCTTGGGTTCGCCAGG CTCCTGGCAAGGGGCTGGAGTGGGTCGCATACATTAGTGATGGCGGTTATAACACCTACTATCC AGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACTCCCTGTACCTGCAA ATGAACAGTCTGAGGGCTGAGGACACAGCCGTCTATTACTGTGCAAGACAAATTCTGCTGCGGT ACTACTTTGACTACTGGGGCCAAGGCACCACTGTCACAGTCTCCTCAGGTGAGTCCTTAAAACA AGCTT

SEQ ID NO:47: Nucleotide sequence of the synthetic HuTIG3 VL gene in pHuTIG3.AA:

GCTAGCACCACCATGGATTCACAGGCCCAGGTTCTTATGCTGCTGCTGCTCTGGGTTTC TGGAACCTGTGGGGACATTCAGATGACAACAGTCTCCATCCTCCCTGTCTGCCTCAGTTGGAGAC AGGGTTACTAT CAC C T G CAAG T C CAG T CAGAG TCTTCTGTATAGTAG CAAAT CAAAAGAAC TACT TGGCCTTGGCGTACCAGCAGAAACCACTACTGGAAGGATTCACT

- 40 041301

GGAATCTGGGGTCCCTAGTCGCTTCTCAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATC AGCAGTCTGCAGCCTGAAGACTTCGCAGTTTATTACTGTCAGCAATATCATAGCTATCCCTGGA CCTTCGGCGGAGGCACCAAGGTGGAAATCAAACGTAAGTAGAATCCAAAGAATTC

SEQ ID NO:48: Аминокислотная последовательность зрелой гамма тяжелой цепи HuTIG3-IgGl.АА, кодируемод в pHuTIG3.AA: EVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTYYPDTV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG К

SEQ ID NO:49: Аминокислотная последовательность зрелой каппа легкой цепи HuTIG3-IgGl.АА, кодируемод в pHuTIG3.AA: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGV PSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:50: Amino acid sequences of the extracellular region of human TIGIT with Q35A mutation:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTAVNWEQQDQLLAICNADLGWHISPS FKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPTG

SEQ ID NO:51: Amino acid sequences of the extracellular region of human TIGIT with N37A mutation:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVAWEQQDQLLAICNADLGWHISPS FKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPTG

SEQ ID NO:52: Amino acid sequences of the extracellular region of human TIGIT with Q39A mutation:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWAQQDQLLAICNADLGWHISPS FKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPTG

SEQ ID NO:53: Amino acid sequences of the extracellular region of human TIGIT with N49A mutation:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICAADLGWHISPS FKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPTG

SEQ ID NO:54: Amino acid sequences

- 41 041301 extracellular region of human TIGIT with D51A mutation:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNAALGWHISPS

FKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPTG

SEQ ID NO:55: Amino acid sequences of the extracellular region of human TIGIT with F86A mutation:

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPS

FKDRVAPGPGLGLTLQSLTVNDTGEYACIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPTG

SEQ ID NO:56: Amino acid sequence of mature heavy chain of anti-human PD-L1 antibody

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTY YADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRWSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG

SEQ ID NO:57: Amino acid sequence of mature light chain of anti-human PD-L1 antibody

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLFTPPTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYVTHNKSSFQACE

SEQ ID NO:58: Amino acid sequence of signal, HA tag and linker peptides fused to mature human TIGIT:

MRWCLLLIWAQGLRQAPLASGYPYDVPDYAGGGGSGGGGSMMTGTIETTGNISAEKGGS

IILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTG EYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLWICTAVIVWALTRKKK ALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYR SLGNCSFFTETG

SEQ ID NO:59: Amino acid sequence of human TIGIT extracellular region fused to human immunoglobulin γΐ chain Fc region (hTIGIT-Fc):

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPS

- 42 041301

FKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPTGT GGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:60: HuTIGl-IgGl.Q mature gamma heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIY YADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

SEQ ID NO:61: HuTIGl-IgG4.P mature gamma heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIY

YADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK

SEQ ID NO:62: HuTIG3-IgGl.Q mature gamma heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTY YPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

SEQ ID NO:63: HuTIG3-IgG4.P mature gamma heavy chain amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTY YPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK

SEQ ID NO:64: Amino acid sequence of mature HuTIGl-IgGl.Q and HuTIGl-IgG4.P light chain kappa light chain:

DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLIYSGSTLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYVTHGLNSPVACE

SEQ ID NO:65: Amino acid sequence of mature kappa light chain HuTIG3-IgGl.Q and HuTIG3-IgG4.P:

DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGV PSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSGETYSLTLVGLSKADY PACEVK

Claims (33)

CLAIM 1. An anti-TIGIT antibody that binds to a TIGIT polypeptide containing a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 11, - 43 041301 Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 12, Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13, Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 15, Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 16 and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 17. 2. An anti-TIGIT antibody that binds to a TIGIT polypeptide comprising a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: : 21, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 23, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 24, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 25. 3. An anti-TIGIT antibody that binds to a TIGIT polypeptide comprising a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 27, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 28, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: : 29, a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 31, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 32, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 33. 4. The anti-TIGIT antibody of claim 1, comprising a mature heavy chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 35 and a mature light chain variable region that is at least 90% identical to the sequence of SEQ ID NO: 37. 5. The anti-TIGIT antibody of claim 4, wherein the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 40, provided that C-terminal lysine may or may not be present, and the mature light chain variable region linked to the light chain constant region containing SEQ ID NO: 41. 6. The anti-TIGIT antibody of claim 4, wherein the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 60, provided that C-terminal lysine may or may not be present, and the mature light chain variable region linked to a light chain constant region containing SEQ ID NO: 64. 7. The anti-TIGIT antibody of claim 4, wherein the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 61, provided that C-terminal lysine may or may not be present, and the mature light chain variable region linked to a light chain constant region containing SEQ ID NO: 64. 8. An anti-TIGIT antibody according to any one of claims 1 to 3, wherein the anti-TIGIT antibody is a chimeric antibody, a humanized antibody, or a veneered antibody. 9. A monoclonal antibody that binds to a TIGIT polypeptide containing a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 13 , a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 17; where the monoclonal antibody has one or more of the following properties: (a) inhibits the binding of TIGIT to CD 155, (b) increases natural T cell activation in the presence of antigen presenting cells expressing CD155, as measured by IL-2 production compared to natural T cell by activation in the presence of antigen-presenting cells expressing CD155 and in the absence of antibody, (c) increases antigen-specific T-cell activation as measured by IL-12 production compared to antigen-specific T-cell activation in the absence of antibody, (d) increases natural killer cell activation , as measured by production of any of IL-2, IL-6, TNFa, or IFNy compared to natural killer cell activation in the absence of antibody, (e) increases T cell production of at least one pro-inflammatory cytokine compared to T cell production at least one pro-inflammatory cytokine in the absence of an antibody, and (f) reduces production by T cells and at least one anti-inflammatory cytokine compared to T cell production of at least one anti-inflammatory cytokine in the absence of the antibody. 10. The monoclonal antibody of claim 9, wherein the IC ₅₀ for inhibition of TIGIT binding to CD155 is about 15-100 ng/mL. 11. Monoclonal antibody according to claim 9, where natural T-cell activation in the presence of anti - 44 041301 body increased by about 1.5-3 times compared with the absence of antibodies. 12. The monoclonal antibody of claim 9, wherein the antigen-specific T-cell activation in the presence of the antibody is increased by about 1.5-3 times compared to the absence of the antibody. 13. A monoclonal antibody according to claim 9, wherein the activation of natural killer cells in the presence of the antibody is increased by about 1.5-3 times compared to the absence of the antibody. 14. The monoclonal antibody of claim 9, wherein the production of at least one pro-inflammatory cytokine by T cells in the presence of the antibody is increased by about 1.5-3 times compared to the absence of the antibody. 15. The monoclonal antibody of claim 9, wherein T cell production of at least one anti-inflammatory cytokine is reduced by about 1.5 to 3 times in the presence of the antibody compared to the absence of the antibody. 16. A method of treating or preventing a cancer, wherein the cancer is a solid tumor or a hematologic cancer, comprising administering to an individual suffering from or at risk of developing a cancer, an antibody according to any one of the preceding claims in an effective regimen or in a therapeutically effective amount . 17. The method of claim 16, wherein the individual is further administered with tumor-infiltrating T cells that are activated by the antibody; or a vaccine that induces an immune response against cancer that is enhanced by an antibody; or natural killer cells whose cytotoxicity against cancer is enhanced by an antibody; or a second antibody against an antigen expressed on the surface of cancer cells, wherein the effector-mediated cytotoxicity of the second anti-cancer antibody is enhanced by the antibody; or a second antibody against an antigen expressed on the surface of an immune cell; or one or more therapies selected from the group consisting of chemotherapy, radiation, cell therapy and surgery; or an inhibitor of one or more immune checkpoint receptors or ligands is administered. 18. The method of claim 16 wherein the solid tumor is lung cancer. 19. The method according to claim 17, where the vaccine contains an antigen or fragment thereof expressed on the surface of malignant cells. 20. The method of claim 17 wherein the immune cell is a T cell or natural killer cell. 21. The method of claim 17 wherein the antigen expressed on the surface of the immune cell is CTLA-4, PD-1 or PD-L1. 22. The method of claim 17 wherein the inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab (lambrolizumab), and atezolizumab. 23. A method of treating an individual infected with a pathogen selected from the group consisting of a virus, a bacterium, a fungus and a protozoan, comprising administering to the individual an antibody according to any one of the preceding claims in an effective mode or in a therapeutically effective amount. 24. The method of claim 23, wherein the subject is also administered a second antibody against the pathogen, wherein the effector-mediated cytotoxicity of the second antibody against the pathogen is enhanced by the antibody; or wherein the individual is further administered with one or more antiviral agents, antiparasitic agents, antibacterial agents, or antifungal agents. 25. The method of claim 23 wherein the pathogen is HIV, HCV, hepatitis virus, herpes virus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus , rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, mollusk virus, poliovirus, rabies virus, JC virus, arbovirus encephalitis virus, chlamydia virus, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci, gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria bacillus, Salmonella bacillus and bacteria that cause cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease. 26. A method of treating a cancer, wherein the cancer is a solid tumor or a hematologic cancer, comprising administering to an individual suffering from the cancer a therapeutically effective amount of an antibody according to any one of the preceding claims. 27. The method of claim 26, wherein the individual is further administered with tumor-infiltrating T cells that are activated by the antibody; or administering a vaccine that induces an immune response against the cancer that is enhanced by the antibody; or administering natural killer cells whose cytotoxicity against cancer is enhanced by the antibody; or administering a second antibody against an antigen expressed on the surface of the cancer cells, wherein the effector-mediated cytotoxicity of the second cancer antibody is enhanced by the antibody; or a second antibody is administered against an antigen expressed on the surface of an immune cell; or one or more therapies selected from the group consisting of chemotherapy, radiation, cell therapy and surgery; or an inhibitor of one or more immune checkpoint receptors or ligands is administered. 28. The method of claim 26 wherein the solid tumor is lung cancer. 29. The method of claim 27, wherein the vaccine comprises an antigen expressed on the surface of malignant cells, or a fragment thereof. 30. The method of claim 27 wherein the immune cell is a T cell or natural killer cell. 31. The method of claim 27 wherein the antigen expressed on the surface of the immune cell is CTLA-4, PD-1 or PD-L1. 32. The method of claim 27 wherein 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, BTLA, VISTA, CD96, A2aR, A2bR, Arginases, CD39, CD73, IDO and TDO. 33. The method of claim 27 wherein the inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab (lambrolizumab), and atezolizumab.