Antibodies to tigit

Abstract

To provide antibodies to TIGIT.SOLUTION: The invention provides monoclonal antibodies that specifically bind to TIGIT. The monoclonal antibodies have the capacity for substantial activation of T cells and natural killer cells by inhibiting binding of TIGIT to CD155. The monoclonal antibodies can be used for treatment of cancer and infectious disease, among other applications. The invention provides, inter alia, an antibody that competes with any of TIG1, TIG2 and TIG3 for binding to human TIGIT.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 304,045, filed March 4, 2016, and U.S. Provisional Patent Application No. 62 / 413,025, filed October 26, 2016, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Provided herein are, inter alia, monoclonal antibodies that specifically bind to immune checkpoint molecules, thereby resulting in substantial activation of immune cells, and their use for the treatment of cancer and infectious diseases, among other applications. [Background technology]

[0003] Antigen-specific immune responses are complex biological processes controlled by multiple layers of positive and negative regulators. T cells first bind to major antigens on antigen-presenting cells via their T cell receptors (TCRs). Stimulated by recognition of their cognate peptide antigens presented by histocompatibility complex (MHC) molecules Optimal T cell activation requires the “secondary signal” provided by costimulatory molecules such as CD28. The immune response also requires co-stimulatory molecules, such as the TNF receptor superfamily. It is positively regulated by checkpoint molecules such as OX40, GITR, and 4-1BB, which belong to the For example, checkpoints are negatively regulated by PD-1 and CTLA-4. The function of checkpoint molecules is to prevent unwanted overreactions of the body's immune system, but they also limit the immune system's ability to effectively fight cancer and infectious diseases. Blockade of PD-1 or CTLA-4 function with antagonistic monoclonal IgG antibodies is effective in the immunotherapy of cancer in humans. (For reviews, see Pardoll, Nat. Rev. Cancer, 12:252-264, 2012; Ma Honey et al., Nat. Rev. Drug Discov. 14:561-584, 2015; Shin et al., Curr. Opin. Immunol. 33 :23-35, 2015; see Marquez-Rodas et al., Ann. Transl. Med. 3:267, 2015).

[0004] 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 integrated transcriptional motif (ITIM) domains, mediates the expression of T cells in activated and natural T cells. It is expressed on a subset of killer (NK) cells (Yu et al., Nat. Immunol. 10:48-57, 2009). GIT expresses CD155 (also called PVR and necl-5), CD112 (also called PVRL2 and nectin-2), It is known that CD4+ interacts with CD113 (also called PVRL3 and nectin-3) and possibly with 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 its high affinity ligand CD155 expressed on T cells suppresses the functions of T cells and NK cells. (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., February 3, 2016. Epu b) TIGIT has also been reported to indirectly inhibit T cells by modulating cytokine production by dendritic cells (Yu et al., supra).

[0005] Tumors are depleted of infiltrating T cells and NK cells are forced to activate checkpoints to evade the immune response. These tumors constitute a highly inhibitory microenvironment that is silenced by target molecules such as PD-1 and TIGIT (Johnston et al., Cancer Cell. 26:926-937, 2014; Chauvin et al., J. Clin. Invest. 125:2046-2058, 2015). , 2015; Inozume et al., J. Invest. Dermatol., Epub 12 Oct. 2015). On CD8+ T cells It has been reported that high-level expression of TIGIT in AML subjects correlates with poor clinical outcomes in AML subjects (Kong et al., Clin. Cancer Res., Epub 13 Jan. 2016). It has been reported that the functional defect of + CD8+ T cells was reversed by siRNA-mediated knockdown of TIGIT expression (Kong et al., supra). It has also been reported that effector CD8+ T cells during HIV infection of blood and SIV infection of lymphoid tissues showed higher TIGIT levels (Chew et al., PLOS Patho gens, 12:e1005349, 2016). In addition, ex vivo antibody blockade of TIGIT inhibits virus-specific CD4 It has been reported to restore 8+ T cell effector responses. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Pardoll, Nat. Rev. Cancer, 12:252-264, 2012 [Non-Patent Document 2] Mahoney et al., Nat. Rev. Drug Discov. 14:561-584, 2015 [Non-Patent Document 3] Shin et al., Curr. Opin. Immunol. 33:23-35, 2015 [Non-Patent Document 4] Marquez-Rodas et al., Ann. Transl. Med. 3:267, 2015 [Non-Patent Document 5] Mercier et al., Front. Immunol. 6:418, 2015 [Non-Patent Document 6] Yu et al., Nat. Immunol. 10:48-57, 2009 [Non-Patent Document 7] Martinet et al., Nat. Rev. Immunol.15:243-254, 2015 [Non-Patent Document 8] Joller et al., J. Immunol. 186:1338-1342, 2011 [Non-Patent Document 9] Stanietsky et al., Eur. J. Immunol. 43:2138-2150, 2013 [Non-Patent Document 10] Li et al., J. Biol. Chem. 289:17647-17657, 2014 [Non-Patent Document 11] Zhang et al., Cancer Immunol. Immunother., February 3, 2016, Epub [Non-Patent Document 12] Johnston et al. Cancer Cell. 26:926-937, 2014 [Non-Patent Document 13] Chauvin et al., J. Clin. Invest. 125:2046-2058, 2015 [Non-Patent Document 14] Inozume et al., J. Invest. Dermatol., Epub 12 Oct 2015 [Non-Patent Document 15] Kong et al., Clin. Cancer Res., Epub 13 January 2016 [Non-Patent Document 16] Chew et al., PLOS Pathogens, 12:e1005349, 2016 Summary of the Invention

[0007] The present invention relates to, inter alia, a method for the production of human TIGIT that competes with either TIG1, TIG2 or TIG3 for binding to human TIGIT. For specific binding to TIGIT, the antibody TIG1 has the mature sequence of SEQ ID NO: 14. and the mature light chain variable region of SEQ ID NO: 10, antibody TIG2 is characterized by a mature light chain variable region of SEQ ID NO: 22 and a mature heavy chain variable region of SEQ ID NO: 18, and antibody TIG3 is characterized by a mature light chain variable region of SEQ ID NO: 30 and a mature heavy chain variable region of SEQ ID NO: 26. Some antibodies bind to the same epitope on human TIGIT as TIG1, TIG2 or TIG3. Some antibodies inhibit binding of human TIGIT to CD155. Some antibodies comprise three light chain CDRs and three heavy chain CDRs substantially from the corresponding three light chain CDRs and three heavy chain CDRs from TIG1. Some antibodies Some antibodies contain three light chain CDRs and three heavy chain CDRs of TIG1, TIG2 or TIG3. TIG1 (light chains of SEQ ID NOs: 15 to 17 and heavy chains of SEQ ID NOs: 11 to 13), TIG2 (light chains of SEQ ID NOs: 23 to 25 and heavy chains of SEQ ID NOs: 19 to 21), or TIG3 (light chains of SEQ ID NOs: 31 to 33 and heavy chains of SEQ ID NOs: 27 to 29) The three heavy chain CDRs defined by Kabat and the three light chain CDRs defined by Kabat are included.

[0008] Some monoclonal antibodies bind to an epitope of human TIGIT that includes residues 35 and 37 of SEQ ID NO:1 and / or residues 49 and 51 of SEQ ID NO:1. Some monoclonal antibodies bind to an epitope of human TIGIT that includes residues 35, 37, 49 and 51 of SEQ ID NO:1. Some monoclonal antibodies bind to an epitope of human TIGIT that includes residues 35-51 of SEQ ID NO:1 and 5 or more residues on either side from SEQ ID NO:1. Some monoclonal antibodies bind to a peptide consisting of residues 35-51 of SEQ ID NO:1. Some such monoclonal antibodies bind to a peptide consisting of residues 35-51 of SEQ ID NO:1. It binds to an epitope consisting of 3 to 20 consecutive residues of SEQ ID NO:1.

[0009] Some antibodies are chimeric, humanized, veneered, or human. Some antibodies have a human IgG1 kappa isotype. Some antibodies have a human IgG4 kappa isotype. Antibodies may be intact or single chain antibodies, Fab or F(ab'). 2 It may be a fragment.

[0010] The present invention further provides a pharmaceutical composition comprising any of the above antibodies and a pharma- ceutically acceptable carrier.

[0011] The invention further provides methods of treating or preventing cancer in a subject, comprising administering to a subject having or at risk of cancer an effective regimen of any of the above-described antibodies. In some methods, the subject has acute myeloid leukemia or adult T-cell leukemia.

[0012] In other embodiments, the present invention contemplates the use of the antibodies described herein in combination with immune checkpoint inhibitors. Blockade of checkpoints has been shown to be a promising approach in human cancer therapy. Examples of immune checkpoints (ligands and receptors) that are candidates for blockade and that are selectively upregulated in various tumor cell types include PD-1 (programmed cell death inhibitor); Protein 1), PD-L1 (programmed 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 inhibitor of T cell activation (VISTA), CD96, A2aR (adenosine A2a receptor), A2bR (adenosine A2b receptor), CD73 (ecto-5'-nucleotidase), CD39 (ENTPD1, ​​NTPDase1), arginase, indoleamine-pyrrole2,3-dioxygenase (IDO), tryptophan-2,3-dioxygenase (TPDase), tryptophan-2,3-dioxygenase (TTPA ... tryptophan 2,3-dioxygenase (TDO), and killer cell inhibitory receptors. Immune checkpoint inhibitors, and combination therapies using them, are discussed in detail elsewhere herein.

[0013] The present invention further provides a method of treating a subject infected with a pathogen, comprising administering to the subject an effective regimen of any of the above-mentioned antibodies. In some methods, the pathogen is HIV or SIV. In other methods, the pathogen is a virus, a bacterium, a fungus, or a protozoan.

[0014] In a further embodiment, the TIGIT polypeptide is linked to one or more amino acid residues, including D51. an anti-TIGIT antibody that binds to TIGIT polypeptide, the TIGIT polypeptide having an amino acid sequence corresponding to SEQ ID NO:1; Provided herein are anti-TIGIT antibodies having the following structure: In some embodiments of any of the embodiments disclosed herein, the antibody is a chimeric, humanized, or veneered antibody. In some embodiments, the antibody is a human antibody. In some embodiments of any of the embodiments disclosed herein, the antibody is L44 , I47, or H55. In some of the embodiments, 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 of all of the embodiments disclosed herein, the antibody binds to the same epitope as TIG1 on 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 NOs: 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 having at least 90% sequence identity to SEQ ID NO: 35 and a mature light chain variable region having at least 90% sequence identity to 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 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 comprises the amino acid sequence of SEQ ID NO: 35 with the proviso that a C-terminal lysine may or may not be present. The mature light chain variable region is linked to a heavy chain constant region comprising SEQ ID NO:40, and the mature light chain variable region is linked to a light chain constant region comprising SEQ ID NO:41.

[0015] In a further aspect, provided herein are antibodies to TIG1, TIG2, and TIG3 for binding to human TIGIT. or TIG3, for specific binding to CD155. To this end, the antibody TIG1 is characterized by a mature light chain variable region of SEQ ID NO: 14 and a mature heavy chain variable region of SEQ ID NO: 10, the antibody TIG2 is characterized by a mature light chain variable region of SEQ ID NO: 22 and a mature heavy chain variable region of SEQ ID NO: 18, and the antibody TIG3 is characterized by a mature light chain variable region of SEQ ID NO: 30 and a mature heavy chain variable region of SEQ ID NO: 26. In some embodiments, the antibody binds to the same epitope on human TIGIT as either TIG1, TIG2 or TIG3. In some embodiments of any of the above embodiments, the antibody inhibits binding of human TIGIT to CD155. In some embodiments, the antibody comprises three light chain CDRs and three heavy chain CDRs corresponding to the three light chain CDRs and three heavy chain CDRs of any one of TIG1, TIG2 or TIG3. In embodiments, the antibody comprises the three heavy chain CDRs and the three light chain CDRs of any one of TIG1, TIG2 or TIG3. In some embodiments, the antibody comprises the three heavy chain CDRs as defined by Kabat and the three light chain CDRs as defined by Kabat of any one of TIG1 (light chain of SEQ ID NOs: 15-17 and heavy chain of SEQ ID NOs: 11-13), TIG2 (light chain of SEQ ID NOs: 23-25 ​​and heavy chain of SEQ ID NOs: 19-21) or TIG3 (light chain of SEQ ID NOs: 31-33 and heavy chain of SEQ ID NOs: 27-29). In some embodiments of any of the embodiments disclosed herein, the antibody is a chimeric, humanized or veneered antibody. In some embodiments, the antibody is a human antibody. In some embodiments of any of the embodiments disclosed herein, the antibody has a 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, a 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 having at least 90% sequence identity to SEQ ID NO: 35, and at least 90% sequence identity with SEQ ID NO:37 In some embodiments, the mature heavy chain variable region has at least 95 or 99% sequence identity to SEQ ID NO:35 and the mature light chain variable region has at least 95 or 99% sequence identity to SEQ ID NO:37. In some embodiments, the mature heavy chain variable region has at least 95 or 99% sequence identity. and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 35. In some embodiments, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 37. In some embodiments, the mature heavy chain variable region has an amino acid sequence of SEQ ID NO: 38, In some embodiments, 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 some embodiments, the mature heavy chain variable region is linked to a light chain constant region comprising SEQ ID NO: 42, with the proviso that a C-terminal lysine may or may not be present. In some embodiments, the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 60, 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 to a C-terminus comprising SEQ ID NO: 60. In some embodiments of all of the embodiments disclosed herein, the antibody comprises a mature heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 43 and a mature light chain variable region having at least 90% sequence identity to SEQ ID NO: 45. In some embodiments, the mature light chain variable region has at least 90% sequence identity. The mature heavy chain variable region has at least 95 or 99% sequence identity to SEQ ID NO: 43, and the mature light chain variable region has at least 95 or 99% sequence identity to SEQ ID NO: 43. The variant region has at least 95 or 99% sequence identity to SEQ ID NO: 45. In some embodiments, the mature heavy chain variable region has the amino acid sequence of 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 has a C-terminal lysine In some embodiments, the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 48, with a C-terminal lysine optionally present or absent, and the mature light chain variable region is linked to a light chain constant region comprising SEQ ID NO: 49. In some embodiments, the mature heavy chain variable region is linked to a light chain constant region comprising SEQ ID NO: 49, with a C-terminal lysine optionally present or absent. 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 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: 65. In some embodiments, the mature heavy chain variable region is linked to a light chain constant region comprising SEQ ID NO: 65, with the proviso that a C-terminal lysine may or may not be present. In yet another aspect, provided herein is an antibody that binds to an epitope comprising residues 35 and 37 of SEQ ID NO:1 and / or residues 49 and 51 of SEQ ID NO:1, and a mature light chain variable region that is linked to a heavy chain constant region that comprises SEQ ID NO:63 and a mature light chain constant region that comprises SEQ ID NO:65. In some embodiments, the antibody is a monoclonal antibody. or 51 of SEQ ID NO:1. In some embodiments, the antibody binds to a peptide consisting of residues 35-51 of SEQ ID NO:1 and up to five adjacent amino acids on either side from SEQ ID NO:1. 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 consecutive residues of SEQ ID NO: 1. In some embodiments of any of the embodiments disclosed herein, the antibody has the following properties: (a) inhibits binding of TIGIT to CD155, optionally with an IC50 of 15-100 ng / ml; (b) inhibits innate T cell activation in the presence of antigen presenting cells expressing CD155, optionally with an IC50 of 15-100 ng / ml, as measured by IL-2 production. (c) antigen-specific T cell activation was measured by IL-12 production and increased IL-12 production by 1.5 to 3 fold in any of the subjects. (d) increasing natural killer cell activation as measured by the production of either IL-2, IL-6, TNFα or IFNγ, optionally by 1.5 to 3 fold; (e) increasing at least one (f) increasing T cell production of at least one pro-inflammatory cytokine, optionally by 1.5-3 fold; and (f) decreasing T cell production of at least one anti-inflammatory cytokine, optionally by 1.5-3 fold. The above.

[0016] In another aspect, provided herein is a pharmaceutical composition comprising any of the antibodies described herein and a pharma- ceutically acceptable carrier.

[0017] In a further aspect, provided herein is a method of treating cancer or effecting prevention of cancer, comprising administering to a subject having or at risk of cancer an effective regimen provided herein 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 disclosed embodiments, antibody-activated tumor-infiltrating T cells are administered to a subject. In embodiments, a vaccine that induces an antibody-enhanced immune response against cancer is administered to the subject. In some embodiments, the vaccine comprises an antigen or fragment thereof expressed on the surface of a cancer cell. In some embodiments of any of the embodiments disclosed herein, the subject is administered natural killer cells whose cytotoxic activity against the cancer is antibody-enhanced. In some embodiments of any of the embodiments disclosed herein, the subject is further administered a secondary antibody against an antigen expressed on the surface of a cancer cell, whereby the effector-mediated cytotoxicity of the secondary antibody against the cancer is antibody-enhanced. In some embodiments of any of the embodiments disclosed herein, the subject is further administered a secondary antibody against an antigen expressed on the surface of an immune cell. In some embodiments, the immune cell is a T In some embodiments of any of the embodiments disclosed herein, the antigen is CTLA-4, PD-1, or PD-L1. In some of the embodiments, the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell therapy, and surgery. In some embodiments of any of the embodiments, one or more immune checkpoint receptors are An inhibitor of the ligand or ligand is further administered to the subject, hi some embodiments, the inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab (lambrolizumab) and atezolizumab.

[0018] In a further aspect, provided herein is a method of treating a subject infected with a pathogen, comprising administering to the subject an effective regimen or a therapeutically effective amount of any of the antibodies disclosed herein. In some embodiments, the pathogen is a virus, a bacterium, a fungus, or a protozoan ... pneumonococci, meningococci, conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and lyme burgdorferi. In some embodiments of any of the embodiments disclosed herein, the subject is treated with a vaccine that induces an antibody-enhanced immune response against the pathogen. In some embodiments, the vaccine comprises a protein or fragment thereof of the pathogen. In some embodiments of any of the embodiments disclosed herein, a second antibody against the pathogen is further administered to the subject, where the antibody enhances the effector-mediated cytotoxicity of the second antibody against the pathogen. In some embodiments of any of the embodiments disclosed herein, one or more of an antiviral agent, an antiparasitic agent, an antibacterial agent, or an antifungal agent is further administered to the subject.

[0019] In another aspect, provided herein is a method of aiding in the treatment of cancer, comprising administering to a subject 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, antibody-activated tumor-infiltrating T cells are administered to a subject. In some embodiments, a vaccine that induces an antibody-enhanced immune response against the cancer is administered to the subject. In some embodiments, the vaccine comprises an antigen expressed on the surface of a cancer cell or a fragment thereof. In some embodiments of any of the embodiments disclosed herein, the subject is administered natural killer cells whose cytotoxic activity against the cancer is enhanced by the antibody. In some embodiments of any of the embodiments disclosed herein, the subject is administered a secondary antibody against an antigen expressed on the surface of a cancer cell, whereby the effector-mediated cytotoxicity of the secondary antibody against the cancer is enhanced by the antibody. In some embodiments of any of the embodiments disclosed herein, the subject is further administered a secondary antibody against an antigen expressed on the surface of an immune cell. In some embodiments, the immune cell is a T In some embodiments of any of the embodiments disclosed herein, the antigen is CTLA-4, PD-1, or PD-L1. In some of the embodiments, the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation, cell therapy, and surgery. In some embodiments of any of the embodiments, one or more immune checkpoint receptors are In some embodiments, one or more immunosuppressants of the antibody or ligand are further administered to the subject. The immune checkpoint receptor or ligand is 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.

[0020] Each of the aspects and embodiments described herein can be used together unless expressly or unambiguously excluded from the context of that embodiment or aspect. [Brief description of the drawings]

[0021] [Figure 1]FIG. 1 is a schematic diagram of the expression vectors for recombinant TIGIT and CD155 proteins. [Diagram 2] FIG. 1 shows inhibition of TIGIT-CD155 interaction by anti-TIGIT antibodies. [Diagram 3] FIG. 1 shows the amino acid sequence of TIG1VH. [Figure 4] FIG. 1 shows the amino acid sequence of TIG1VL. [Diagram 5] FIG. 1 shows the amino acid sequence of TIG2VH. [Figure 6] FIG. 1 shows the amino acid sequence of TIG2VL. [Figure 7] FIG. 1 shows the amino acid sequence of TIG3VH. [Figure 8] FIG. 1 shows the amino acid sequence of TIG3VL. [Figure 9A] FIG. 1 shows the nucleotide sequence of the HuTIG1VH gene and the encoded amino acid sequence. [Figure 9B] FIG. 1 shows the nucleotide sequence of the HuTIG1VL gene and the encoded amino acid sequence. [Figure 10] FIG. 1 shows the schematic structure of the expression vector pHuTIG1.AA. [Figure 11] Figure 1 shows the results of ELISA analysis of binding of HuTIG1-IgG1.AA to human TIGIT. [Figure 12] FACS analysis of binding of HuTIG1-IgG1.AA and HuTIG3-IgG1.AA to human TIGIT. [Figure 13] Graph showing blocking of the interaction between human TIGIT and human CD155 by HuTIG1-IgG1.AA and HuTIG3-IgG1.AA. [Figure 14A] FIG. 1 shows the nucleotide sequence of the HuTIG3VH gene and the encoded amino acid sequence. [Figure 14B] FIG. 1 shows the nucleotide sequence and encoded amino acid sequence of the HuTIG3VL gene. [Figure 15] Figure 1 shows ELISA analysis of binding of HuTIG3-IgG1.AA to human TIGIT. [Figure 16] FIG. 1 shows increased IL-2 production by TIG1 upon antigen-specific recall stimulation. [Figure 17] FIG. 1 shows increased CD4+ and CD8+ T cell proliferation by TIG1 upon antigen-specific recall stimulation. [Figure 18] FIG. 1 shows the expression of CD155 on K562 cells (upper panel) and the expression of TIGIT on NK cells (lower panel). [Figure 19] FIG. 1 shows the enhancement of NK cell-mediated cytotoxicity by TIG1 against K562 target cells. [Figure 20] FIG. 1 shows that HuTIG1-IgG1.AA enhances cytokine effector responses. [Figure 21] FIG. 1 shows expression of human TIGIT on the cell surface by the Jurkat-TIGIT cell line. [Figure 22] FIG. 1 shows that HuTIG1-IgG1.AA and HuTIG3-IgG1.AA do not elicit CDC activity. [Diagram 23] FIG. 1 shows that HuTIG1-IgG1.AA and HuTIG3-IgG1.AA increased intrinsic T cell activation in an in vitro T cell antagonist activity assay for anti-human TIGIT antibodies. [Figure 24] FIG. 1 shows that monocyte-derived dendritic cells (moDCs) differentiated by GM-CSF / IL-4 expressed CD112, CD155, and PD-L1. [Diagram 25] Figure 1 shows enhanced secretion of IFNγ upon addition of HuTIG1-IgG1.AA, anti-PD-L1 antibody, and HuTIG1-IgG1.AA in combination with anti-PD-L1 antibody. [Figure 26] Figure 26A shows the expression of TIGIT and CD96 on human lymphoid and myeloid cells, and Figure 26B shows representative histograms of anti-TIGIT staining at various expression levels. [Figure 27] FIG. 13. HuTIG1-IgG1.AA epitope mapping by flow cytometry. [Figure 28] FIG. 1 shows a ribbon diagram of the extracellular IgV domain of human TIGIT with numbered epitope residue side chains displayed in stick representation. [Figure 29A-B] FIG. 1 shows the results of CD4+ T cell subset analysis for TIGIT expression. [Fig. 30A-B] FIG. 1 shows the results of CD8+ T cell subset analysis for TIGIT expression. [Diagram 31] FIG. 1 shows enhanced NK cell-mediated cytotoxicity by HuTIG1-IgG1.AA and HuTIG3-IgG1.AA against K562 target cells. [Diagram 32] Figure 32A shows the expression of TIGIT on tumor-infiltrating lymphocytes from dissociated tumor samples, and Figure 32B shows representative histograms of anti-TIGIT staining at various expression levels. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention specifically binds to the extracellular domain of TIGIT, a member of the immunoglobulin superfamily that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail. Specifically provided is a monoclonal antibody that can inhibit the binding of TIGIT to CD155, thereby activating T cells and / or NK cells. The monoclonal antibody can be used to treat cancer and infectious diseases, among other applications.

[0023] I. Definition Monoclonal antibodies or other biological entities, such as fragments of TIGIT, are typically isolated. This means that the antibody or other biological entity is A monoclonal antibody is typically at least 50% w / w pure from interfering proteins and other contaminants that may result from its production or purification, but may also be purified to facilitate its use. The present invention does not exclude the possibility that the monoclonal antibody may be mixed with an excess of a pharma- ceutically acceptable carrier or other vehicle intended to prevent the antibody from being contaminated. The monoclonal antibody may be at least 60, 70, 80, 90, 95 or 99% w / w pure with respect to interfering proteins and contaminants from production or purification. In some cases, the isolated monoclonal antibody or other biological entity agent is the predominant macromolecular species remaining following purification.

[0024] Specific binding of a monoclonal antibody to its target antigen is at least 10, as determined, for example, by the assay of Example 15. 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 The affinity (association constant or Ka) of Specific binding means binding that is greater than or equal to non-specific binding to at least one unrelated target. Specific binding is detectably higher and distinguishable on a larger scale. Specific binding is the result of the formation of a specific spatial fit between specific functional groups (e.g., lock and key type), whereas non-specific binding is usually the result of van der Waals forces. However, specific binding does not necessarily imply that a monoclonal antibody binds to one and only one target.

[0025] The basic antibody structural unit is a tetramer of subunits. Each tetramer consists of one "light" subunit. The polypeptides contain two identical pairs of polypeptide chains, one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region refers to a light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region that is primarily responsible for effector function.

[0026] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. The variable and constant regions within light and heavy chains consist of the following amino acid numbers: The heavy chains are joined by a "J" region of about 12 or more amino acids, and the heavy chains also contain a "D" region of about 10 or more amino acids. (See generally, Fundamental Immunology (Paul, W., 2nd ed., Raven Press, NY, 1999). 89), Chapter 7) (incorporated by reference in its entirety for all purposes).

[0027] The mature variable regions of each light / heavy chain pair form the antigen-binding site. Each antibody has two binding sites. Except in the case of bifunctional or bispecific antibodies, the two binding sites are identical. The chains all exhibit the same general structure of relatively conserved framework regions (FR) linked by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, thereby enabling binding to a specific epitope. Both light and heavy chains contain, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is based on the sequence of the sequences in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, , Bethesda, Md. (1987 and 1991), or Chothia and Lesk, J. Mol. Biol. 196:901-9 17 (1987), Chothia et al., Nature 342:878-883 (1989). Kabat defines the sequence of the heavy chains as follows: Alternatively, a commonly used numbering convention (Kabat numbering) is provided in which corresponding residues between different light chains are assigned the same numbers.

[0028] The term "antibody" includes intact antibodies and binding fragments thereof. Thus, any reference to an antibody should be understood to refer to the antibody in its intact form or to a binding fragment, unless the context requires otherwise. Typically, a fragment will contain separate heavy and light chains, Fab, Fab', F(ab') and / or Fab'-binding fragments, which compete with the intact antibody from which it is derived for specific binding to a target. 2 , F(ab) c , Dab, Nano The fragments include, but are not limited to, scFv, diabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, bis-scFv, triabodies, and tetrabodies. Fragments can be produced by recombinant DNA technology. They may be produced by the synthesis of recombinant immunoglobulins or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. (e.g. Songsivilaiand Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Ko See Stelny et al., J. Immunol., 148:1547-53 (1992).

[0029] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes may be formed from consecutive amino acids or by the tertiary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are usually retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are usually lost on treatment with denaturing solvents. Epitopes generally contain at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0030] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that reveals the ability of one antibody to compete with the binding of another antibody to a target antigen. X-ray crystallography of the antibody binding to its antigen can be used to define the epitope of the antibody and determine the site of attachment. Alternatively, two antibodies can be used to identify residues that interact with one another to reduce or eliminate binding of the other. Two antibodies have the same epitope if all amino acid mutations in the antigen that bind to the other reduce or eliminate binding of the other. Two antibodies have some amino acid mutations that reduce or eliminate binding of one antibody. If one of the two epitopes reduces or eliminates binding of the other, then they have overlapping epitopes.

[0031] Competition between antibodies is determined by an assay in which an antibody inhibits specific binding of a reference antibody to a common antigen under test conditions (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). The test antibody is administered in excess (e.g., at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 18-fold, 19-fold, 25-fold, 26-fold, 27-fold, x, 9x, 10x, 15x, 20x, 25x, 30x, 35x, 40x, 45x, 50x, 60x, 70x, 80x , 90-fold, 100-fold or more (including values ​​in between) inhibits binding of the reference antibody by at least 50%, but preferably 75%, 90% or 99%. In other embodiments, a test antibody competes with a reference antibody if the excess test antibody inhibits binding of the reference antibody by 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 a competitive binding assay. Antibodies identified by (i) (competing antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to adjacent epitopes that are close enough to the epitope bound by the reference antibody that there is steric hindrance. Preferably, competition is assessed as in Example 14.

[0032] The term "subject" includes human and other mammalian subjects. In some cases, the methods of the invention find use in laboratory animals, veterinary applications, and development of animal models of disease, such as, but not limited to, rodents, including mice, rats, hamsters, and primates, such as monkeys. In some embodiments, the subject is receiving, or is a candidate for receiving, either a prophylactic or therapeutic treatment.

[0033] As used herein, "corresponds to" or "corresponding to" or "corresponding to" an amino acid residue of a reference amino acid sequence. The amino acid residues of the target amino acid sequence are determined by the amino acid residues of the target sequence being the same as those of the reference sequence. It indicates a homologous or equivalent position to the recited residue in an amino acid sequence. It is within the skill of the art to recognize that the position of a particular amino acid residue in a polypeptide, such as a TIGIT polypeptide, corresponds to a homologous reference sequence. For example, it is possible to determine whether the position corresponds to the position of the Basic local alignment search tool (BLAST), ClustalW2, structure-based sequence alignment program (Structure based sequences alignment program) The sequence of the TIGHT polypeptide is aligned to the sequence of the reference sequence using a sequence analysis program such as Strategic Analysis of Polymorphisms (STRAP). In addition, the crystal structure coordinates of the reference sequence can be used to aid in determining the three-dimensional structure of the residues of the homologous polypeptide (Stengel et al., Proc. Natl. Acad. Sci. USA, 109:5399-5404, 2012). In another embodiment, equivalent residues can be identified by determining homology at the tertiary structure level. Such methods can be used to identify the amino acid residues of the TIGIT polypeptide variant according to the corresponding amino acid residue position numbering of the reference sequence. Residues can be numbered. For example, each of the TIGIT variants or epitopes of interest can be numbered. Use the amino acid sequence of SEQ ID NO:1 to determine the amino acid residue position numbering of the amino acid residues. In some embodiments, an amino acid sequence may have about 80%, 81%, 82%, 83%, 84%, , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0034] For the purposes of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids within the same class. Non-conservative substitutions involve the substitution of a member of one of these classes for another. Equivalent to exchange.

[0035] Percent sequence identity is based on maximally aligned antibody sequences according to the Kabat numbering convention. After alignment, the entire mature portion of the antibody region of interest (e.g., the heavy or light chain) is determined. When comparing a region of interest (variable region) to the same region of a reference antibody, sequence identity between the region of interest and the reference antibody is Percent identity is the number of positions occupied by the same amino acid in both the subject and reference antibody regions, divided by the total number of aligned positions in these two regions, without considering gaps, and multiplied by 100 to convert it to a percentage.

[0036] A composition or method "comprising" one or more recited elements is not specifically recited. For example, a composition that includes an antibody may include other components not included in the antibody. The antibody may contain only one or may contain antibodies in combination with other components.

[0037] Unless otherwise clear from the context, reference to a range includes all integers within the range, and all subranges defined by such integers.

[0038] II.Target molecule Unless otherwise indicated, TIGIT refers to human TIGIT. An exemplary human sequence has been assigned Swiss-Prot accession number Q495A1. The complete human GITIT sequence has 244 amino acids, of which amino acids 1-21 are the signal peptide and 22-244 constitute the mature protein (SEQ ID NO:1). Approximately residues 22-141 constitute the extracellular domain (SEQ ID NO:3). Approximately residues 142-162 constitute the transmembrane domain, and approximately residues 163-244 constitute the cytoplasmic domain.

[0039] Unless otherwise indicated, CD155 refers to the human form of the protein. An exemplary human sequence for human CD155 is designated Swiss-ProtP15151, which is a 417 amino acid protein. of which approximately residues 1-20 are a signal peptide, 21-343 are an extracellular domain (SEQ ID NO: 6), 344-367 are a transmembrane domain, and 368-417 are a cytoplasmic domain. be.

[0040] Unless otherwise clear from the context, a reference to one of the above proteins is By this term is meant at least the extracellular domain of a protein, and usually the entire protein except for a cleavable signal peptide.

[0041] III. Antibodies of the Invention A. Binding Specificity and Functional Properties The present invention relates to a monoclonal antibody that binds to an epitope in the extracellular domain of the TIGIT protein. Three such exemplary mouse antibodies, designated TIG1, TIG2 and TIG3, are provided. The sequences of the heavy and light chain mature variable regions of these antibodies are set forth in SEQ ID NOs: 10 and 14, 18 and 22, and 26 and 30, respectively, and TIG1, TIG2, and TIG3 specifically bind to the extracellular domain of human TIGIT.

[0042] Some antibodies of the invention bind to the same or overlapping epitopes as the antibodies designated TIG1, TIG2, or TIG3. Other antibodies with such binding specificities may be used to bind to TIGIT, or to a desired epitope. and screening the resulting antibodies for binding to the extracellular domain of TIGHT, optionally in competition with TIG1, TIG2, or TIG3. Antibodies against the mutagenized form of the TIGIT antigen can be produced by screening. The antibody can also be screened to identify antibodies that exhibit the same or similar binding profile as TIG1, TIG2 or TIG3 for a collection of mutational changes. This can be a systematic exchange substitution, that is, substitution of one residue at a time or more widely spaced with alanine (or serine if alanine is already present) throughout the entire extracellular domain, or throughout the section where the epitope is known to reside.

[0043] Example 16: Residues 35, 37, 49 and 51 of SEQ ID NO:1 form an epitope for the TIG1 antibody The alanine substitution for any one of these residues is The substitution essentially abolishes antibody binding. Thus, the present invention relates to a method for the preparation of a medicament comprising the steps of: and 37 and / or an epitope of human TIGIT comprising residues 49 and 51 of SEQ ID NO:1; Preferably, other antibodies that bind to an epitope that includes all of these residues. The epitope may be linear (e.g., 3-20, 3-17, or 5-10 consecutive residues) or may be conformational. Some such antibodies bind to residues 35-51 of SEQ ID NO:1. and a peptide consisting of five or fewer adjacent amino acids from SEQ ID NO:1 on either side. Some such antibodies bind to a peptide consisting of residues 35 to 51 of SEQ ID NO:1. Some such antibodies can be generated by immunization with such peptides. Example 19 also reveals that residue 90 of SEQ ID NO:1 is important for binding of humanized (Hu)TIG1 antibodies to TIGIT, in addition to residues 35, 37, 49 and 51 of SEQ ID NO:1. Other embodiments In one embodiment, the antibody (e.g., a HuTIG1 antibody) binds to an epitope that includes one or more residues corresponding to amino acid positions 35, 37, 49, 51, and / or 90 of SEQ ID NO:1. In a further embodiment, the antibody (e.g., a HuTIG1 antibody) does not bind to one or more residues corresponding to amino acid positions 34, 39, 44, 47, 52, 55, 86, 88, 92, and / or 96 of SEQ ID NO:1.

[0044] Antibodies having the binding specificity of a selected murine antibody (e.g., TIG1, TIG2 or TIG3) can also be produced using a variant of the phage display method. See Winter, WO 92 / 20791. This method is particularly suitable for the production of human antibodies. In this method, a selected Either the heavy or light chain variable region of the mouse antibody produced can be used as the starting material. For example, if the light chain variable region is selected as the starting material, the members can be selected from the same light chain variable region (i.e. A phage library was constructed that displays the heavy chain variable regions (i.e., mouse starting material) and different heavy chain variable regions. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. 8 M -1 , preferably at least 10 9 M -1 A phage showing the heavy chain fragment is then selected. The variable regions serve as starting material for constructing further phage libraries, in which each phage displays the same heavy chain variable region (i.e., the region identified from the first phage library) and a different light chain variable region. For example, the antibody can be obtained from a library of rearranged human variable light chain regions. Again, phages that show strong specific binding to TIGIT are selected. The resulting The resulting antibodies will usually have the same or similar epitopic specificity as the murine starting material.

[0045] Some antibodies have a mature heavy chain variable region, including CDRs H1, H2 and H3, and a mature light chain region, including CDRL1, L2 and L3, entirely or substantially from TIG1. A mature heavy chain variable region comprising CDRH1, H2 and H3, and CDRs L1, L2 and L3 substantially from TIG2. Some antibodies have a mature light chain region that includes CDRH1, L3, which is derived entirely or substantially from TIG3. and a mature heavy chain variable region comprising CDRL1, H2, and H3, and a mature light chain region comprising CDRL1, L2, and L3. The CDRs may be defined by any conventional definition, including the Kabat, Chothia, Kabat·Chothia hybrid, AbM, or Contact definitions, as shown in the table below:

[0046] TIFF2025032326000002.tif43142

[0047] Other antibodies can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of exemplary antibodies such as TIG1, TIG2, or TIG3. Also included in the present invention are 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% identical to TIG1, TIG2 or TIG3 in terms of their functional properties and / or that differ from the respective antibodies by a small number of amino acid substitutions (e.g., conservative substitutions), deletions or insertions of minor functional importance. Amino acids within the variable region frameworks that are likely to be important for binding can be identified as described in the section on humanization below. Approximately 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 CDR of TIG1, TIG2 or TIG3 and preferably at least one of the nucleotides defined by Kabat that is 1 to 5% identical to any of the nucleotides defined by Kabat. Also included are monoclonal antibodies that have all six CDRs.

[0048] The antibody preferably has (i) the property of inhibiting the binding of human TIGIT to human CD155, (ii) other (iii) the property of increasing antigen-specific T cell responses; (iv) the property of activating natural killer cells; (v) the property of stimulating innate T cell activation; and (vi) the property of stimulating the production of one or more immunostimulatory cytokines and / or one or more immunosuppressive cytokines by T cells and other cells of the immune system. Examples of methods for measuring these characteristics include A representative assay is provided in the Examples.

[0049] Preferred antibodies completely or partially inhibit the binding of TIGIT to CD155. Some antibodies have an IC of about 25-300ng / ml, 25-75ng / ml, 25-50ng / ml, 40-75ng / ml, 50-75ng / ml, 50-90ng / ml, 50-100ng / ml, 75-100ng / ml, 50-150, 75-175ng / ml, 100-200ng / ml, 125-225ng / ml, 100-250ng / ml, 150-300ng / ml, 175-250ng / ml, 200-300ng / ml, 25-275ng / ml, 250-300ng / ml, 49+ / -10%ng / ml, 65+ / -10%ng / ml or 76+ / -10%ng / ml, when measured as in the Examples. 50 In other embodiments, the antibody can inhibit such interactions at a concentration of at least about 25 ng / ml, 50 ng / ml, 75 ng / ml, 100 ng / ml, 125 ng / ml, 150 ng / ml, 175 ng / ml, 200 ng / ml, 225 ng / ml, 250 ng / ml, 275 ng / ml, or 300 ng / ml or more. Any of the above ICs 50 (including concentrations between these values) can completely or partially inhibit the binding of TIGIT to CD155. Some antibodies can increase antigen-specific T cell responses by 1.5 to 3 fold, for example, about 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 fold or more, when measured as in the Examples. Some antibodies can inhibit the production of IL-2, IL-6, and TNFα by NK cells when measured as in the Examples. and IFNγ, and IFNγ, and , 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 Some antibodies can increase intrinsic T cell activity, as measured in the Examples. The antibody can increase the expression of the antibody by 1.5 to 3 fold, for example, by 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 by 3 fold or more. Some antibodies can inhibit cancer or infectious disease as shown in animal models or clinical trials. Animal models of cancer are widely available in which human cancer cells are injected into immunodeficient laboratory animals, such as mice or rats.

[0050] Humanization or chimerization of an antibody increases its in vivo half-life relative to the starting murine antibody. The resulting half-life may be, for example, 10 to 50 days in humans. Pharmacokinetic studies such as those described by Eur Jof Immunol 24:542 (1994) It can be measured.

[0051] B. Non-human antibodies Other non-human monoclonal antibodies against TIGIT, e.g., mouse, guinea pig, primate The production of rabbit, chicken or rat antibodies can be achieved, for example, by immunizing an animal with TIGIT or a fragment thereof, or with cells bearing TIGIT. See Harlow and Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such immunogens can be prepared from natural sources, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused or complexed to a carrier protein. Optionally, the immunogen can be administered with an adjuvant. Several types of adjuvants can be used, as described below. Complete Freund's adjuvant, followed by incomplete adjuvant, is preferred for immunization of laboratory animals. Rabbits or guinea pigs are generally used for the production of polyclonal antibodies. Mice are generally used for the production of monoclonal antibodies. Antibodies are generated that specifically bind to TIGIT. Optionally, the antibodies are screened for specific binding to a specific region of TIGIT. Such screening will involve the use of antibodies and TIGIT deletions. This can be accomplished by determining binding to a collection of mutants and determining which deletion mutants bind to the antibody. Binding can be assessed, for example, by Western blot, FACS or ELISA.

[0052] C. Humanized Antibodies Reduction or elimination of the HAMA (human anti-mouse antibody (which also applies to human anti-rat antibody or human anti-rabbit antibody or human anti-hamster antibody, etc.)) response is a significant aspect of the clinical development of suitable therapeutic agents. See, e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transp. lantation (1986), 41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. l.ResponseMod.(1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immuno l. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res. 1990), 50:1495. The invention described herein provides antibodies that have been humanized to reduce or eliminate the HAMA response. Variants of these antibodies can further be obtained using routine methods known in the art, some of which are further described below.

[0053] Humanized antibodies are antibodies in which the CDRs from a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences. and genetically engineered antibodies that are rafted (see, e.g., Queen, U.S. Pat. No. 5,530,101 and the same references). (See, e.g., U.S. Pat. No. 5,585,089, Winter, U.S. Pat. No. 5,225,539, Carter, U.S. Pat. No. 6,407,213, Adair, U.S. Pat. No. 5,859,205 and U.S. Pat. No. 6,881,557, Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequence may be, for example, a mature human antibody sequence, a complex of such sequences, , consensus sequences of human antibody sequences, or germline region sequences. Thus, a humanized antibody has some or all of the CDRs, which are completely or substantially from the donor antibody, and A humanized heavy chain is an antibody having at least one, two, and usually all three CDRs that are completely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, that are substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs that are completely or substantially from a donor antibody light chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, that are substantially from human heavy chain variable region framework and constant region sequences. all CDRs and, if present, substantially human light chain variable region framework and constant regions The humanized antibodies, other than nanobodies and dAbs, comprise a humanized heavy chain and a humanized light chain. As elsewhere in this application, a CDR in a subject antibody is considered substantially identical if at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. Generally, the CDRs are from the corresponding CDRs in the reference antibody, but the CDRH2s in the subject antibody as defined by Kabat have at least about 65%, 66%, 67% or more of the corresponding residues (as defined by Kabat). ,68%,69%,70%,71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,8 A substantially identical CDR is 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical between the respective CDRs of the reference antibody. The antibody chain variable region framework sequences or antibody chain variable region CDRs are derived from the corresponding CDRs. A constant region is substantially a human variable region framework sequence or a human constant region if at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the corresponding residues as defined by Kabat are identical, respectively. It is from the area.

[0054] A humanized antibody often incorporates all six CDRs (preferably as defined by Kabat) from a non-human (e.g., murine) antibody, although it is possible to incorporate less than all of the CDRs (e.g., For example, at least three, four, or five CDRs can be used to generate a humanized antibody (see, e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biol. 1999:1331, 2003). logy, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al. , Journal of Immunology, 164:1432-1441, 2000).

[0055] For some antibodies, all that is required to retain binding in a humanized antibody is the alignment of the CDRs. Only a portion of the CDR residues are required for binding, i.e., a subset of the CDR residues, called SDRs. The CDR residues that do not contact the antigen and are not within the SDRs have been identified by molecular modeling and / or by experiment, or As described in Gonzales et al., Mol. Immunol. 41:863, 2004, the Chothia hypervariable loop ( From the region of the Kabat CDR located outside the (e.g., residues H60-H65 in CDR H2 are not required. At positions where one or more donor CDR residues are absent or where an entire donor CDR is deleted, in such humanized antibodies, the amino acid occupying that position will be the amino acid occupying the corresponding position (according to Kabat numbering) in the acceptor antibody sequence. The number of such acceptor substitutions that an amino acid contains within its CDRs depends on competing considerations. Such substitutions may be advantageous in terms of reducing the number of murine amino acids in the humanized antibody and, therefore, reducing potential immunogenicity. However, substitutions may also lead to changes in affinity, so it is preferable to avoid significant decreases in affinity. The location of substitutions within the CDRs and the amino acids to be substituted are also selected empirically. You can choose.

[0056] Although the acceptor may be identical in sequence to the selected human framework sequence, whether from a human immunoglobulin or from a human consensus framework, the present invention contemplates that the acceptor sequence may contain pre-existing amino acid substitutions compared to the human immunoglobulin sequence or the human consensus framework sequence. These pre-existing substitutions are preferably minimal, usually no more than 4, 3, 2 or 1 amino acid difference compared to the human immunoglobulin sequence or the human consensus framework sequence.

[0057] The human acceptor antibody sequence can optionally be selected from among the many human antibody sequences known to provide a high degree of sequence identity (e.g., 65-85% identity) between the human acceptor sequence variable region framework and the corresponding variable region framework of the donor antibody chain.

[0058] Certain amino acids from the human variable region framework residues can be selected for substitution based on their potential effect on CDR conformation and / or binding to antigen, studied by modeling, by examining the properties of amino acids at particular positions, or by experimental observation of the effects of substitution or mutagenesis of particular amino acids.

[0059] For example, when an amino acid differs between a non-human variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be replaced with the equivalent framework amino acid from the non-human antibody, such that the amino acid from the non-human antibody is: (1) Direct non-covalent binding to antigens; (2) adjacent to the CDR regions; (3) or interacts with the CDR region (e.g., is within about 6 angstroms of the CDR region). Where this is reasonably expected, substitutions may be made.

[0060] Other candidates for substitution are acceptor human framework amino acids that are not normally found in that position in human immunoglobulins. These amino acids can be replaced with amino acids from the equivalent position of the non-human donor antibody or from the equivalent position of a more typical human immunoglobulin. Other candidates for substitution are acceptor human framework amino acids that are not normally found in that position in human immunoglobulins.

[0061] A preferred humanized antibody has a mature heavy chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 35, and a mature light chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO: 37. Preferably, there are any mutations with respect to variable region framework residues other than those identified as likely to be important for binding (see paragraph

[0043] ). Any mutations are preferably conservative amino acid substitutions. A preferred antibody comprises a mature heavy chain variable region having the sequence of SEQ ID NO: 35 and a mature light chain variable region having the sequence of SEQ ID NO: 37. For expression of a full-length antibody, the mature heavy chain variable region is The mature light chain variable region is preferably linked to a heavy chain constant region consisting of or comprising SEQ ID NO: 41, with the proviso that a nucleotide sequence 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 comprising SEQ ID NO: 41.

[0062] Another preferred humanized antibody has a mature heavy chain variable region that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO:43, and a humanized antibody that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% identical to SEQ ID NO:45. , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or have mature light chain variable regions that are less than 100% identical. Preferably, there are any mutations with respect to variable region framework residues other than those identified as likely to be important for binding ( (See paragraph

[0043] and the Examples.) Any mutations are conservative amino acid substitutions. 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 expression of a full length antibody, the mature heavy chain variable region may be SEQ ID NO: 48, with the proviso that the 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 comprising SEQ ID NO:49.

[0063] D. Chimeric and Veneer Antibodies The present invention further provides non-human antibodies, in particular chimeric and veneered forms of the TIG1, TIG2 and TIG3 antibodies of the examples.

[0064] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., murine) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the non-human antibody and are about two-thirds human in sequence.

[0065] A veneered antibody is a peptide that is synthesized by combining some, and usually all, of the CDRs of a non-human antibody with a non-human variable region framework. A type of humanized antibody is one in which some of the variable region framework residues that may contribute to B or T cell epitopes are retained, but other variable region framework residues that may contribute to B or T cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), are replaced with residues from the corresponding positions in a human antibody sequence. The result is The CDRs are entirely or substantially from a non-human antibody, and the variable regions of the non-human antibody are replaced Veneer forms of TIG1, TIG2 or TIG3 antibodies are included in the present invention.

[0066] E. Human Antibodies Human antibodies against TIGIT can be obtained by various techniques, which are described below. Antibodies are selected by competitive binding experiments, by the phage display method of Winter, supra, or to have the same epitope specificity as a particular mouse antibody, e.g., one of the mouse monoclonal antibodies described in the Examples. Human antibodies can also be screened for a particular epitope specificity by using only a fragment of TIGIT as the target antigen and / or by screening antibodies against a collection of deletion mutants of TIGIT.

[0067] Methods for producing human antibodies include the trioma method of Oestberg et al., Hybridoma 2:361-367 (1983), Oestberg, U.S. Pat. No. 4,634,664, and Engleman et al., U.S. Pat. No. 4,634,666; the use of transgenic mice containing human immunoglobulin genes (e.g., Lonberg et al., WO 93 / 12227 (1993); U.S. Pat. No. 5,877,397; U.S. Pat. No. 5,874,299; U.S. Pat. No. 5,814,318; U.S. Pat. No. 5,789,650; U.S. Pat. No. 5,770,429; U.S. Pat. No. 5,661,016 ... Patent No. 5,633,425, U.S. Patent No. 5,625,126, U.S. Patent No. 5,569,825, U.S. Patent No. 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherl apati, WO 91 / 10741 (1991); and phage display methods (e.g., Dow er et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047, U.S. Pat. No. 5,877,218, U.S. Pat. (See U.S. Patent Nos. 5,871,907, 5,858,657, 5,837,242, 5,733,743 and 5,565,332).

[0068] F. Selection of Constant Regions The heavy and light chain variable regions of a chimeric, humanized (including veneered) or human antibody 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. Examples include For example, human isotopes IgG1 and IgG3 have complement-mediated cytotoxicity, whereas human isotopes IgG2 and IgG4 do not. The light chain constant region can be lambda or kappa. For immunotherapy against cancers or pathogens that do not express TIGIT, human IgG2 or IgG4, or endogenous IgG1 or endogenous IgG4 are used. Attenuated forms of human IgG1 with reduced effector function are often preferred. Human IgG4 is often preferred to contain the S288P (Eu numbering) modifying mutation on the heavy chain to prevent Fab arm exchange. However, in cancer cells expressing TIGIT (e.g., T-cell or NK-cell tumors), For tumor removal or for immunosuppression, human IgG1 or IgG3 are often preferred.

[0069] Human constant regions exhibit allotypic and isoallotypic variation among different individuals, i.e., constant regions may differ at one or more polymorphic positions from one individual to another. Isoallotypes are in that sera that recognize a soallotype bind to the nonpolymorphic regions of one or more other isotypes. Reference to a human constant region includes constant regions with any naturally occurring allotypes or with any permutation of residues occupying the polymorphic positions of the naturally occurring allotypes.

[0070] One or several amino acids at the amino or carboxy termini of the light and / or heavy chains, e.g., the C-terminal lysine of the heavy chain, are missing or derivatized in some or all of the molecules. Substitutions can be made within the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA, 1999, 2001). 03:4005, 2006), or can extend the half-life in humans (e.g., (See, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include: and / or Gln at position 250 and / or Leu at position 428 (Eu numbering) to increase the half-life of the antibody. can be.

[0071] Some antibodies of the invention are modified by introducing constant region mutations such that they have reduced effector function, e.g., CDC and ADCC or ADCP, compared to the same antibody without the mutations. Preferably, each or all of these effector functions are reduced by at least 50%, 75%, 90% or 95% compared to the antibody without the mutation. This example illustrates how to measure CDC. Other assays are described in Shields et al., 2001 J. Biol. Chem., vol. 276, pp. 6591-6604; Chappel et al. , 1993 J. Biol. Chem., vol. 268, pp. 25124-25131; Lazar et al., 2006 PNAS, 103; 4005-401 It is described by 0.

[0072] Substitutions at any or all of positions 234, 235, 236 and / or 237 may be used to inhibit Fcγ receptors, particularly FcγRI. Reduces affinity for receptors (see, e.g., U.S. Patent No. 6,624,821). Lanin is the preferred residue for substitution, and L234A / L235A reduces effector function Other combinations of mutations with reduced effector function include L234A / L235A / G237A, E233P / L234V / L235A / G236, A327G / A330S / P331S, K322A, L234A, and Optionally, the amino acids 234, 236 and / or 237 in human IgG2 may be selected from the group consisting of 235A, L234F / L235E / P331S and 237 in human IgG2. The 237 position is substituted with alanine and the 235 position is substituted with glutamine (see, for example, U.S. Pat. No. 5,624,821). Amino acid substitutions at positions 233-236 reduce complement binding (see Tao et al., J. Exp. Med. 178:661 (1993); and Canfield and Morrison, J. Exp. Med. 173:1483 (1991)). Substitution of IgG2 residues and IgG4 residues at positions 327, 330, and 331 with human IgG1 greatly reduces ADCC and CDC (see, e.g., Armour KL. et al., 1999 Eur J Immunol. 29(8):2613-24; and Shi Elds RL. et al., 2001. J. Biol. Chem. 276(9):6591-604). The 297G (Eu numbering) mutation reduces glycosylation and thereby reduces effector function.

[0073] G. Expression of Recombinant Antibodies Chimeric, humanized (including veneered) and human antibodies are generally produced by recombinant expression. Recombinant polynucleotide constructs generally include expression control sequences operably linked to the coding regions 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. After the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the recovery and purification of the recombinant antibody.

[0074] Mammalian cells are the preferred hosts for the expression of immunoglobulin-encoding nucleotide segments or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of preferred vectors capable of secreting intact heterologous proteins are available. Suitable host cell lines are known in the art and include CHO cells. Cells include various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody producing melanomas including Sp2 / 0 and NS0. Preferably, the cells are non-human. Expression vectors for these cells contain expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, For example, ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sites. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. Co et al., J. See Immunol. 148:1149 (1992).

[0075] Once expressed, antibodies can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0076] IV. Therapeutic Applications The antibodies can be used to enhance immune responses in the treatment of cancer and infectious diseases. Disorders that can be treated with the antibodies of the present invention include, but are not limited to, cancers, including hematological tumors and solid tumors. Such cancers may or may not express TIGIT or CD155. Antibodies against TIGIT are effective against cancers that do not express TIGIT, because inhibition of the interaction between CD155 and TIGIT stimulates immune responses against such cancers. Examples of hematological tumors include acute myeloid leukemia, adult T-cell leukemia, and leukemia. , T-cell large granular lymphocyte leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic Examples of solid tumors include leukemias, lymphomas, and myelomas, including myeloid leukemia, acute monocytic leukemia, Hodgkin's and non-Hodgkin's lymphoma, and multiple myeloma. Examples of solid tumors include, but are not limited to, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, pancreatic cancer, gastric cancer, esophageal cancer, hepatocellular carcinoma (liver cancer), renal cell carcinoma (kidney cancer), head and neck tumors, mesothelioma, melanoma, sarcoma, and brain tumors (e.g., glioblastoma, leukemia, and leukemia). Examples of tumors include gliomas.

[0077] The methods of the invention can be carried out in an adjuvant setting. An "adjuvant setting" is when a subject has a history of a proliferative disease, particularly cancer, and has undergone surgery, radiation therapy, and / or chemotherapy. "Adjuvant setting" refers to a clinical setting in which subjects have generally (but not necessarily) responded to therapy, including but not limited to, chemotherapy, chemotherapy alone, chemotherapy alone or in combination with chemotherapy alone. However, due to a history of proliferative disease, these subjects are considered to be at risk for developing the disease. Treatment or administration in an "adjuvant setting" refers to a subsequent treatment modality. In some embodiments, provided herein is a method of treating cancer or effecting prevention of cancer, comprising administering to a subject having or at risk of cancer a therapeutically effective amount of any of the antibodies disclosed herein in an adjuvant setting.

[0078] The methods provided herein can also be performed in a "non-adjuvant setting," i.e., the methods can be performed prior to primary / definitive treatment. In some aspects, the subject has been previously treated. In other aspects, the subject has not been previously treated. In some aspects, the treatment is a first-line therapy. In some embodiments, provided herein is a method of treating cancer or effecting prevention of cancer, comprising administering to a subject having or at risk of cancer a therapeutically effective amount of any of the antibodies disclosed herein in a non-adjuvant setting.

[0079] Other disorders that can be treated with the antibodies of the invention include viruses, bacteria, fungi, protozoa and other pathogens (e.g., hepatitis (types A, B or C), herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, Kozak virus, coronavirus (cornovirus), respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, and the like). Infectious diseases include: rabies, dengue virus, papilloma virus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, HIV, SIV and arboviral encephalitis viruses, chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis and lyme disease. can be done.

[0080] A. Antibody Administration The antibodies described herein can be administered in an effective regimen, meaning a dose, route of administration, and frequency of administration that delays the onset of the disorder, reduces the severity of the disorder, prevents further deterioration of the disorder, and / or improves at least one sign or symptom of the disorder. A regimen in which the subject is already suffering from the disorder can be referred to as a therapeutically effective regimen. A regimen in which the subject is at high risk of the disorder compared to the general population, but has not yet experienced symptoms, can be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy can be observed in an individual subject, compared to historical controls, or compared to past experience in the same subject. In other cases, therapeutic or prophylactic efficacy can be demonstrated in a population of treated subjects compared to a control population of untreated subjects in preclinical or clinical trials.

[0081] In some embodiments, any of the methods described herein comprises administration to a subject in need thereof of a therapeutically effective amount of one or more of the antibodies described herein. A "therapeutically effective dosage" or "prophylactic dose" of a method (e.g., any of the anti-TIGIT antibodies described herein) may be used to treat a patient with a disease that is characterized by a high level of urinary retention and / or urinary incontinence. An "effective dose" is an amount sufficient to effect a beneficial or desired result. For therapeutic uses, beneficial or desired results include reducing one or more symptoms resulting from cancer, reducing one or more symptoms resulting from cancer, Achieving clinical outcomes, such as improving the quality of life of an affected subject, reducing the dose of other drug therapies required to treat the cancer, enhancing the effectiveness of other drug therapies, such as by targeting, slowing disease progression, and / or extending survival. An effective dosage can be administered in one or more administrations. For purposes of this invention, An effective dosage of a cancer therapy is an amount sufficient to directly or indirectly provide therapeutic or prophylactic treatment. As understood in a clinical context, a therapeutically effective dosage of an anti-cancer therapy may or may not be achieved in combination with another anti-cancer therapy.

[0082] Exemplary dosages for any of the antibodies described herein are about 0.1-20 mg / kg or 0.5-5 mg / kg body weight (e.g., about 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg). g / kg) or as a fixed dosage, 10 to 1500 mg (e.g., less than 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, or or 1500 mg or more (including any value between these numbers). It will depend on the subject's condition, response to previous treatment, if any, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic, among other factors.

[0083] Administration may be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intratumoral, topical, intranasal or intramuscular. Administration into the systemic circulation by intravenous or subcutaneous administration is preferred. Intravenous administration may be by infusion over a period of time such as, for example, 30 to 90 minutes.

[0084] Dosing frequency depends on, among other factors, the half-life of the antibody in the circulation, the condition of the subject, and the route of administration. Frequency may range from daily to weekly, depending on changes in the subject's condition or progression of the disorder being treated. The frequency may be once a week, once a month, once a year, or at irregular intervals. Exemplary frequencies for intravenous administration are between once a week and once a year over the course of continuous treatment, although more or less frequent dosing is possible. Exemplary dosing frequencies for subcutaneous administration are daily to once a month, although more or less frequent dosing is possible. is also possible.

[0085] The number of doses administered depends on whether the disorder is acute or chronic and on the response of the disorder to treatment. For acute disorders, or acute exacerbations of chronic disorders, between 1 and 10 doses are administered. For acute disorders, or acute exacerbations of chronic disorders, a single bolus dose, optionally in divided form, may be sufficient. Treatment may be repeated for recurrence of acute disorders or acute exacerbations. For chronic disorders, the antibody may also 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 life of the subject.

[0086] Treatments containing anti-TIGIT antibodies improve median progression-free survival or overall survival in subjects with cancer. 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109 ... The disease can be alleviated by increasing the number of TIGIT antibodies by 4%, 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%, or either of these periods can be extended by 2 weeks, 1, 2 or 3 months, or preferably 4 or 6 months, or even 9 months or 1 year. Additionally or alternatively, the anti-TIGIT antibody The treatment includes determining whether the subject has a complete response rate, partial response rate, or objective response rate (complete + partial) compared to the control. At least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% compared to the control , 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%, 100%, 101%, 1 The TIGIT antibody may be increased by 1%, 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%. Control subjects receive the same treatment as the subjects receiving the anti-TIGIT antibody, except for the anti-TIGIT antibody. Thus, control subjects receive only a placebo. Alternatively, if a subject receiving an anti-TIGIT antibody also receives a placebo and any chemotherapeutic agent other than the anti-TIGIT antibody, the subject may receive a combination of a placebo and such chemotherapeutic agent.

[0087] The anti-TIGIT antibodies disclosed herein enhance NK cell-mediated cytotoxicity of CD155-expressing cells (such as, but not limited to, K562 cells) by at least about 10%, 11%, 12%, 13%, or 5% relative to the amount of NK cell-mediated cytotoxicity of CD155-expressing cells in the absence of one of the anti-TIGIT antibodies disclosed herein. ,14%,15%,16%,17%,18%,19%,20%,21%,22%,23%,24%,25%26% 27%,28%,29%, It can be increased by any of the following percentages: 30%, 31%, 32%, 33%, 34%, 35% or more.

[0088] Typically, in a clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial), a subject The increase in median progression-free survival and / or median response rate in subjects treated with an anti-TIGIT antibody compared to a control group is statistically significant, for example at the p=0.05 or 0.01 or even 0.001 level. Complete and partial response rates are assessed, for example, by the National Cancer Institute and / or the Food and Drug Administration. The outcome is determined by objective criteria commonly used in cancer clinical trials, such as those established or approved by the National Cancer Institute, including, for example, tumor volume, tumor number, metastases, survival time, and quality of life measures, among others.

[0089] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic and comply with GMP regulations. The pharmaceutical composition may be provided in a unit dosage form (i.e., a single dosage amount for administration). The pharmaceutical composition may be prepared using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The pharmaceutical composition can be formulated in a suitable aqueous solution. The formulation depends on the route of administration selected. For injection, the antibody 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 can contain formulatory agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the antibody can be formulated in a suitable aqueous solution. It may be in lyophilized form for constitution with a vehicle, e.g., sterile, pyrogen-free water, before use. The concentration of the antibody in the liquid formulation may vary, e.g., from about 10 mg / ml to about 150 mg / ml. The concentration in some formulations is about 25-50 mg / ml.

[0090] B. Combination Therapy The present invention relates to a method for treating or preventing the development of a medicament for the treatment of a disease comprising administering to a patient a therapeutically effective amount of one or more active therapeutic agents (e.g., chemotherapeutic agents) or other prophylactic or therapeutic modalities. The present invention contemplates the use of anti-TIGIT antibodies in combination with therapeutic agents (e.g., radiation). In such combination therapies, the various active agents often have different, complementary mechanisms of action. Such combination therapies may allow for a reduction in the dose of one or more of the agents, thereby reducing the This may be particularly advantageous in reducing or eliminating adverse effects associated with one or more of the agents. Such combination therapy may also have a synergistic therapeutic or prophylactic effect against the underlying disease, disorder or condition.

[0091] As used herein, "combination" is intended to include therapies that can be administered separately, e.g., formulated separately for separate administration (e.g., as provided in a kit), as well as therapies that can be administered together in a single formulation (i.e., a "co-formulation").

[0092] In certain embodiments, any of the anti-TIGIT antibodies disclosed herein are administered sequentially or In other embodiments, the antibodies are administered sequentially, e.g., where one agent is administered before one or more other agents. In other embodiments, the antibodies are administered simultaneously, e.g., where two or more agents are administered simultaneously or are administered at about the same time, and these two or more drugs may be present in two or more separate formulations or may be combined in a single formulation (i.e., a co-formulation). Whether the agents are administered sequentially or simultaneously, they are considered to be administered in a combination for purposes of the present invention.

[0093] The antibodies of the present invention may be combined with at least one other (active) agent in any suitable manner under the circumstances. In one embodiment, at least one active agent and a composition according to the present invention Treatment with at least one anti-TIGIT antibody is maintained for a period of time. In another embodiment, treatment with at least one active agent is tapered or discontinued (e.g., if the subject is stable), while treatment with an anti-TIGIT antibody of the present invention is maintained at a constant dosing regimen. In a further embodiment, treatment with at least one active agent is tapered or discontinued (e.g., if the subject is stable), and treatment with an anti-TIGIT antibody of the present invention is also tapered (e.g., if the subject is stable). , lower doses, less frequent dosing or shorter treatment regimens). Then, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable), and treatment with an anti-TIGIT antibody of the present invention is increased (e.g., at a higher dose or more frequently). In yet another embodiment, treatment with at least one active agent is maintained and treatment with an anti-TIGIT antibody of the present invention is reduced or discontinued (e.g., a lower dose, less frequent dosing, or a shorter treatment regimen). Treatment with at least one active agent, as well as treatment with an anti-TIGIT antibody of the present invention, is reduced or discontinued (eg, a lower dose, less frequent dosing, or a shorter treatment regimen).

[0094] Treatment with the antibodies of the invention can be combined with other treatments effective against the disorder being treated. When used to treat a proliferative condition, cancer, tumor, or precancerous disease, disorder, or condition, the antibodies of the invention can be combined with chemotherapy, radiation (e.g., localized radiation therapy or total body irradiation), or other treatments that may be effective against the disorder being treated. It can be combined with radiation therapy, stem cell treatment, surgery, or treatment with other biologics. do.

[0095] In certain embodiments, the present invention provides a method for tumor inhibition of tumor growth, comprising administering to the patient a compound as described herein in combination with a signal transduction inhibitor (STI) to achieve additive or synergistic inhibition of tumor growth. The present invention provides a method for treating a patient with TIGIT comprising administering to the patient an anti-TIGIT antibody as disclosed herein. Signal transduction inhibitors selectively block one or more steps in a signal transduction pathway. The signal transduction inhibitors (STIs) of the present invention include (i) BCR-Abl kinase inhibitors (e.g., imatinib mesylate (GLEEVEC)), (ii) epidermal growth factor (EGF) receptor inhibitors, including kinase inhibitors and antibodies, (iii) HER-2 / neu receptor inhibitors (e.g., HERCEPTIN), (iv) Akt kinase inhibitors (e.g., HERCEPTIN), and (v) HER-2 / neu receptor inhibitors (e.g., HERCEPTIN). (v) inhibitors of the myrrh kinase or Akt pathway (e.g., rapamycin), (v) cell cycle kinase inhibitors (e.g., flavopiridol), and (vi) phosphatidylinositol kinase inhibitors. Agents involved in immune modulation can also be used in combination with the anti-TIGIT antibodies disclosed herein to inhibit tumor growth in cancer patients.

[0096] Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and Piposulfan; Aziridines such as Benzodepa, Carboquone, Metullo and Uredop PA; Ethyleneimine and methylolmelamine (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolmelamine); Nitrogenmaster amides, such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fentanyl acetate, Mustine, Lomustine, Nimustine, Ranimustine; Antibiotics, such as Acrasinomycin alacinomysins, actinomycin, autramycin, azace , bleomycin, cactinomycin, calicheamicin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercapto Purines, thiamiprine, thioguanine; pyrimidine analogues, e.g., ancitabine, azathioprine; Cytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., calstenoside, anti-adrenals such as Aminoglutethimide, Mitotane, Trilostane; folic acid supplements such as Folinicacid; Aceglatone; Aldophosphatidylcholine; Amidoglycosides; Aminolevulinic acid; Amsacrine; Bestravsil; Bisantrene; Ethyl Datrexate (edatraxate); Defofamine; Demecolcine; Diaziquone; Elformitin; Elliptinium acetate; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Lazo Xanthane;Schizophyllan;Spirogermanium;Tenuazonic acid;Triazicon;2,2',2''-Triazicon Trichlorotriethylamine;Urethane;Vindesine;Dacarbazine;Mannomustine;Mitob Lonitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (Ara-C); Cyclophosphamide; Thiotepa; Toxoids (i.e., taxanes), such as paclitaxel, taxol, and doxetaxel; Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopropionate; Phosphorus; methotrexate; platinum and platinum coordination complexes, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxant vincristine;vinorelbine;navelbine;novantrone;teniposide;daunomycin;aminopterin;xeloda;ibandronate;CPT11;topoisomerase inhibitors;camp Tothecin, difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecita vinca; antimetabolites (e.g., azathioprine); anthracyclines; plant alkaloids (including, e.g., vinca alkaloids); as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0097] Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens [including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, ketoxifene, onapristone, and toremifene]; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as well as pharmacologic acceptable salts, acids, or derivatives of any of the above. In certain embodiments, combination therapy includes the administration of hormones or related hormonal agents.

[0098] Additional Treatment Modalities That Can Be Used in Combination with the Anti-TIGIT Antibodies Disclosed herein These include radiation therapy, monoclonal antibodies against tumor antigens, monoclonal antibody-toxin conjugates, T cell adjuvants, bone marrow transplantation, or antigen-presenting cells (e.g., dendritic cell therapy), or As used herein, the term "cell-based therapies" includes cell-cell based therapies. "Cell therapy" refers to any therapy that involves the introduction of cells to achieve a desired therapeutic effect, such as repairing damaged or replete mature cell populations or tissues, or enhancing an immune response against pathogens or proliferating cells (e.g., cancer cells). In other embodiments, cell therapy involves the introduction of undifferentiated cells (e.g., stem cells or other pluripotent or totipotent cells) that can differentiate into the specific cell types required to achieve a desired therapeutic effect, such as repairing damaged or replete mature cell populations or tissues, or enhancing an immune response against pathogens or proliferating cells (e.g., cancer cells). In some embodiments, the antibodies of the invention can also be administered together with tumor-infiltrating T cells, or with natural killer cells, optionally expanded or selected for homing to tumors ex vivo. The antibodies of the invention contribute to the activation of these cells.

[0099] A preferred combination is an antibody of the present invention and a second antibody against 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 antibody of the present invention for the treatment of cancer include Herceptin® (trastuzumab) against the HER2 antigen, Avastin® (bevacizumab) against VEGF, or EGF. Antibodies against receptors include Erbitux® (cetuximab) and Vectibix® (panitumumab). Other agents that can be administered include antibodies or other inhibitors of PD-1, PD-L1, CTLA-4, 4-1BB, BTLA, VISTA, TIM-3, and LAG-3, or other downstream signaling inhibitors, such as mTOR and GSK3β 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), tarsinib, sucralobacter ... Examples of secondary antibodies or other agents in the combination therapy include rifabutinib, cobimetinib, and nivolumab. The choice of secondary antibody or other agent in the combination therapy depends on the cancer being treated. Optionally, the cancer is tested for expression or preferential expression of the antigen to guide the selection of the appropriate antibody. The isotype of the secondary antibody is preferably human IgG1 to promote effector functions such as ADCC, CDC, and phagocytosis. be.

[0100] The antibodies of the invention can also be administered with a vaccine to elicit an immune response against the cancer, the immune response being enhanced by the antibodies of the invention. The vaccine is effective to induce an immune response and can include an antigen expressed on the surface of the cancer, or a fragment thereof, optionally linked to a carrier molecule.

[0101] The present invention relates to an anti-TIGIT antibody as described herein in combination with an immune checkpoint inhibitor. The use of

[0102] The enormous number of genetic and epigenetic changes that characterize all cancers results in a diverse set of antigens that the immune system can use to distinguish tumor cells from their normal counterparts. In the case of T cells, this is initiated by antigen recognition by the T cell receptor (TCR). The ultimate magnitude (e.g., cytokine production or proliferation levels) and quality (e.g., survival) of the response The type of immune response that occurs (such as the pattern of cytokine production) is determined by co-stimulatory and inhibitory signals. Under normal physiological conditions, immune checkpoints regulate the immune system's response to pathogenic infections and to prevent autoimmunity ( , maintaining self-tolerance) and also for protecting tissues from injury. The expression of immune checkpoint proteins can be dysregulated by tumors as an important immune resistance mechanism.

[0103] Examples of immune checkpoints (ligands and receptors) that are candidates for blockade and that are selectively upregulated in various tumor cell types include PD-1 (programmed cytoplasmic receptor 1 (PD-1) and PD-2 (programmed cell death receptor 2 (PD-2)). Cell death protein 1), PD-L1 (programmed 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 inhibitor of T cell activation (VISTA), CD96 , A2aR (adenosine A2a receptor), A2bR (adenosine A2b receptor), CD73 (ecto-5'-nucleoside receptor) tidase), CD39 (ENTPD1, ​​NTPDase1), arginase, indoleamine-pyrrole2,3-dioxygenase (IDO), tryptophan-2,3-dioxygenase tryptophan 2,3-dioxygenase (TDO), and killer cell inhibitory receptors. Based on their structural features, they can be divided into two classes: i) killer cell immunoglobulin-like receptors (KIRs), and ii) C-type lectin receptors (members of the type II transmembrane receptor family). The receptors (e.g., the 2B4 (also known as CD244) receptor) and ligands (e.g., certain B7 family inhibitory ligands, e.g., B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1)) are involved in the regulation of cell proliferation and cell death. Other less well-defined immune checkpoints have been described in the literature, including both IL-1, B7x and VCTN1) (see Pardoll, (April 2012) Nature Rev. Cancer 12:252-64).

[0104] The present invention also contemplates the use of any of the anti-TIGIT antibodies disclosed herein in combination with inhibitors of the immune checkpoint receptors and ligands described above, as well as immune checkpoint receptors and ligands yet to be described. While several inductors are currently available, others are in late-stage development. For example, the fully humanized CTLA-4 monoclonal antibody ipilimumab (YERVOY, Bristol-Myers Squibb) received regulatory approval in the United States when it was approved for the treatment of melanoma in 2011. A fusion protein containing CTLA-4 and an antibody (CTLA-4-Ig, abatacept (ORENCIA, Bristol-Myers Squibb)) has been shown to be effective in treating rheumatoid arthritis. Other fusion proteins have been used to treat kidney transplant patients who are sensitized to the Epstein-Barr virus. Anti-PD-1 antibodies (e.g., Nivolumab) have been used to treat kidney transplant patients who are sensitized to the Epstein-Barr virus. lambrolizumab (Bristol-Myers Squibb) and pembrolizumab (Merck) are in development. Anti-PD-L1 antibodies (e.g., MPDL3280A (Roche)) are also under evaluation. Lorisumab has shown promise in patients with melanoma, lung and kidney cancer.

[0105] Similar combination therapy can be used to treat or prevent infectious diseases, such as viral, bacterial, fungal and parasitic diseases, disorders and conditions, and disorders related thereto. For example, the antibody of the present invention can be combined with an antibody against a pathogen or a vaccine against a pathogen, such as palivizumab against Rous sarcoma virus. The vaccine can be a protein of the pathogen or a fragment thereof that is effective in inducing an immune response. The antibody of the present invention enhances the immune response of the antibody or vaccine against the pathogen. The antibody of the present invention can also be administered with ex vivo expanded T cells or natural killer cells.

[0106] Such combination therapies may involve the use of antiviral agents that target various viral life cycle stages and have different mechanisms of action, including, but not limited to: inhibition of viral uncoating; antiviral drugs (e.g., amantadine and rimantadine); reverse transcriptase inhibitors (e.g., acyclovir, zidovudine and lamivudine); drugs that target integrase; drugs that block transcription factor binding to viral DNA; drugs that affect translation (e.g., antisense molecules) (e.g., fomivirsen); drugs that modulate translation / ribozyme function; protease inhibitors; viral assembly modulators (e.g., rifampicin); antiretroviral agents, e.g., nucleosides Analogue reverse transcriptase inhibitors (e.g., azidozymidine (AZT), ddl, ddC, 3TC, d4T), etc.; non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz, nevirapine); nucleotide analogues reverse transcriptase inhibitors; and agents that prevent the release of viral particles (e.g., zanamivir and oseltamivir). Treatment and / or prevention of certain viral (e.g., HIV) infections include , often requiring a collection of antiviral drugs (a "cocktail").

[0107] Other anti-TIGIT antibodies contemplated for use in combination with any of the anti-TIGIT antibodies disclosed herein include Antivirals include abacavir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevirertet, cidofovir, combivir, darunavir, delavirdine, didanosine, and docosanol. , edoxudine, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, phosphonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, various interferons (e.g., peginterferon alfa-2a), lopinavir, robin, These include, but are not limited to, rido, maraviroc, moroxydine, methisazone, nelfinavir, nexavar, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, ritonavir, pyramidine, saquinavir, stavudine, telaprevir, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, and zalcitabine.

[0108] The present invention relates to the use of any of the anti-TIGIT antibodies disclosed herein in combination with an anti-parasitic agent. Such agents include, but are not limited to, thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide, bithionol, oxamniquine, metrifonate, ivermectin, albendazole, eflornithine, melarsoprol, pentamidine, benznidazole, nifurtimox, and nitroimidazole. Those skilled in the art will be aware of other agents that may find utility in treating parasitic disorders.

[0109]

[0023] Embodiments of the present invention contemplate the use of any of the anti-TIGIT antibodies disclosed herein in combination with an agent useful for the treatment or prevention of bacterial disorders. Antibacterial agents can be classified in a variety of ways, including by their chemical structure and by their spectrum of activity. Examples of antibacterial agents include those that target the bacterial cell wall (e.g., cephalosporins, antibacterial agents (e.g., rosporins and penicillins), or those that target cell membranes (e.g., polymyxins), or Antibacterial agents that interfere with essential bacterial enzymes (e.g., sulfonamides, rifamycins, and quinolines) are the most common. Most antibacterial agents that target protein synthesis (e.g., tetracyclines, Antimicrobials (aminoglycosides and macrolides) are bacteriostatic, whereas drugs such as aminoglycosides are bactericidal. Another means of categorizing antimicrobial agents is based on their target specificity: "narrow spectrum" agents target specific types of bacteria (e.g., gram-positive bacteria such as Streptococcus), whereas "broad spectrum" agents have activity against a wider range of bacteria. Those of skill in the art will know which types of antibacterial agents are suitable for use in particular bacterial infections.

[0110]

[0023] Embodiments of the present invention contemplate the use of any of the anti-TIGIT antibodies disclosed herein in combination with an agent useful for the treatment or prevention of fungal disorders. Antifungal agents include polyenes, (e.g., amphotericin, nystatin, and pimaricin), azoles (e.g., fluconazole, These include arylamines (eg, naftifine and terbinafine) and morpholines (eg, amorolfine), and antimetabolites (eg, 5-fluorocytosine).

[0111] The present invention includes pharma- ceutically acceptable salts, acids, or derivatives of the agents (and members of classes of agents) listed above.

[0112] Antibodies against TIGIT may be used in combination with any of the secondary antibodies or agents described. Alternatively, they can be used in combination as components of a pharmaceutical composition, in which the drug can be combined with one or more pharma- ceutically acceptable carriers as described above.

[0113] V. Other Applications Anti-TIGIT antibodies can be used to detect TIGIT in clinical diagnostic or treatment contexts or in research. For example, the antibodies can be used to detect TIGIT levels in T cells, natural killer cells, and lymphocytes as an indication that a subject is suffering from a treatable cancer or infectious disease. The presence of TIGIT on cancer cells can be detected. Expression of TIGHT on T cells, natural killer cells, and / or cancer cells in mice indicates that the cancer or also provides an indication that an infectious disease can be treated with the antibodies of the invention. They can also be sold as research reagents for experimental studies in detecting natural killer cells and cancer cells and their responses to various stimuli. For such use, the monoclonal antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes or radioisotopes and provided in the form of kits with all the necessary reagents to perform an assay for TIGIT. The antibody can be isolated, for example, by affinity chromatography. It can also be used to purify the product.

[0114] VI. Kit Antibodies against TIGIT may be used in combination with any of the secondary antibodies or agents described. The invention disclosed herein can be combined as a component of a kit, comprising one or more of the antibodies disclosed herein and one or more pharma- ceutically acceptable excipients or carriers (e.g., but not limited to, phosphate buffered saline, water, sterile water, polyethylene glycol, ethyl acetate ... In one embodiment, one or more kits containing the carrier, diluent or excipients are provided, which may include one or more of the following: glycols, polyvinylpyrrolidone, lecithin, peanut oil, sesame oil, emulsions, e.g., oil / water emulsions or water / oil emulsions, microemulsions, nanocarriers, and various types of wetting agents. Additives, e.g., alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like, may also be included in the kits of the invention along with carriers, diluents or excipients. In one embodiment, pharma- ceutically acceptable carriers suitable for use in the antibody compositions disclosed herein are sterile, pyrogen-free, and / or otherwise free from associated infectious diseases and other undesirable effects. The kits are safe for administration to subjects without the risk of untoward side effects. In the kits, each agent can be provided in a separate vial, along with instructions for combination and subsequent administration or instructions for separate administration. The kits can also include written instructions for the proper handling and storage of any of the anti-TIGIT antibodies disclosed herein. Cut.

[0115] Every maximum numerical limit given throughout this specification is intended to include every lower numerical limit, as if such lower numerical limit were expressly written herein. Every minimum numerical limit given throughout this specification will include every higher numerical limit, as if such higher numerical limit were expressly written herein. Every numerical range given throughout this specification will include every narrower range that falls within such broader range, as if such narrower numerical ranges were all expressly written herein.

[0116] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item was specifically and individually indicated to be incorporated by reference. Where different versions of a sequence have been linked to an accession number at different times, the version linked to that accession number as of the effective filing date of this application is intended. Effective filing date means the earlier of the actual filing date or the filing date of a priority application, as appropriate, with reference to the accession number. Similarly, where different versions of publications, websites, etc. have been published at different times, the version published closest to the effective filing date of this application is intended, unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the invention may be used in combination with any other, unless otherwise specifically indicated.

[0117] Although the invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be understood that certain changes and modifications can be practiced within the scope of the appended claims. EXAMPLES

[0118] [Example] The following examples describe the production, characterization, and humanization of monoclonal antibodies against human TIGIT. This application also discusses exemplary methods by which binding characteristics of the antibodies described herein can be determined.

[0119] [Example 1] Expression of recombinant human TIGIT protein Gene cloning, mutagenesis, and plasmid construction in this study were carried out using the methodology described by Sambrook and Russell (Molecular Cloning, A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory, New York, NY). aboratory Press, ColdSpring Harbor, NY), Kostelny et al. (Int. J. Cancer 93:556-565, 2001), and Cole et al. (J. Immunol. 159:3613-3621, 1997). This was carried out using standard molecular biology techniques.

[0120] The mammalian expression vector pFCm404 (FIG. 1) for the production of the extracellular domain of human TIGIT fused to the Fc domain of the human immunoglobulin γ1 chain (hTIGIT-Fc, SEQ ID NO: 59) contains the following genetic components: Proceeding clockwise from the SalI site of pFcm404 in FIG. 1, the plasmid contains a transcription unit for hTIGIT-Fc starting with the human cytomegalovirus (CMV) major immediate early promoter and enhancer (CMV-P in FIG. 1). The CMV promoter is followed by a signal peptide (sp, SEQ ID NO: 2), the extracellular domain of human TIGIT (hTIGIT, SEQ ID NO: 3), a polypeptide linker (SEQ ID NO: 4), and the nucleotide sequence of the human TIGIT gene (SEQ ID NO: 5). 4), and the coding region for the human γ1 Fc region (SEQ ID NO: 5), followed by the coding region for the human γ1 heavy chain gene. The hTIGIT-Fc gene is followed by the SV40 early promoter (SV40-P), There is a puromycin N-acetyltransferase gene (puro) for resistance to puromycin, a segment containing the SV40 polyadenylation site (SV40-A). Finally, pFCm404 contains a bacterial origin of replication (pUCori) and a β-lactamase gene (β-lactamase). Contains a portion of the plasmid pUC19. The arrow in the figure indicates the direction of transcription. Relevant restriction enzyme sites is shown in the figure.

[0121] The coding region of the extracellular region of human TIGIT in pFCm404 was replaced with a DNA fragment encoding the extracellular region of human CD155 (hCD155, SEQ ID NO: 6), resulting in a new expression vector, pFCm406 (Figure 1). The extracellular region of human CD155 fused to the human γ1 Fc region (hCD155-Fc, SEQ ID NO: 7) is expressed from pFCm406.

[0122] The coding region between the AgeI and EagI sites of the γ1Fc region in pFCm404 was cloned with the FLAG peptide (FLAG , SEQ ID NO: 8), followed by a DNA fragment encoding the glycosylphosphatidylinositol attachment signal of human CD55 (GIP, SEQ ID NO: 9), resulting in a new expression vector pFCm407 (Figure 1) was obtained. The extracellular domain (hTIGIT-GPI) is expressed on the cell surface.

[0123] To obtain cell lines that stably produce hTIGIT-Fc and hCD155-Fc in the culture supernatant, expression vectors pFCm404 and pFCm406 were transfected into mouse melanoma cell line NS0 (European Collection The chromosomes of the chromosome 1 gene of the chromosome 2 gene of Animal Cell Cultures, Salisbury, Wiltshire, UK were then incubated at 37°C. NS0 cells in DME medium containing 10% fetal bovine serum (FBS) in a 7.5% CO2 incubator. were grown as described by Bebbington et al. (Bio / Technology 10:169-175, 1992). The cells were then stably transfected into NS0 by electroporation. Before transfection, the expression vector was linearized using FspI. Approximately 10 cells were transfected. 7 Each well was transfected with 10 μg of linearized plasmid, suspended in DME medium containing 10% FBS, and seeded into several 96-well plates. After 24 hours, selection medium (DME medium containing 10% FBS and 3 μg / ml puromycin) was applied. Approximately 10 days after the start of selection, goat anti-human gamma chain antibody for coating and HRP conjugate for detection of Fc fusion protein were added. Cultures were examined for production of Fc fusion proteins by ELISA using goat anti-human gamma chain. The supernatant was assayed.

[0124] NS0 stable transfectants producing high levels of Fc fusion protein were hybridized The cells were adapted to and amplified in SFM medium (ThermoFisher Scientific, Waltham, MA). The cells were grown to exhaustion in roller bottles. After centrifugation and filtration, the culture supernatant was applied to a Protein A Sepharose column (GE Healthcare, Piscataway, NJ). The column was loaded with phosphate-buffered saline. After washing with saline (PBS), the Fc fusion protein was eluted with 0.1 M glycine-HCl (pH 3.0). After neutralization with Tris-HCl (pH 8), the buffer of the eluted Fc fusion protein was exchanged into PBS by dialysis. .

[0125] To obtain a cell line expressing hTIGIT-GPI on the cell surface, the expression vector pFCm407 was stably transfected into NS0 cells. The expression of hTIGIT-GPI on the cell surface was monitored by flow cytometry using rat anti-FLAG monoclonal antibody and PE-labeled goat anti-rat IgG, an Fc-specific antibody. The NS0 cell line expressing high levels of hTIGHT-GPI on the cell surface (NS0-hTIGIT) was used for screening of anti-TIGIT antibodies.

[0126] [Example 2] Generation and characterization of anti-TIGIT monoclonal antibodies Mouse hybridomas producing anti-human TIGIT monoclonal antibodies were purchased from JNBiosciences ( GenomONECF EX Cell Fusion Reagent (Cosmo Bio, Carlsbad, CA) was used. The hybridoma was produced according to standard hybridoma techniques such as those used by the company Eppendorf (CA). The fusion protein and NS0-hTIGIT cells were used.

[0127] The mouse antibodies secreted into the culture supernatant of the hybridoma cells were subjected to a series of screening to determine the following properties: (1) binding to purified hTIGIT-Fc fusion protein; (2) binding to NS0-hTIGIT cells; (3) does not bind to NS0 cells; (4) binds to human peripheral blood monoclonal antibodies treated with phytohemagglutinin. (5) hCD155-Fc fusion protein and NS0-hTIGIT bind to CD3+ T cells derived from human T cells. We identified antibodies with binding properties that block cell binding. The first property, binding to hTIGIT-Fc, was tested by ELISA using HRP-conjugated goat antibodies against mouse kappa and lambda chains (SouthernBiotech, Birmingham, AL) for identification of 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) for detection of cell-bound mouse antibodies. The fifth property, as described below, was examined by flow cytometry using a PE-labeled goat anti-mouse gamma chain antibody (SouthernBiotech) for detection of cell-bound mouse antibodies. Three mouse monoclonal antibodies (TIG1, TIG2, and TIG3) were found to have all five of these properties. The TIG1, TIG2, and TIG3 monoclonal antibodies were purified by protein A column chromatography as described above. TIG1, TIG2, and TIG3 were purified from the culture supernatant of hybridoma cells and have the mouse IgG2b / kappa, IgG2a / kappa, and IgG2a / kappa isotypes, respectively.

[0128] The ability of TIG1, TIG2, and TIG3 to block the interaction between TIGIT and CD155 was analyzed by flow cytometry. NS0-hTIGIT cells were incubated with various concentrations of anti-TIGIT monoclonal antibodies as competitors. The cells were incubated with a subsaturating concentration of hCD155-Fc in the presence (or absence) of monoclonal antibodies. Detection of hCD155-Fc bound to NS0-hTIGIT cells was performed using DyLight488-conjugated goat anti-human IgG, an Fc-specific antibody (JacksonImmunoResearch, West Grove, PA). As reported, TIG1, TIG2, and TIG3 are expressed by D155-Fc binding to hCNSO-hTIGIT cells. The half-maximal inhibition of the interaction between hCD155-Fc and NSO-hTIGIT cells was observed in a dose-dependent manner. Inhibitory concentration (IC 50 ) were 49ng / ml for TIG1, 197ng / ml for TIG2, and 100ng / ml for TIG3. The concentration of CD155 required to block more than 95% of TIGIT binding was 460 ng / ml. The antibody concentrations were 0.63 μg / ml for TIG1, 1.25 μg / ml for TIG2, and 2.5 μg / ml for TIG3.

[0129] [Example 3] V gene sequencing of anti-TIGIT monoclonal antibodies The heavy and light chain (VH and VL, respectively) sequences of the mouse anti-TIGIT antibody were determined according to the procedure described by Tsurushita et al. (Methods 36:69-83, 2005). , Carlsbad, CA) according to the supplier's protocol for approximately 10 7Total RNA was extracted from cells using the SMARTerRACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA). Oligo dT primed cDNA for 5'-PAGE was synthesized using SMARTerRACE cDNA Amplification Kit (CA) according to the supplier's protocol. Phusion DNA polymerase (ThermoFisher Scientific, Waltham, MA) was used to polymerize the light chain using a 3' primer (Tsurushita et al., supra) and a 5' primer that anneal specifically to the light chain constant region, respectively. VH and VL cDNA were amplified by polymerase chain reaction using mAb (Asp-Asp). The VL gene was cloned using the CloneJet PCR Cloning Kit (Thermo Fisher Scientific). The clones were then sequenced using the primers provided in the CloneJet PCR Cloning Kit. Several VH and VL clones were sequenced and unique sequences were identified that were homologous to typical mouse heavy and light chain variable regions.

[0130] The amino acid sequence of the mature VH of TIG1 (SEQ ID NO: 10) is shown in Figure 3. The acid sequences are NFGMH (SEQ ID NO:11), FISSGSSSIYYADTVKG (SEQ ID NO:12), and MRLDYYAMDY (SEQ ID NO:13), respectively.

[0131] The amino acid sequence of the mature VL of TIG1 (SEQ ID NO: 14) is shown in Figure 4. The acid sequences are RASKSISKYLA (SEQ ID NO: 15), SGSTLQS (SEQ ID NO: 16), and QQHNEYPWT (SEQ ID NO: 17), respectively.

[0132] The amino acid sequence of mature TIG2 VH (SEQ ID NO: 18) is shown in Figure 5. The acid sequences are EYTMH (SEQ ID NO: 19), GINPNNGGTSYNQKFKG (SEQ ID NO: 20), and PGWYNYAMDY (SEQ ID NO: 21), respectively.

[0133] The amino acid sequence of mature TIG2VL (SEQ ID NO:22) is shown in Figure 6. The CDR1, 2 and 3 amino acid sequences of TIG2 VL are KASQGVSTAVA (SEQ ID NO:23), SASYRYT (SEQ ID NO:24) and QQHYITPWT (SEQ ID NO:25), respectively.

[0134] The amino acid sequence of mature TIG3 VH (SEQ ID NO: 26) is shown in Figure 7. The acid sequences are DYDMS (SEQ ID NO:27), YISDGGYNTYYPDTVKG (SEQ ID NO:28), and QILLRYYFDY (SEQ ID NO:29), respectively.

[0135] The amino acid sequence of mature TIG3VL (SEQ ID NO:30) is shown in Figure 8. The acid sequences are KSSQSLLYSSNQKNYLA (SEQ ID NO:31), WASTRES (SEQ ID NO:32), and QQYHSYPWT (SEQ ID NO:33), respectively.

[0136] The assignment of CDR sequences and the numbering of amino acid positions in Figures 3 to 8 are according to Kabat et al. (1991). The CDR1, CDR2 and CDR3 sequences in the figures are underlined.

[0137] [Example 4] Construction and expression of humanized TIG1 antibody Humanization of TIG1 VH and VL was performed according to the procedure of Tsurushita et al. (Methods 36:69-83, 2005). As described by ueen et al. (Proc. Natl. Acad. Sci. USA. 86:10029-10033, 1989) Briefly, a three-dimensional molecular model of the variable region of TIG1 was first constructed using a proprietary algorithm from JN Biosciences. The molecular model was then used to identify framework amino acid residues that are important in forming the structure of the complementarity determining regions (CDRs) of TIG1. and cDNA-derived human VH and VL amino acids with high affinity to TIG1 VH and VL, respectively. Finally, the CDR sequences, together with the framework amino acid residues important for maintaining the CDR structure, were grafted from TIG1 VH and VL into the corresponding selected human framework sequences. did.

[0138] The amino acid sequence of the designed humanized TIG1 VH (HuTIG1 VH) is: MDSRLNLVFLVLILKGVQCEVQLVESGG GLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVSS (SEQ ID NO: 34). Mature HuTIG1 VH amino acid sequence, EVQ LVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVSS (SEQ ID NO:35) starts at position 20 of SEQ ID NO:34.

[0139] The amino acid sequence of the designed humanized TIG1 VL (HuTIG1 VL) is: MRFQVQVLGLLLLWISGAQCDIQMTQSP The mature HuTIG1VL amino acid sequence is DIQMTQSPSSLSAS VGDRVTITCRASKSISKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKVEIK (SEQ ID NO:37) starts at position 21 of SEQ ID NO:36.

[0140] The gene encoding HuTIG1VH was cloned into a 3′-transcriptome containing a splice donor signal, The fragment was synthesized as an exon containing a SpeI site at the 5' end and a HindIII site at the 3' end of the fragment. The nucleotide sequence of the synthetic HuTIG1VH gene flanked by SpeI and HindIII sites (SEQ ID NO:3) 8) is shown in FIG. 9A along with the deduced amino acid sequence (SEQ ID NO:34).

[0141] The gene encoding HuTIG1VL was inserted into the 3' end of the coding region with a splice donor and the 5' end of the fragment. The exon was synthesized with an NheI site at one end and an EcoRI site at the 3' end of the fragment. The nucleotide sequence of the synthetic HuTIG1VL gene (SEQ ID NO: 39), flanked by the putative nucleotide sequence and EcoRI sites, was The amino acid sequence (SEQ ID NO:36) is shown in FIG. 9B.

[0142] A mammalian expression vector pHuTIG1.AA (FIG. 10) for production of the humanized IgG1 / kappa form of the murine anti-human TIGIT monoclonal antibody TIG1 (HuTIG1-IgG1.AA) was constructed containing the following genetic components: Proceeding clockwise from the SalI site in FIG. 10, the vector contains the human cytomegalovirus (CMV) major immediate early promoter and endogenous ribozyme (ERK) promoter to initiate transcription of the antibody heavy chain gene. The heavy chain transcription unit starts with a CMV promoter (CMV-P in the figure). After the CMV promoter, the humanized form of the heavy chain variable region of TIG1 (HuTIG1VH) is encoded, flanked by SpeI and HindIII sites. The human genome contains exons that encode CH1, hinge, CH2, and CH3 exons and an intervening intron. There is a genomic sequence containing the γ1 heavy chain constant region and the polyadenylation site of the human γ1 heavy chain gene. The CH2 region encoded by pHuTIG1.AA has amino acid substitutions at positions 234 and 235 (Eu numbering) to alanine residues (L234A / L235A) to remove effector function (Hezareh et al., J. Virol. 75:12161-12168, 2001). After the heavy chain gene sequence, the light chain transcription unit is The gene begins with a promoter and enhancer (CMV-P), followed by an exon encoding the humanized form of the light chain variable region of TIG1 (HuTIG1Vl) flanked by NheI and EcoRI sites, preceded by a portion of an intron. The genomic sequence contains a human kappa chain constant region (Cκ), and the Cκ exon is followed by the polyadenylation site of the human kappa chain gene. The light chain gene is then followed by the SV40 early promoter (SV40-P), the puromycin N-acetyltransferase gene (puro) for resistance to puromycin, and a segment containing the SV40 polyadenylation site (SV40-A). Finally, pHuTIG1.AA is designed to encode a bacterial origin of replication (pUCori) and β-lactamase. The figure contains a portion of the plasmid pUC19, including the gene (β-lactamase). The transcription direction is indicated.

[0143] The amino acid sequence of the mature gamma heavy chain of HuTIG1-IgG1.AA encoded by pHuTIG1.AA is EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGLEWVAFISSGSSSIYYADTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARMRLDYYAMDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 40).

[0144] The C-terminal lysine may or may not be present.

[0145] The amino acid sequence of the mature kappa light chain of HuTIG1-IgG1.AA encoded by pHuTIG1.AA is DIQMTQSPSSLSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 41).

[0146] The expression vector pHuTIG1.AA was transfected into the Chinese hamster ovary cell line CHO-K1 (ATCC, Manassas, CA). The HuTIG1-IgG1.AA gene was introduced into the chromosomes of CHO-K1 cells (Fig. 1b, VA) to obtain a cell line that stably produces HuTIG1-IgG1.AA. The cells were cultured in SFM4CHO medium (GE Healthcare Life Sciences) at 37°C in a 7.5% CO2 incubator. The plasmid was grown in a 200-well plate (Thermo Fisher Scientific, Logan, UT). Stable transfection into CHO-K1 was performed by electroporation. Prior to transfection, pHuTIG1.AA was linearized using FspI. In a typical experiment, approximately 10 7 The cells were transfected with 20 μg of linearized plasmid and suspended in SFM4CHO medium. The cells were appropriately diluted and then plated on several 96-well plates. After 48 hours, 100 μl / well of SFM4CHO medium containing 20 μg / ml of puromycin was added for isolation of stable transfectants. Approximately 10 days after initiation, culture supernatants of the expressing strains were assayed for antibody production.

[0147] Expression of HuTIG1-IgG1.AA was measured by sandwich ELISA. In a typical experiment, ELISA plates were coated with goat anti-human IgG Fc specific polyclonal antibody, washed with washing buffer (PBS containing 0.05% Tween 20), and incubated with ELISA buffer (containing 2% skim milk and 0.05% Tween 20). After washing with washing buffer, the test was diluted appropriately in ELISA buffer. The samples were applied to the ELISA plate. The appropriate humanized IgG1 / κ antibody was used as a standard. The ELISA plate was incubated for 1 hour at room temperature, washed with washing buffer, and then the HRP conjugate was added. Bound antibodies were detected using a denatured goat anti-human kappa chain polyclonal antibody. Plates were incubated for 0.5 h at room temperature and washed with wash buffer, after which color development was initiated by the addition of 100 μl / well ABTS substrate (Sigma-Aldrich) and stopped with 100 μl / well 2% oxalic acid. Absorbance was measured at 405 nm.

[0148] CHO-K1 stable transfectants highly producing HuTIG1-IgG1.AA were grown in SFM4CHO until cell viability was less than 50%. After centrifugation and filtration, the culture supernatant was diluted with Protein A The column was loaded with PB After washing with S, the antibody was eluted with 0.1 M glycine-HCl (pH 3.0). The buffer for the eluted antibody was 1 MT The solution was neutralized with ris-HCl (pH 8) and then exchanged into PBS by dialysis. The antibody concentration was determined by measuring the absorbance at 280 nm (1 mg / ml = 1.4 OD).

[0149] [Example 5] Characterization of HuTIG1-IgG1.AA Binding of HuTIG1-IgG1.AA to human TIGIT was examined by ELISA. ELISA plates were coated with 5 μg / ml hTIGIT-Fc in PBS (100 μl / well) overnight at 4° C., washed with wash buffer (PBS containing 0.05% Tween 20) and blocked with 200 μl / well SuperBlock blocking buffer (Thermo Fisher Scientific). After washing with wash buffer, plate-bound To bind human TIGIT, HuTIG1-IgG1.AA was added in two-fold serial dilutions starting at 2.5 μg / ml in SuperBlock blocking buffer. The ELISA plate was incubated for 1 hour at room temperature. After basting and washing with wash buffer, bound antibodies were detected with 100 μl / well of HRP-conjugated goat anti-human kappa chain polyclonal antibody (SouthernBiotech) diluted 1 / 2,000 in ELISA buffer. After incubation for 30 min at room temperature and washing with wash buffer, color development was detected with 100 μl / well of HRP-conjugated goat anti-human kappa chain polyclonal antibody (SouthernBiotech) diluted 1 / 2,000 in ELISA buffer. The wells were initiated with ABTS substrate and stopped with 100 μl / well 2% oxalic acid. Absorbance was measured at 405 nm. As shown in FIG. 11, HuTIG1-IgG1.AA inhibited human TIGIT in a dose-dependent manner. The half-maximal effective concentration (EC 50 ) was 65 ng / ml, indicating that HuTIG1-IgG1.AA is a good binder to human TIGIT. .

[0150] The binding of HuTIG1-IgG1.AA to human TIGIT was also examined by flow cytometry. NS0-hTIGIT cells (8x10 5 NS0-hTIGIT cells (100 μg / ml) were incubated for 20 min at 4° C. in the presence (or absence) of various concentrations of HuTIG1-IgG1.AA in 2-fold serial dilutions starting at 10 μg / ml in 160 μl of FACS buffer (PBS containing 0.5% bovine serum albumin and 0.05% sodium azide). After washing with FACS buffer, HuTIG1-IgG1.AA bound on NS0-hTIGIT cells was detected with a PE-labeled goat anti-human IgG antibody in FACS buffer. After 20 min incubation and washing with FACS buffer, the cells were analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences, San Jose, CA). The cells were then subjected to a cytometry experiment to obtain mean channel fluorescence (MCF) at each antibody concentration. A plot of MCF (vertical axis) against concentration (horizontal axis) is shown. EC 50 The value was 60 ng / ml, indicating that HuTIG1-IgG1.AA is a good binder to human TIGIT expressed on the cell surface. .

[0151] 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. 6 100 ng / ml of cells were incubated with a subsaturating concentration (125 ng / ml) of FITC-labeled hCD155-Fc fusion protein (hCD155-Fc-FITC) in the presence (or absence) of various concentrations of HuTIG1-IgG1.AA in 2-fold serial dilutions starting at 10 μg / ml in 200 μl of FACS buffer for 20 min at 4° C. After washing with FACS buffer, the cells were analyzed by flow cytometry using a FACScan flow cytometer (BD Biosciences, San Jose, CA). To investigate the binding of hCD155-Fc-FITC to the cells, MCF was obtained at each antibody concentration. As shown in Figure 13, HuTIG1-IgG1.AA blocked the interaction between hCD155-Fc-FITC and TIGIT on cells in a dose-dependent manner. The half-maximal inhibitory concentration (EC 50 ) was 67 ng / ml, indicating that HuTIG1-IgG1.AA is an effective blocker of the interaction between CD155 and TIGIT.

[0152] [Example 6] Construction and expression of humanized TIG3 antibody Humanization of TIG3 VH and VL was performed according to the general procedure described in Example 4. The amino acid sequence of TIG3 VH (HuTIG3VH) is MNFGLRLIFLVLTLKGVNCEVQLVESGGGLVQPGGSLRLSCAAS. GFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSS (SEQ ID NO: 42). Mature HuTIG3 VH amino acid sequence, EVQLVESGGGLVQPGGSLR LSCAASGFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSS (SEQ ID NO:43) starts at position 20 of SEQ ID NO:42.

[0153] The amino acid sequence of the designed humanized TIG3VL (HuTIG3 VL) is: MDSQAQVLMLLLLWVSGTCGDIQMTQSP The mature HuTIG3VL amino acid sequence is SSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGTKVEIK (SEQ ID NO: 44). SSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGTKVEIK (SEQ ID NO:45) starts at position 21 of SEQ ID NO:44.

[0154] The gene encoding HuTIG3VH was inserted into the 3' end of the coding region with a splice donor and the 5' end of the fragment. It was synthesized as an exon containing a SpeI site at one end 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 in Figure 14A along with the deduced amino acid sequence (SEQ ID NO:42).

[0155] The gene encoding humanized TIG3VL (HuTIG3 VL) was spliced ​​to the 3' end of the coding region. The fragment was synthesized as an exon containing an 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 HuTIG3VL gene (SEQ ID NO: 47) was compared with the deduced amino acid sequence (SEQ ID NO: This is shown in Figure 14B along with row number 44).

[0156] The mammalian expression vector pHuTIG3.AA for production of the humanized IgG1 / kappa form of the anti-human TIGIT monoclonal antibody TIG3 (HuTIG3-IgG1.AA) was constructed by modifying pHuTIG1.AA as follows: (1) replacing the HuTIG1 VH gene between the SpeI and HindIII sites with the HuTIG3 VH gene; and (2) the HuTIG1VL gene between the NheI and EcoRI sites was replaced with the HuTIG3VL gene. The CH2 region encoded by uTIG3.AA has amino acid substitutions at positions 234 and 235 (Eu numbering) to alanine residues (L234A / L235A) to eliminate effector function.

[0157] The amino acid sequence of the mature gamma heavy chain of HuTIG3-IgG1.AA encoded by pHuTIG3.AA is EVQLVESGGGLVQPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVAYISDGGYNTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQILLRYYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 48).

[0158] The C-terminal lysine may or may not be present.

[0159] The amino acid sequence of the mature kappa light chain of HuTIG3-IgG1.AA encoded by pHuTIG3.AA is DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYHSYPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:49).

[0160] Transient injection of pHuTIG3.AA into 100 ml of logarithmic-phase Expi293 cells (ThermoFisher Scientific) Transfections were performed according to the supplier's protocol. CHO-K1 stable transfectants highly producing HuTIG3-IgG1.AA were generated by electroporation of pHuTIG3.AA as described above and grown in SFM4CHO until cell viability reached 50%. HuTIG3-IgG1.AA was purified from the cell supernatant of each of the transiently transfected Expi293 cells and As described above, the antibody was stably transfected into CHO-K1 cells using a protein A affinity column. It was affected.

[0161] [Example 7] Characterization of HuTIG3-IgG1.AA Binding of HuTIG3-IgG1.AA to human TIGIT was assessed by ELISA as described in Example 5. As shown in Figure 15, HuTIG3-IgG1.AA bound to human TIGIT in a dose-dependent manner. The half-maximal effective concentration (EC 50 ) was 85 ng / ml, indicating that HuTIG3-IgG1.AA is a good binder to human TIGIT.

[0162] The binding of HuTIG3-IgG1.AA to human TIGIT was also examined by flow cytometry as described in Example 5. As shown in Figure 12, HuTIG3-IgG1.AA binds to human TIGIT. The half-maximal effective concentration (EC 50 ) The antibody titer was 370 ng / ml, indicating that HuTIG3-IgG1.AA is a good binder of human TIGIT. This indicates that.

[0163] The activity of HuTIG3-IgG1.AA to block the interaction between TIGIT and CD155 was examined as described in Example 5. Analysis by flow cytometry using sea urchin NS0-hTIGIT cells and FITC-labeled hCD155-Fc As shown in Figure 13, HuTIG3-IgG1.AA blocked the interaction between TIGIT and CD155 in a dose-dependent manner. Half-maximal inhibition of HuTIG3-IgG1.AA to block TIGIT-CD155 interaction Concentration (IC 50 ) was 279 ng / ml, indicating that HuTIG3-IgG1.AA is an effective blocker of the interaction between CD155 and TIGIT.

[0164] [Example 8] IL-2 cytokine responses to tetanus toxoid in human T cells are enhanced by TIGIT blockade The ability of mouse anti-TIGIT antibodies to enhance antigen-specific T cell responses was assessed using an in vitro antigen-specific recall assay against tetanus toxoid (see, e.g., Piersma et al., Vaccine. 2006, 12th April 2006). See, J Immunol. 2009 Aug. 15; 183(4):2827-36. This allows the immune system to induce CD4+ and CD8+ T cell memory responses. A booster shot to vaccinate against tetanus toxoid achieves adequate vaccine protection. Alternative measures of efficacy (e.g., Plotkin et al., Clin Vaccine Immunol. 2010 July;17(7):1055-106) 5; see Goulon et al., November 1972, Presse Med. 1:3049-3050) as an anti-tetanus A serum level of 0.1 IU / mL of toxoid is indicative of a sustained immune response.

[0165] Peripheral blood mononuclear cells (PBMCs) were obtained from human volunteers vaccinated with tetanus toxoid (iQBiosciences), and the volunteers were confirmed to have tetanus blood titers greater than 1.0 IU / ml. was confirmed by ELISA (Genway). 800,000 PBMCs were cultured in RPMI1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS) in a 96-well round-bottom plate (Nunc). Tetanus toxoid (List Biological Laboratories) was added at 2 μg / ml 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 μg / ml or 10 μg / ml for 4 days in a 5% CO2 incubator at 4 °C. PBMCs were spiked with 2 μg / ml tetanus toxoid without any antibodies. The supernatant was collected, and IL-2, a cytokine that is an indicator of T cell activation, was analyzed by flow cytometry (BD FACSC) using a cytokine bead array (BD Pharmingen, CBA Th1 / Th2 Cytokine Kit). As shown in Figure 16, the effect of tetanus toxoid stimulation alone was IL-2 production was 31 pg / ml. IL-2 production was 3.3 μg / ml and 10 μg / ml of mouse anti-TIGIT In the presence of the antibody, tetanus toxoid-stimulated IL-2 production was further increased to 70 pg / ml and 86 pg / ml, respectively. Tetanus toxoid-stimulated IL-2 production was increased in the presence of 3.3 μg / ml and 10 μg / ml of the murine anti-PD-1 antibody EH12.2H7. were also increased to 39 pg / ml and 57 pg / ml, respectively. These data indicate that TIG1 has the functional capacity to enhance antigen-specific T cell cytokine responses.

[0166] [Example 9] Human T cell proliferative responses to tetanus toxoid are enhanced by TIGIT blockade The ability of mouse anti-TIGIT antibodies to enhance antigen-specific T cell responses was assessed using an in vitro antigen-specific recall assay against tetanus toxoid (see, e.g., Piersma et al., Vaccine. 2006, 12th April 2006). See, J Immunol. 2009 Aug. 15; 183(4):2827-36. The study was conducted using human volunteers vaccinated with tetanus toxoid. PBMCs were obtained as described in Example 8 (iQBiosciences). PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) (ThermoFisher), a reagent used to label cells in vitro, and tracked for multiple generations by flow cytometry using dye dilution. 250,000 CFSE-labeled PBMCs were cultured in AIM-V medium (Invitrogen) in 96-well round-bottom plates (Nunc). Each well was cultured in the presence of 2.5 μg / ml, 5 μg / ml, or 10 μg / ml of mouse anti-PD-1 antibody (Biolegend clone EH12.2H7) or TIG1. Tetanus toxoid (AstarteBiologics, LLC) was added at 2 μg / ml to the cells. The cultures were incubated at 37°C in a 5% CO2 incubator. On day 4, IL-2 [50 units / ml] (Peprotech) was added to the cultures, and on day 6, CFSE dilution was performed for CD4+ T cells or CD8+ T cells. PBMCs were collected for proliferation measurements by flow cytometry (BD FACSCalibur) in vitro.

[0167] As shown in Figure 17 (top panel), tetanus toxoid induced 4.5% and 6.5% proliferation of human CD4+ T cells from donors 1 and 2, respectively. Addition of TIG1 further enhanced CD4+ T cell proliferation at all antibody concentrations tested (2.5 μg / ml, 5 μg / ml, and 10 μg / ml). Similar proliferative effects were observed for anti-PD-1 antibodies in these dose ranges.

[0168] As shown in Figure 17 (lower panel), tetanus toxoid induced 15% and 15.5% proliferation of human CD8+ T cells from donors 1 and 2, respectively. Addition of TIG1 further enhanced CD8+ T cell proliferation at all antibody concentrations tested (2.5 μg / ml, 5 μg / ml, and 10 μg / ml). Similar proliferative effects were observed for antibody EH12.2H7 at these dose ranges. These results demonstrate that TIG1 has the ability to enhance antigen-specific T cell proliferative responses.

[0169] [Example 10] NK cell-mediated cytotoxicity of human primary effector cells and K562 target cells by TIGIT blockade Increasing Harm Human NK cells target target cells lacking MHCI, such as the chronic myeloid leukemia (CML) cell line K562. (See, e.g., Nagel et al., Cancer Res 1981; 41:2284-2288; Andersson et al., Int JCancer. 1979 Feb;23(2):143-7.; Lozzio et al., Leuk Res 1979;3(6):363-70). Commercially available antibody (anti-CD155 clone SKII.4 from Biolegend). Using a ELISA kit (ATCC), expression of CD155 (PVR) on K562 cells was confirmed. As shown in FIG. 18 (top panel), 98% of K562 cells were found to express high levels of CD155 (black). Histograms of the immunoreactivity of human CD56-positive NK cells in PBMCs (iQBiosciences) using commercial antibodies (anti-TIGIT clone MBSA43 from eBiosciences and anti-CD56 clone HCD56 from Biolegend). TIGIT expression was confirmed for three representative human donors (FIG. 18, lower panel).

[0170] NK cell-mediated lysis of K562 cells was measured with or without TIG1. The following day, K562 cells were CFSE-labeled and incubated with PBMCs at 1:100 (target) in the presence of 10 μg / ml TIG1 for 4 h at 37°C in a 5% CO2 incubator. Target: PBMCs; 100 PBMCs containing approximately 5% NK cells were co-cultured with 1 K562 cell. After the incubation period, cells were harvested and stained with 5 nM Sytox Red (ThermoFisher). Killed target cells were identified by (CFSE+Sytox Red+). K562 target lysed by NK cells The percentage of target cells was determined by flow cytometry (FACSCalibur; FlowJo Analysis). As shown in Figure 19, 2% of K562 cells were lysed without TIG1. When TIG1 was added, 4.4% of K562 cells were lysed. The addition of TIG1 enhanced NK cell-mediated natural cytotoxicity of K562 cells by 2-fold, indicating that TIG1 has a role in the inhibition of target cell proliferation by a subset of human effector cells. The results demonstrate the ability to increase the levels of cell killing.

[0171] [Example 11] SEB-induced human T cell cytokine production is enhanced by TIGIT blockade Superantigens such as SEB (StaphylococcusEnterotoxin B) It activates T cells by linking MHC class II molecules on antigen-presenting cells to the νβ element of the TCR. SEB activates T cells in human blood, resulting in the production of cytokines including interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and interleukin gamma (IFNγ) (see, e.g., Krakauer et al., Toxins (Basel). August 2010;2(8):1963-1983). Compared to a typical recall antigen-induced T cell response in which 0.1-0.001% of T cells may be activated, SEB has been shown to activate up to 10-20% of T cells in human blood, depending on the fraction of νβ3, νβ12, νβ14, and νβ17-bearing T cells found in each particular blood donor. Therefore, SEB can be used in a T cell-based cytokine secretion assay to determine the level of target modification by TIGIT blockade using human whole blood cells (WBCs).

[0172] Freshly isolated blood without signs of hemolysis should be collected using sodium heparin within 4 hours of collection. A 250 μl sample of WBC was obtained from the Stanford Blood Center and placed in a 96-well round-bottom plate (Nunc). The cells were then aliquoted into wells and stimulated with SEB (Toxin Technology) at a final concentration of 1 μg / ml 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 briefly centrifuged to separate the cells from the harvested cells. After plasma sample collection, expression levels of IL-2, IL-6, TNFα and IFNγ were measured by cytokine bead assay (Biolegend, LEGENDplex™ human CD8 / NK panel) by flow cytometry (BD FACSCalibur) and analyzed by Biolegend software for quantitative measurements.

[0173] To determine the change in expression levels for each of IL-2, IL-6, TNFα, and IFNγ, cytokine levels in the presence of the test antibody (plus SEB) were compared to those in the absence of the antibody for each donor. The mean values ​​were divided by the cytokine levels in the presence of anti-TIGIT. A 2-fold change (e.g., a 2-fold change detected in the presence of anti-TIGIT) means that the absolute concentration of the cytokine measured in the experiment is twice the amount found in the SEB-stimulated control condition. As shown in Figure 20, SEB-stimulated IL-2, IL-6, TNFα or IFNγ production by healthy donor blood cells was enhanced 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 WBCs and its modulation by HuTIG1-IgG1.AA is a readout for enhanced cytokine effector responses. HuTIG1-IgG1.AA was able to enhance immune responses as demonstrated by stimulation of IL-2, IL-6, TNFα and IFNγ production in the assays shown here.

[0174] [Example 12] Characterization of HuTIG1-IgG1.AA and HuTIG3-IgG1.AA-mediated complement-dependent cytotoxicity Complement dependent cytotoxicity (CDC) refers to the lysis of cells expressing its target molecule in the presence of complement (see, eg, Gazzano-Santoro et al., J. Immunol. Methods, 202:163). Activation of the classical complement pathway occurs via the release of antibodies (with appropriate subunits) that bind to their cognate antigens on the cell surface. Complement activation is initiated by the binding of the first component of the complement system (C1q) to the antigen (antibody of the complement receptor β-blocker). To investigate the mechanism of action of the IgG antibody, we used the human T cell Jurkat dual reporter parental cell line (DualJurkat; Invivogen ) was engineered to stably express human TIGIT on its surface (Jurkat-TIGIT). As shown in Figure 21, flow cytometry using a commercially available PE-labeled anti-TIGIT antibody (clone MSBA43 from eBiosciences) was performed. -Cytometry confirmed the expression of TIGIT on the surface of Jurkat-TIGIT cells. Therefore, Jurkat-TIGHT was used as target cells that constitutively express membrane-bound TIGIT and therefore can undergo antibody-bound CDC activity in the presence of complement.

[0175] The CDC assay was performed using Jurkat-TIGIT cells and human complement, with up to 50 μg / ml The reactivity with Jurkat cells and the reactivity with Jurkat cells were examined in the presence of HuTIG1-IgG1.AA, HuTIG3-IgG1.AA, or rabbit antithymocyte globulin (ATG) (Fresenius Biotech GmbH) at different concentrations. Complement-dependent cytotoxicity activity recorded for cut cells (see, e.g., Eiermann et al., J Hematother Stem Cell Res. 2001 June;10(3):385-90.; Ayuk et al., AntiCancer Research 29:135 ATG was used as a positive control for the 1990-nCoV assay (see 5-1360 (2009)). 50,000 Jurkat-TIGIT cells in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS) were plated in a 96-well circular plate. Treatment antibodies were added starting at a highest concentration of 50 μg / ml, followed by a 3-fold dilution series, and incubated with the cells for 1 hour at 37°C in a 5% CO2 incubator. After this 1 hour incubation, human complement was added and incubated for an additional 3 hours in a 5% CO2 incubator at 37°C. After the 3 hour incubation was completed, 5 μg / ml propidium iodide (ThermoFisher) was added and the samples were run through a flow cytometer. Analysis was performed by cytometry (BD FACSCalibur) to detect iodide protease activity as a readout for cell death. The percentage of Lopidium positives was determined.

[0176] To determine normalized changes, the percentage of viability was measured by flow cytometry and the percentage of propidium iodide positive cells was assessed as dead cells. Values ​​for untreated samples were normalized to 100%. The percentage of viability of samples in the presence of antibody was divided by the normalized untreated one to obtain the fold change in viability. As shown in Figure 22, both HuTIG1-IgG1.AA and HuTIG3-IgG1.AA at 50 μg / ml or less did not induce CDC activity. These data demonstrate that neither binding of HuTIG1-IgG1.AA to TIGIT nor binding of HuTIG3-IgG1.AA to TIGIT results in complement-mediated cell death of T cells.

[0177] [Example 13] Jurkat dual reporter cell line characterization of HuTIG1-IgG1.AA and HuTIG3-IgG1.AA Five copies of the FN-κB consensus transcription response element and three copies of the c-Rel binding site A secreted luciferase reporter driven by the IFNβ minimal promoter fused to Using a Jurkat-TIGIT reporter cell line that contains the gene intrachromosomally, we have identified human TIGIT The functional consequences of blocking the receptor were analyzed. Jurkat-TIGIT cells express secreted embryonic alkaline phosphatase (ESP) under the control of the ISG54 minimal promoter with five IFN-stimulated response elements. The secreted embryonic alkaline phosphatase (SEAP) reporter gene is also expressed. .

[0178] The assay includes two cell lines representing T effector cells (Jurkat-TIGIT) and antigen-presenting cells. T effector cells express luciferases that are activated downstream of the T cell receptor (TCR). Antigen-presenting cells (artificial antigen-presenting cells, or "aAPCs") stably express a ferase reporter gene that binds and activates T effector cells, such as Jurkat-TIGIT, in an antigen-independent manner. The aAPC cells were engineered to stably express T cell activation protein (T cell activator protein 1 (T cell activator protein 2 ... When T effector cells are co-cultured with their aAPC counterparts, the interaction of TIGIT with CD155 inhibits T effector cell activation and reduces luciferase expression. Addition of an anti-TIGIT blocking antibody allows expression of luciferase activity. An inhibitory signal is released.

[0179] In this assay, 16,000 aAPCs expressing human CD155 and T cell activation protein (TCR Activator CD155 CHO-K1) (Promega) were seeded onto 96-well half-area plates and incubated for 37 h. Incubate for 24 hours at 37 °C in a 5% CO2 incubator. The next day, start with 10 μg / ml. 50,000 human Jurkat-TIGIT cells were co-cultured with TCR activator CD155 CHO-K1 cells for an additional 24 hours in the absence or presence of HuTIG1-IgG1.AA or HuTIG3-IgG1.AA using a two-fold dilution series. The supernatant was collected and the secreted luciferase was measured as relative luminescence units using QUANTI-Luc (Invivogen) and a multimode plate reader (Perkin Elmer EnSpire). To determine the fold change in relative light units, the relative light units of Jurkat-TIGIT co-cultured with TCR activator CD155CHO-K1 in the presence of various concentrations of anti-TIGIT antibodies were measured using a fold change ratio of 1:1 with the non-antibody concentration. Thus, by way of example, a 2-fold increase (e.g., as detected in the presence of anti-TIGIT) would indicate that the relative light output measured in the experiment was greater than that measured in the presence of a TCR activator alone. This means that the amount is twice that found in the stimulated control condition.

[0180] As shown in FIG. 23, both HuTIG1-IgG1.AA and HuTIG3-IgG1.AA increased intrinsic T cell activation signaling by approximately 1.5-2.5 fold increase as determined by the increase in relative luminescence units induced in the presence of 0.6 μg / ml to 10 μg / ml of each blocking TIGIT antibody. These data demonstrate that anti-TIGIT antibodies have antagonist activity by blocking the function of TIGIT and increase innate T cell activity.

[0181] [Example 14] Competitive binding with TIGIT Using flow cytometry, we identified antibodies that compete with TIG1 for binding to human TIGIT. The Pierce FITC Antibody Labeling Kit (Thermo Fisher Scientific) was used according to the manufacturer's instructions. Label TIG1 with the fluorescent dye FITC using the protocol described in. The resulting binding of FITC-labeled TIG1 is investigated by flow cytometry using NS0-hTIGIT cells, co-cultured with various concentrations of FITC-labeled TIG1 in 200 μl of FACS buffer (PBS containing 0.5% bovine serum albumin and 0.05% sodium azide), starting at 10 μg / ml, in two-fold serial dilutions. Incubate 100,000 NS0-hTIGIT cells for 30 min at 4 °C in 2 ml of FACS buffer. After washing with , NS0-hTIGIT cells were suspended in 0.2 ml of FACS buffer and subjected to flow cytometry using a FACScan flow cytometer (BD Biosciences, San Jose, CA) and incubated at 4°C for 1 h at 20 ... Obtain mean channel fluorescence (MCF) at 100 nm. Determine the subsaturating concentration for FITC-labeled TIG1 at which 90% of the maximal fluorescence level is achieved.

[0182] A subsaturating concentration of FITC-labeled TIG1 was incubated with 100,000 NS0-hTIGIT cells in 200 μl of FACS buffer. Incubate in the presence (or absence) of a 200-fold higher concentration of test antibody for 30 minutes at 4°C in a 5% CO2 buffer. For example, if 0.1 μg / ml of FITC-labeled TIG1 is used to bind to NS0-hTIGIT cells, add 20 μg / ml of test antibody to the cell suspension. As a background control, incubate 100,000 NS0-hTIGIT cells without any antibody. After washing with 2 ml of FACS buffer, NS0-hTIGIT cells are suspended in 0.2 ml of FACS buffer and subjected to flow cytometry analysis. .

[0183] The MCF [MCFA] of NS0-hTIGIT cells incubated with FITC-labeled TIG1 was normalized to 100%. The MCF of NS0-hTIGIT cells incubated without antibody [MCF B] is normalized to 0%. The MCF of NS0-hTIGIT cells incubated with FITC-labeled TIG1 and test antibody [MCFC] is calculated by the following formula: Normalize by: 100x([MCF C]-[MCF B]) / ([MCF A]-[MCF B]). FITC-labeled TIG1 and test antibodies A test antibody is determined to compete with TIG1 for binding to human TIGIT if the normalized MCF of NS0-hTIGIT cells incubated with is less than 20%.

[0184] The same assay can be used to identify antibodies that compete with TIG2 or TIG3.

[0185] [Example 15] Affinity measurement Antigen-binding affinity of monoclonal antibodies can be determined by label-free optical surface plasmon resonance (SPR) bioassays. Sensors, e.g., BiacoreT100 (GE Healthcare Life Sciences, Marlborough, MA), Pr oteOnXPR36 (BioRad, Hercules, CA), Octet RED384 (ForteBio, Menlo Park, CA) and IBI S MX96 (Wasatch Microfluidics, Salt Lake City, UT) (Yang (Anal. Biochem. 508:78-96, 2016). Antigen binding affinity for each of TIG1, TIG2, and TIG3 is measured as described. Protein A / G (Thermo Fisher Scientific) is immobilized on the flow cell of a CM5 sensor chip (GE Healthcare Life Sciences) using a standard binding protocol. Test antibodies (TIG1, TIG2, or TIG3) are captured by Protein A / G on the CM5 sensor chip. Various concentrations of Recombinant human TIGIT protein, e.g., His-tagged recombinant human TIGIT (Creative BioMatrix) A 100-milliliter (RT, Shirley, NY) was used in the flow for binding of the test antibodies on the sensor chip. BIAevaluation software (GE Healthcare Life Sciences) was used to measure the binding of the test antibodies. The association rate (ka) and dissociation rate (kd) of the interaction between the antibody and human TIGIT were obtained. 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) is calculated by dividing the dissociation rate (kd) by the association rate (ka).

[0186] The BioRad ProteOn XPR36 system was used to measure the concentration of 10 mM HEPES, 150 mM NaCl, pH 7.4, 0.005% Tw Biosensor studies were performed on HuTIG1-IgG1.AA in een-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 GE anti-human IgG antibody ~10,000 RU. His-tagged soluble recombinant human TIGIT (catalog no. TIT-H52H3; AcroBiosystems, Newark, DE) was tested in a 3-fold dilution series starting at 100 nM as the highest concentration down to 1.2 nM. Local Rmax was used to measure the different densities. A global fit to the data from three antibody surfaces was performed to a 1:1 interaction model. The resulting association rate (ka) and dissociation rate (kd) were (4.42±0.02)×10, respectively. 5 M -1 s -1 and (4.09±0.02)x10 -4 s -1 The calculated dissociation constant (Kd) is 925±7 pM.

[0187] [Example 16] Epitope mapping Localizing the epitopes recognized by TIG1, TIG2, and TIG3 in the TIGIT molecule For this purpose, overlap extension PCR method (Higuchi, R., 1989, "PCR Technology: Principles and Applications for DNA Amplification", Erlich, HA, ed., Stockton Press, New York, Single amino acid substitution mutants were generated in the extracellular domain of TIGIT (SEQ ID NO: 3) by site-directed mutagenesis using the ELISA kit (Kan. 1999, NY, pp. 61-70). The following six mutants in the extracellular domain of TIGIT were used in the assay: 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 of each mutant name and the number in the middle indicate the number of mutants. The letters on the right side of the figure indicate the amino acid residues in wild-type TIGIT, the positions counting from the N-terminal methionine residue in the extracellular domain of TIGT (SEQ ID NO: 3), and the amino acids in the mutants. Each of these six mutants has an amino acid substitution near the interface of the TIGIT-CD155 complex (Stengel, Proc. Natl. Acad. Sci. USA , 109:5399-5404, 2012).

[0188] The mammalian expression vector pFCm179 contains (i) the puromycin N-acetyltransferase gene fused to the E. coli xanthine guanine phosphoribosyltransferase gene pFCm404 (Figure 1) has the same structure as pFCm404, except that (i) the extracellular domain of human TIGIT is replaced by human IL-15, and (ii) the extracellular domain of human TIGIT is replaced by human IL-15. DNA fragments encoding the six alanine substitution mutants of the extracellular domain of human TIGIT were individually introduced 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, carry the Q35A, N37A, Q39A, N49A, D51A and F86A mutants in the TIGIT coding region, respectively, and express a single nucleotide polymorphism specific for each mutant. Except for the amino acid substitutions, the TIGIT-Fc fusion protein sequence is the same as that encoded by pFCm404. Express the column.

[0189] 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 in a 7.5% CO2 incubator at 37°C. The polyethylenimine method (Durocher et al., Nucl. Acids Res. 30:e9, HEK293 cells were individually transfected with expression vectors (pFCm404, pFCm179-Q35A, pFCm179-N37A, pFCm179-Q39A, pFCm179-N49A, pFCm179-D51A and pFCm179-F86A) of wild-type and mutant TIGIT-Fc fusion proteins using the ELISA kit (Kanagawa et al., 2002). The production levels of TIGIT-Fc fusion proteins in the culture supernatants were measured by ELISA. Briefly, ELISA plates were stained with 100 μl / well of goat anti-human IgG Fc specific polyclonal antibody (Sigma-Aldrich) diluted 1 / 2,000 in PBS. Coat overnight at 4°C, wash with wash buffer, and then with 200 μl / well of ELISA buffer for 20 min. After washing with washing buffer, the culture supernatant of transiently transfected HEK293 cells, appropriately diluted in ELISA buffer (100 μl / well), was applied to the ELISA plate. Purified hTIGIT-Fc was used as a standard. The ELISA plate was incubated for 30 min at room temperature, washed with wash buffer, and then ELISA was performed using 100 μl / well of HRP-conjugated goat anti-human IgG polyclonal antibody (SouthernBiotech) diluted 1 / 1,2000 in ELISA buffer. Bound Fc fusion proteins were detected. Plates were incubated for 30 min at room temperature and washed with wash buffer, after which color development was initiated by the addition of 100 μl / well ABTS substrate (Sigma-Aldrich) and stopped with 100 μl / well 2% oxalic acid. Absorbance was measured at 405 nm.

[0190] Binding of each of the mouse TIG1, TIG2 and TIG3 antibodies to the TIGIT mutants was examined by ELISA. ELISA plates were filled with 100 μl / well of goat anti-human IgG Fc specific, diluted 1 / 2,000 in PBS. The cells were coated with polyclonal antibody (Sigma-Aldrich) overnight at 4°C, washed with washing buffer, and blocked with 200 μl / well of ELISA buffer for 20 min at room temperature. After washing with washing buffer, cells were incubated with either wild-type TIGIT or six TIGIT mutants in ELISA buffer (100 μl / well). 0.5 μg / ml of transiently expressed TIGIT-Fc fusion protein with one of the following was applied in duplicate to the ELISA plate for incubation at room temperature for 30 min. As a negative control For the assay, culture supernatants of non-transfected HEK293 cells were used. After washing with wash buffer, 100 μl / well of test antibody (TIG1, TIG2 or TIG3) at 200 ng / ml in ELISA buffer (100 μl / well) was applied. The ELISA plate was incubated for 30 min at room temperature, washed with wash buffer, and then binding was performed using 100 μl / well of HRP-conjugated goat anti-mouse kappa chain polyclonal antibody (Bethyl Laboratories, Montgomery, TX) diluted 1 / 2,000 in ELISA buffer. The combined antibodies were detected. The plates were incubated for 0.5 hours at room temperature and washed with washing buffer. Afterwards, color development was initiated by the addition of 100 μl / well ABTS substrate (Sigma-Aldrich) and 100 μl / well The wells were stopped with 2% oxalic acid and the absorbance was measured at 405 nm.

[0191] [Table 1]

[0192] For each of TIG1, TIG2, and TIG3, the relative binding to each TIGIT mutant was calculated using the following formula: The average absorbance [AbsA] for the wild-type TIGIT-Fc fusion protein was normalized to 100%. The average absorbance [Abs B] for the cell supernatant of untransfected HEK293 cells was normalized to 0%. The average absorbance [AbsC] for the mutant TIGIT-Fc fusion protein was calculated by the following formula: Normalization was performed: 100x([AbsC]-[Abs B]) / ([Abs A]-[Abs B]). The results are summarized in Table 1. The relative binding was less than 5% when the amino acid residues at positions 35, 37, 49, and 51 were substituted with alanine, indicating that the amino acid residues at these four positions are important for the binding of TIG1 to TIGIT. None of the TIGIT mutants used in this assay abolished the binding of TIG2 and TIG3 to TIGIT.

[0193] [Example 17] Enhanced secretion of IFNγ upon addition of HuTIG1-IgG1.AA, anti-PD-L1 antibody, and HuTIG1-IgG1.AA in combination with anti-PD-L1 antibody Blood was layered using density gradient medium (Lymphoprep; STEMCELL; 07801) and, after centrifugation, the PBM Human PBMCs were first isolated from the buffy coat by collecting 100% PBS from the interphase. In the next step, human CD14 cells were isolated from PBMCs by positive selection for CD14 (EasySep Human CD14 Positive Selection kit; STEMCELL; Cat. no. 18058) according to the manufacturer's protocol. Monocytes were isolated and cultured for 6 days in RPMI medium supplemented with 5% FBS, 0.1 μg / ml GM-CSF (R&D; Catalog No. 215-GM-050 / CF) and 0.1 μg / ml IL-4 (R&D; Catalog No. 204-IL-050 / CF). Differentiated monocyte-derived dendritic cells (moDCs) were harvested and analyzed by flow cytometry for the expression of CD155, CD112 and PD-L1. This was checked by cytometry (Figure 24).

[0194] Human CD4+ cells from four different donors were negatively depleted. tion) (RosetteSephuman CD4 T cell enrichment cocktail; STEMCELL; #15062) Cells were isolated from the fawn coat and purity was determined using flow cytometry.

[0195] moDCs and CD4+ cells were combined at a 1:4 ratio (25,000 moDCs, 100,000 CD4+ cells) for 4 days. , HuTIG1-IgG1.AA (5 μg / ml or or 15 μg / ml), or anti-PD-L1 antibody (10 μg / ml; clone 243.55.S1; SEQ ID NOs: 56 and 57), or were cultured for 4 days in the presence of both HuTIG1-IgG1.AA (15 μg / ml) and anti-PD-L1 antibody (10 μg / ml), and then analyzed using Cytometric BeadArray (CBA; Human IFNγFlex Set; BD; Cat. No. 560111). IFNγ was quantified. Human IgG1Fc (25 μg / ml; BioXcell; Cat. No. BE0096) was used as a control. Used.

[0196] As shown in Figure 25, HuTIG1-IgG1.AA and anti-PD-L1 antibody blockade independently increased IFNγ production compared to the appropriate isotype control antibody. The antibody combination significantly and synergistically increased IFNγ production compared to either monotherapy alone This has been increased to.

[0197] [Example 18] HuTIG1-IgG1.AA and HuTIG3-IgG1.AA detect TIGIT expressed on human lymphoid and myeloid cells Total human PBMCs isolated from 10 donors were co-stained with directly conjugated antibodies to visualize T cell, B cell, NK cell and myeloid lineage subsets. Dead cells were excluded using the ua Dead CellStain kit. Conjugated to Alexa647 dye. The expression of TIGIT was determined using the HuTIG1-IgG1.AA and HuTIG3-IgG1.AA antibodies. CD96 expression was determined using an anti-CD96 antibody (clone NK92.39). In Figure 26A, plus (+) signs indicate expression above background and (+++) represents the highest expression observed. Plus / minus (+ / -) signs indicate that only a subset of cells within the designated population express TIGIT or CD96 above background and minus (-) signs indicate a lack of expression above background. In all cases, background was determined using the target antibody (all That is, the fluorescent antibodies used in the staining panel other than HuTIG1-IgG1.AA, HuTIG3-IgG1.AA, and anti-CD96 antibody The total photopigment was determined using a modified Fluorescence Minus One (FMO) method. Alternatively, the target antibody was replaced with an IgG-specific isotype control antibody of the same conjugation. Figure 26B shows representative histograms of anti-TIGIT antibody staining in PBMCs at various expression levels used to assign CD96 expression levels in Figure 26A. Light histograms represent improved FMO. Dark histograms represent HuTIG1-IgG1.AA or HuTIG3-IgG1.AA staining.

[0198] Dissociated tumor cells from melanoma, colorectal cancer, non-small cell lung cancer, and renal clear cell carcinoma were purchased from commercial sources and stained with directly conjugated antibodies to identify T cell and antigen-presenting cell subpopulations. The subsets were visualized. Leukocytes were identified using a pan-isoform anti-CD45 antibody (clone HI30). Dead cells were excluded using the Live / deadFixable Aqua DeadCell Stain kit. The expression of TIGIT was determined using the HuTIG1-IgG1.AA antibody conjugated to Alexa647 dye. In all cases, background was measured using a modified FluorescenceMinusOne (FMM), which adds all fluorochromes used in the staining panel except the target antibody (i.e., HuTIG1-IgG1.AA). The expression levels of HuTIG1-IgG1.AA were determined using the FMO (Improved FMO) method. Rather, the target antibody was replaced with an IgG-specific isotype control antibody of the same conjugation. Figure 32B shows representative histograms of anti-TIGIT staining for tumor infiltrating lymphocytes (TILs) at various expression levels shown in Figure 32A. The light histograms represent the improved FMO. The dark histograms represent the HuTIG1-IgG1.AA staining.

[0199] [Example 19] Epitope mapping of anti-human TIGIT antibody HuTIG1-IgG1.AA by flow cytometry Human TIGIT cDNA was inserted into pcDNA3.1(+), and the 21-residue native signal peptide was inserted followed by the mature TIGIT gene. The protein (SEQ ID NO: 58) was cloned to have a 9-residue hemagglutinin (HA) peptide tag followed by a 10-residue peptide linker in front of the N-terminus of the protein. Targeted amino acid residues (T34, Q35, N37, E39, L44, I47, N49, D51, L52, H55, F86, I88, H90, Y92, and T96) were mutated to alanine, one amino acid at a time. Plasmids were purified and transiently transfected into CHO-K1 cells using Fugene6 (Promega) transfection reagent according to the manufacturer's recommendations. Expression of HA-tagged human TIGIT mutants was detected with anti-HA antibody. This was confirmed by.

[0200] For flow cytometric analysis of anti-hTIGIT antibody binding epitopes, HA-tagged human TIGIT proteins with or without single amino acid mutations were transiently transfected into CHO-K1 cells. After 24 hours, the transfected cells were incubated in HBSS buffer (ThermoFisher S The cells were suspended in 100% PBS (Chem. Sci., Cat. No. 14175095) and incubated with various concentrations of HuTIG1-IgG1.AA antibody in a volume of 50 μL. After 1 h of incubation at 4°C, the cells were washed and , 50 μL of Alexa488-conjugated anti-human IgG (H+L) secondary antibody (ThermoFisher Scientific, A second incubation with IgG1 (catalog no. A-11013) was performed to isolate antibody-bound cells. Cells were incubated with anti-HA antibody (Sigma, Cat. No. H7411) as a positive control, and non-transfected CHO-K1 was used as a negative control. Cells were analyzed on an AttuneNxT cytometer (ThermoFisher Scientific) and analyzed using FlowJo software. For each mutant, the saturating antibody binding MFI (mean fluorescence intensity) was calculated as 100% POC ( The MFI of the non-primary antibody-bound control was normalized to 0% POC. The data was then fitted using GraphPad Prism 7 curve fitting using the following equation: The linear regression was performed by fitting a nonlinear sigmoid curve: Y = lower limit + (upper limit - lower limit) / (1 + 10 (LogEC 50 -X)×Hill勾配) ).

[0201] In this assay, HuTIG1-IgG1.AA inhibited EC 50 = 1.28 nM to wild-type human TIGIT. Five of the 15 single alanine TIGIT mutants bound to HuTIG1-IgG1.AA with greater than a 10-fold reduction in binding affinity. Human TIGIT with N49A, D51A, H90A, N37A or Q35A mutations (SEQ ID NO: 1) No. 3) are, respectively, EC 50 All of these residues are located in one of the β-sheet frontal regions of the TIGITIgV extracellular domain. They are located together in successive sites (Figure 28).

[0202] [Example 20] Ex vivo immunophenotyping of T cell subsets from whole blood of non-human primates administered HuTIG1-IgG1.AA Type Checking Pharmacokinetic studies were performed by Charles River Laboratories (Reno, NV). The objectives of this study were For direct assessment of TIGIT receptor occupancy, 10 mg / kg of anti-TIGIT (HuTIG1-IgG1.AA) antibody was administered. Whole blood samples from cynomolgus monkeys treated with a single dose were to be investigated ex vivo.

[0203] Three experimentally naive female cynomolgus monkeys (#67, #68, and #69) were administered a single intravenous dose of 10 mg / kg of HuTIG1-IgG1.AA antibody. They received whole blood in sodium heparin samples within 24 hours of the last sampling time point, corresponding to pre-dose, 1, 2, 7, 14, 21, and 28 days post-dose. For whole blood flow cytometry studies, fluorescently labeled anti-human TIGIT (clone MBSA43) from BioLegend was used to detect the expression of TIGIT on cynomolgus blood lymphocyte cell subsets. Expression levels were determined (as anti-human TIGHT has been shown to cross-react with non-human primates) (PLoSPathog. 2016 Jan;12(1);BioLegend). Live cells were acquired by FACSCalibur. , and analyzed using FlowJo software.

[0204] Anti-TIGIT (MBSA43) inhibited CD4+ and CD8+ T cell subsets upon staining of pre-injection whole blood samples. After administration, anti-TIGIT (MBSA43) was able to bind to both of the different subsets examined. TIGIT expression could not be detected on these T cell subsets by day 14 or day 21, depending on the group, with levels returning to pre-treatment detection levels by day 21 or day 28, depending on the different animal examined (Figures 29B and 30B). This inability to detect TIGIT expression was consistent with no significant changes in CD4+ or CD8+ T cell frequencies suggesting that surface TIGIT was occupied by the treatment antibody (HuTIG1-IgG1.AA). This was not due to T cell subset depletion (Figures 29A and 30A, respectively).

[0205] These data indicate that the anti-TIGIT antibody HuTIG1-IgG1.AA is cross-reactive with cynomolgus monkeys. and anti-human TIGIT (clone MBSA43) from BioLegend. Supports that body occupancy assessment be provided.

[0206] [Example 21] Enhancement of NK cell-mediated cytotoxicity by HuTIG1-IgG1.AA and HuTIG3-IgG1.AA against 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 was important to screen donor PBMCs with high NK cell frequency, sufficient TIGIT surface expression, and sufficient CD226 expression. A peripheral blood LeukoPak (consisting of over 10 billion cells) was obtained and separated using a Miltenyi autoMACSPro Separator according to the manufacturer's instructions (Miltenyi Biotec, Cat. No. 13). NK cell isolation was performed using the same method as described in the 0-092-657 (in Japanese). The purified NK cells were resuspended in medium containing IL-2 [200 units / ml]. The plate was placed in a 5% CO2 incubator at 37°C for 24 hours, after which the wells were harvested. Natural cytotoxicity studies were performed with the cells.

[0207] Lysis of K562 mediated by purified NK cells was measured with or without anti-TIGIT antibody (TIG1) NK cells were cultured in RPMI1640 medium (Invitrogen) at 37°C in a 5% CO2 incubator. The cells were cultured overnight in the presence of IL-2 (200 units / ml) (Peprotech). were labeled with CFSE and incubated with NK cells at 1:20 ( Target: NK, 1 K562 cell vs. 20 NK cells were co-cultured. K562 cells do not express CD20, so Tuxanthin was used as a negative control. After the incubation period, cells were harvested and stained with 5 nM SytoxRed (ThermoFisher) to distinguish dead target cells (CFSE+Sytox Red+). The percentage of K562 target cells lysed by NK cells was determined by flow cytometry (FACSCalibur; FlowJoAnalysis).

[0208] As shown in FIG. 31, when purified NK cells were used, the addition of anti-TIGIT antibody reduced the expression of K562 The anti-TIGIT antibodies (HuTIG1-IgG1.AA or HuTIG3-IgG1.AA) have the ability to increase the level of target cell killing by a subset of human effector cells (by 28% or 19%, respectively, relative to "no antibody" or Rituxan normalized percentages). This was intended to prove that there is such a thing. The present invention includes the following embodiments. [1] 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. antibody. [2] An anti-TIGIT antibody described in embodiment 1, which is a monoclonal antibody. [3] The anti-TIGIT antibody according to embodiment 1 or embodiment 2, which is a chimeric, humanized or veneered antibody. antibody. [4] An anti-TIGIT antibody described in embodiment 1, which is a human antibody. [5] An anti-TIGIT antibody described in any of embodiments 1 to 4, which does not bind to one or more amino acid residues including L44, I47, or H55. [6] An anti-TIGIT antibody described in any one of embodiments 1 to 5, comprising a mature light chain variable region of SEQ ID NO: 14 and a mature heavy chain variable region of SEQ ID NO: 10. [7] A method for the production of a nucleotide sequence corresponding to SEQ ID NO: 1, which binds to the same epitope as TIG1, The anti-TIGIT antibody according to any one of aspects 1 to 6. [8] An anti-TIGIT antibody described in any of embodiments 1 to 7, comprising three light chain CDRs comprising SEQ ID NOs: 15 to 17, and three heavy chain CDRs comprising SEQ ID NOs: 11 to 13. [9] A mature heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 35, and Any of embodiments 1 to 7, comprising a mature light chain variable region having at least 90% sequence identity to SEQ ID NO:37. The anti-TIGIT antibody according to any one of the preceding claims.

[10] An anti-TIGIT antibody described in embodiment 9, wherein the mature heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 35 and the mature light chain variable region comprises the amino acid sequence of SEQ ID NO: 37.

[11] The mature heavy chain variable region may or may not have a C-terminal lysine. 40, and the mature light chain variable region is linked to a light chain constant region comprising SEQ ID NO: 41, with the proviso that:

[12] A monoclonal antibody that competes with either TIG1, TIG2, or TIG3 for binding to human TIGIT. and for specific binding to CD155, the antibody TIG1 is a mature IgG1 antibody of SEQ ID NO: 14. antibody TIG1 characterized by a mature light chain variable region of SEQ ID NO: 22 and a mature heavy chain variable region of SEQ ID NO: 18; and antibody TIG3 characterized by a mature light chain variable region of SEQ ID NO: 30 and a mature heavy chain variable region of SEQ ID NO: 26.

[13] It binds to the same epitope on human TIGIT as either TIG1, TIG2 or TIG3. 13. A monoclonal antibody according to embodiment 12.

[14] The antibody of any one of embodiments 1 to 13, which inhibits binding of CD155 to human TIGIT. .

[15] Corresponding to the three light chain CDRs and the three heavy chain CDRs of either TIG1, TIG2 or TIG3 13. The monoclonal antibody of embodiment 12, comprising three light chain CDRs and three heavy chain CDRs.

[16] A recombinant human TIG1 antibody that contains three heavy chain CDRs and three light chain CDRs from either TIG1, TIG2, or TIG3. 13. The monoclonal antibody of embodiment 12.

[17] TIG1 (light chains of SEQ ID NOs: 15 to 17 and heavy chains of SEQ ID NOs: 11 to 13), TIG2 (sequence numbers 23 to 25) 13. The monoclonal antibody according to embodiment 12, comprising the three heavy chain CDRs defined by Kabat and the three light chain CDRs defined by Kabat of either TIG3 (light chain of SEQ ID NOs: 19 to 21 and heavy chain of SEQ ID NOs: 31 to 33 and heavy chain of SEQ ID NOs: 27 to 29).

[18] A monoclonal antibody described in any one of embodiments 12 to 17, which is a chimeric, humanized or veneered antibody.

[19] The monoclonal antibody of embodiment 12, which is a human antibody.

[20] A monoclonal antibody described in any one of embodiments 12 to 19, having a human IgG1 kappa isotype.

[21] A monoclonal antibody described in any one of embodiments 12 to 20, which is an intact antibody.

[22] A monoclonal antibody described in any of embodiments 12 to 20, which is a single chain antibody, Fab or F(ab')2 fragment.

[23] A mature heavy chain variable region having at least 90% sequence identity to SEQ ID NO: 35, and 37. 13. A monoclonal antibody according to any one of claims 1 to 12.

[24] A mature heavy chain variable region having at least 95% sequence identity with SEQ ID NO: 35, 24. The method of claim 23, wherein the chain variable region has at least 95% sequence identity to SEQ ID NO: 37. Monoclonal antibodies.

[25] The monoclonal antibody of embodiment 24, wherein 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.

[26] The mature heavy chain variable region may or may not have a C-terminal lysine.

[27] The monoclonal antibody of embodiment 25, wherein 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, with the proviso that a C-terminal lysine may or may not be present.

[28] The monoclonal antibody of embodiment 25, wherein 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.

[29] The monoclonal antibody of embodiment 25, wherein the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 61, and the mature light chain variable region is linked to a light chain constant region comprising SEQ ID NO: 64, provided that the mature heavy chain variable region has at least 90% sequence identity to SEQ ID NO: 43, and 45. 13. A monoclonal antibody according to any one of claims 1 to 12.

[30] A mature heavy chain variable region having at least 95% sequence identity to SEQ ID NO: 43, 30. The method of claim 29, wherein the chain variable region has at least 95% sequence identity to SEQ ID NO: 45. Monoclonal antibodies.

[31] The monoclonal antibody of embodiment 30, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 43 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 45.

[32] The mature heavy chain variable region may or may not have a C-terminal lysine.

[33] The monoclonal antibody of embodiment 31, wherein the mature heavy chain variable region is linked to a heavy chain constant region comprising 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 linked to a light chain constant region comprising SEQ ID NO: 49.

[34] The monoclonal antibody of embodiment 31, wherein the mature heavy chain variable region is linked to a heavy chain constant region comprising SEQ ID NO: 62, 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: 65, with the proviso that a C-terminal lysine may or may not be present. 63, and the mature light chain variable region is linked to a light chain constant region comprising SEQ ID NO: 65, provided that the mature light chain variable region is linked to an epitope comprising residues 35 and 37 of SEQ ID NO: 1 and / or residues 49 and 51 of SEQ ID NO: 1. A monoclonal antibody that binds to the antibody.

[36] A monoclonal antibody described in embodiment 35, which binds to an epitope including residues 35, 37, 49 and 51 of SEQ ID NO:1.

[37] Residues 35 to 51 of SEQ ID NO:1, and up to 5 adjacent residues from SEQ ID NO:1 on either side. 36. The monoclonal antibody of embodiment 35, which binds to a peptide consisting of amino acids.

[38] The monoclonal antibody according to embodiment 37, which binds to a peptide consisting of residues 35 to 51 of SEQ ID NO:1. Clonal antibodies.

[39] The monoclonal antibody described in embodiment 35, wherein the epitope consists of 3 to 20 consecutive residues of SEQ ID NO:1.

[40] It has the following properties: (a) it inhibits the binding of TIGIT to CD155, optionally with an IC50 of 15-100 ng / ml; and (b) it inhibits innate T cell activation in the presence of antigen-presenting cells expressing CD155, as measured by IL-2 production. (c) measuring antigen-specific T cell activation by IL-12 production; (d) increasing natural killer cell activation as measured by production of either IL-2, IL-6, TNFα or IFNγ, optionally by 1.5-3 fold; (e) increasing T cell production of at least one proinflammatory cytokine, optionally by 1.5-3 fold; (f) increasing at least 40. The monoclonal antibody according to any one of embodiments 12 to 39, further comprising one or more of the following:

[41] A method for treating a subject comprising administering to the subject the antibody of any one of claims 1 to 40 and a pharma- ceutical acceptable carrier. Pharmaceutical compositions.

[42] A method for treating cancer or preventing cancer, comprising administering to a subject having or at risk of cancer an effective regimen or treatment of an antibody according to any one of embodiments 1 to 40. administering an effective amount.

[43] The method of embodiment 42, wherein the cancer is acute myeloid leukemia or adult T-cell leukemia. Law.

[44] The method of embodiment 42 or 43, wherein the subject is administered antibody-activated tumor-infiltrating T cells. 44. The method of embodiment 43.

[45] The method of any one of embodiments 42-44, wherein the subject is administered a vaccine that induces an antibody-enhanced immune response against cancer.

[46] The method of embodiment 45, wherein the vaccine comprises an antigen or a fragment thereof expressed on the surface of a cancer cell.

[47] The method of any one of embodiments 42-46, wherein the subject is administered natural killer cells whose cytotoxic activity against the cancer is enhanced by the antibody.

[48] ​​The method of any of embodiments 42 to 47, wherein the subject is further administered a secondary antibody against an antigen expressed on the surface of the cancer cell, whereby the effector-mediated cytotoxicity of the secondary antibody against the cancer is enhanced by the antibody.

[49] The method of any one of embodiments 42 to 47, wherein the subject is further administered a secondary antibody against an antigen expressed on the surface of the immune cell.

[50] The method of embodiment 49, wherein the immune cell is a T cell or a natural killer cell. .

[51] The method of embodiment 49 or embodiment 50, wherein the antigen is CTLA-4, PD-1, or PD-L1. Law.

[52] The method of any one of embodiments 42 to 51, wherein the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation therapy, cell therapy, and surgery.

[53] The subject is further treated with an inhibitor of one or more immune checkpoint receptors or ligands. The method according to any one of embodiments 42 to 52, wherein the

[54] The method of embodiment 53, wherein the inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab (lambrolizumab), and atezolizumab.

[55] A method for treating a subject infected with a pathogen, comprising the steps of any one of embodiments 1 to 40. The method comprises administering to a subject an effective regimen or a therapeutically effective amount of the antibody described above.

[56] The method of embodiment 55, wherein the pathogen is a virus, bacterium, fungus or protozoan.

[57] Pathogens include HIV, SIV, hepatitis, herpesvirus, adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, Kozak virus, coronavirus (cornovirus), respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arbovirus encephalitis virus, chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci, gonococci, klebsiella, proteus, serratia, pseudomonas, and respirators. 57. The method of embodiment 56, wherein the bacterium is O. gonorrhea, diphtheria, salmonella, bacillus, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme borreliosis.

[58] The method of any one of embodiments 55-57, wherein the subject is treated with a vaccine that induces an antibody-enhanced immune response against a pathogen.

[59] The method of embodiment 58, wherein the vaccine comprises a pathogen protein or a fragment thereof.

[60] The method of any of embodiments 55 to 59, wherein a secondary antibody against the pathogen is further administered to the subject, and the antibody enhances the effector-mediated cytotoxicity of the secondary antibody against the pathogen.

[61] The subject is further treated with one or more of an antiviral, antiparasitic, antibacterial, or antifungal agent. The method according to any one of embodiments 55 to 60, wherein the subject is administered

[62] A method for assisting in the treatment of cancer, comprising administering to a subject having cancer any one of embodiments 1 to 40. A method comprising administering a therapeutically effective amount of the antibody described herein.

[63] The method of embodiment 62, wherein the cancer is acute myeloid leukemia or adult T-cell leukemia. Law.

[64] The method of embodiment 62 or 63, wherein the subject is administered antibody-activated tumor-infiltrating T cells. 64. The method of embodiment 63.

[65] The method of any one of embodiments 62-64, wherein the subject is administered a vaccine that induces an antibody-enhanced immune response against cancer.

[66] The method of embodiment 65, wherein the vaccine comprises an antigen or a fragment thereof expressed on the surface of a cancer cell.

[67] The method of any one of embodiments 62-66, wherein the subject is administered natural killer cells whose cytotoxic activity against the cancer is enhanced by the antibody.

[68] The method of any of embodiments 62-67, wherein the subject is administered a secondary antibody against an antigen expressed on the surface of a cancer cell, whereby the effector-mediated cytotoxicity of the secondary antibody against the cancer is enhanced by the antibody.

[69] The method of any one of embodiments 62 to 67, wherein the subject is further administered a secondary antibody against an antigen expressed on the surface of an immune cell.

[70] The method of embodiment 69, wherein the immune cell is a T cell or a natural killer cell. .

[71] The method of embodiment 69 or embodiment 70, wherein the antigen is CTLA-4, PD-1, or PD-L1. Law.

[72] The method of any one of embodiments 62 to 71, wherein the subject is further administered one or more therapies selected from the group consisting of chemotherapy, radiation therapy, cell therapy, and surgery.

[73] The subject is further treated with an inhibitor of one or more immune checkpoint receptors or ligands. The method according to any one of embodiments 62 to 72, wherein the

[74] The method of embodiment 73, wherein the one or more immune checkpoint receptors or ligands are selected from the group consisting of CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, BTLA, VISTA, CD96, A2aR, A2bR, arginase, CD39, CD73, IDO, and TDO.

[75] The method of embodiment 74, wherein the inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab (lambrolizumab), and atezolizumab.

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Claims

[Claim 1] The invention as depicted in the drawings.

Images