Anti-tigit antibodies

Abstract

Provided are anti-TIGIT antibodies, and antigen binding fragments thereof, that inhibit TIGIT-mediated signaling, as well as combinations comprising the antibodies or antigen binding fragments thereof, and methods of their use.

Description

[0001] This application is a divisional application of a patent application with application number 201880048693.8, application date July 26, 2018, and name “Anti-TIGIT Antibody”. Background Art

[0002] Cancer immunotherapy relies on the regulation of the immune system to improve the recognition and response to tumor cells. This regulation can be achieved through a variety of mechanisms including co-stimulatory molecules present on activated immune cells or via inhibition of co-inhibitory receptors. The activation of the immune response is a complex mechanism involving many cell populations, such as antigen presenting cells that are important for initiating antigen-specific responses and effector cells responsible for destroying tumor cells. There are many mechanisms for regulating the activity of effector cells such as cytotoxic T cells and represent targets of choice in the context of cancer immunotherapy.

[0003] TIGIT (T cell immunoreceptor with Ig and ITIM domains), also known as WUCAM, VSIG9 or Vstm3, is a co-inhibitory receptor preferentially expressed on NK, CD8+ and CD4+ T cells and regulatory T cells (Treg cells, or simply "Treg"). TIGIT is a transmembrane protein that contains the known ITIM domain in its intracellular portion, a transmembrane domain, and an immunoglobulin variable domain on the extracellular portion of the receptor. Several ligands have been described that bind to the TIGIT receptor, of which CD155 / PVR shows the best affinity, followed by CD113 / PVRL3 and CD112 / PVRL2 (Yu et al. (2009) Nat. Immunol. 10:48). DNAM / CD226, a known co-stimulatory receptor also expressed on NK and T cells, competes with TIGIT for binding to CD155 and CD112 but with lower affinity, suggesting that tight control of activation of these effector cells could avoid uncontrolled cytotoxicity against normal cells expressing the CD155 ligand.

[0004] TIGIT expression is increased on tumor infiltrating lymphocytes (TILs) and in disease settings such as HIV infection. Compared with TIGIT-negative counterparts, TIGIT expression marks exhausted T cells with lower effector function (Kurtulus et al. (2015) J. Clin. Invest. 276: 112; Chew et al. (2016) Plos Pathogens. 12). In contrast, compared with TIGIT-negative Treg populations, Treg cells expressing TIGIT show enhanced immunosuppressive activity (Joller et al. (2014) Immunity. 40: 569).

[0005] Like other co-inhibitory receptors expressed on T cells (PD1 or CTLA4) that have been shown to be relevant targets for immunotherapy and whose antagonistic antibodies have been approved for the treatment of human cancer, the development of antagonistic anti-TIGIT antibodies can help turn on the immune system and better fight cancer cells. It has been suggested that antagonistic anti-TIGIT antibodies, either as monotherapy or in combination with a-PD1 antibodies, can achieve strong anti-tumor efficacy in preclinical models (Johnston et al. (2014) Cancer Cell 26:1; WO2016 / 028656; US2016 / 0176963; US2016 / 0376365, all of which are incorporated herein by reference).

[0006] Therefore, antagonistic antibodies specific for TIGIT that can inhibit TIGIT receptor activity provide an opportunity to reduce the immunosuppressive effects associated with the tumor microenvironment and thereby enhance the anti-tumor immune response to tumor cells. Summary of the invention

[0007] The present invention provides anti-TIGIT antibodies that can reduce the immunosuppressive effects of TIGIT-mediated signaling. Specifically, the antibodies or antigen-binding fragments of the present invention can inhibit TIGIT-mediated immunosuppression by preventing ligand binding on T cells (conventional αβT cells and unconventional γδT cells) and NK cells and / or depleting TIGIT-positive Treg cells, and / or by inducing internalization of TIGIT receptors.

[0008] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human TIGIT and comprises a protein selected from Figure 1 A heavy chain variable domain comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the HCDR1, HCDR2 and HCDR3 sequences shown in Figure 2 The light chain variable domains of the light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) of the LCDR1, LCDR2 and LCDR3 sequences shown.

[0009] In certain embodiments, the antibody or antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the combination is selected from Figure 1 The HCDRs of the antibodies in Figure 2 The groups of combinations formed by the LCDRs of the corresponding antibodies.

[0010] In certain embodiments, the antibodies or antigen-binding fragments according to the present invention may comprise a heavy chain variable domain having an amino acid sequence selected from the group consisting of: SEQ ID No: 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 327, 329 and 331, and amino acid sequences that exhibit at least 90%, 95%, 97%, 98% or 99% sequence identity thereto; and optionally a light chain variable domain having an amino acid sequence selected from the group consisting of: SEQ ID The amino acid sequences of Nos:212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 328, 330 and 332, and amino acid sequences that exhibit at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0011] In certain embodiments, the antibody or antigen-binding fragment comprises a combination of a heavy chain variable domain and a light chain variable domain, wherein the combination is selected from Figure 5 The VH of each antibody in the present invention or an amino acid sequence thereof that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity with the ... Figure 5 The VL of the same antibody or a combination thereof formed by an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity.

[0012] The most preferred antibodies and antigen-binding fragments provided herein are those based on the CDRs or complete variable domains of antibody 31282 provided herein.

[0013] As demonstrated herein, these preferred anti-TIGIT antibodies and antigen-binding fragments based on antibody 31282 have particularly surprising and advantageous properties. These properties include: higher affinity for TIGIT expressed on CD8 T cells (from healthy donors or from cancer patients) than each of the previously described tested anti-TIGIT antibodies; IC50 / C155 / PVR competition compared to each of the previously described tested anti-TIGIT antibodies; 50 Better; EC in T cell activation assays compared to each of the previously described anti-TIGIT antibodies tested 50Better; and strongly improve the activity in T cells from the peripheral blood of cancer patients and importantly in tumor infiltrating lymphocytes. In addition, it is surprisingly shown herein that antibodies and antigen-binding fragments according to the present invention, particularly those based on antibody 31282, preferentially deplete Treg cells. That is, Treg cells expressing TIGIT exposed to the provided anti-TIGIT antibodies experience a greater proportion of lysis than conventional CD4 and CD8 T cells. This is surprising because conventional CD4 and CD8 T cells also express TIGIT, but do not experience the same degree of cell lysis when contacted with the antibody. It is also surprisingly shown that antibodies and antigen-binding fragments according to the present invention, particularly those based on antibody 31282, not only promote the pro-inflammatory activity of conventional T cells, but also increase the activity of unconventional γδ T cells.

[0014] Thus, in certain preferred embodiments, provided herein are antibodies or antigen-binding fragments comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein:

[0015] HCDR1 comprises or consists of SEQ ID NO: 16 (YTFTSYYMH),

[0016] HCDR2 comprises or consists of SEQ ID NO: 17 (VIGPSGASTSYAQKFQG),

[0017] HCDR3 comprises or consists of SEQ ID NO: 18 (ARDHSDYWSGIMEV),

[0018] LCDR1 comprises or consists of SEQ ID NO: 61 (RASQSVRSSYLA),

[0019] LCDR2 comprises or consists of SEQ ID NO: 62 (GASSRAT), and

[0020] LCDR3 comprises or consists of SEQ ID NO: 63 (QQYFSPPWT).

[0021] In certain such embodiments, the heavy chain variable domain comprises or consists of an amino acid sequence according to SEQ ID NO: 221 or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto; and the light chain variable domain comprises or consists of an amino acid sequence according to SEQ ID NO: 222 or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0022] In certain preferred embodiments, the anti-TIGIT antibody is antibody 31282 described herein.

[0023] In another aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that cross-competes with an antibody according to the first aspect of the invention (eg, an antibody exemplified herein) for binding to human TIGIT.

[0024] In another aspect, the invention provides an isolated antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody according to the first aspect of the invention (eg, an antibody exemplified herein).

[0025] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to a human TIGIT epitope comprising TIGIT residues Q56 and I109, optionally comprising residues Q56, N58 and I109. In some preferred embodiments, an antibody or antigen-binding fragment thereof that binds to a human TIGIT epitope comprising TIGIT residues Q56, N58, E60, I68, L73, H76 and I109 is provided.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof binds to a human TIGIT epitope consisting of TIGIT residues Q56, N58, E60, I68, L73, H76, and I109.

[0027] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human TIGIT without competing with CD155 for binding to TIGIT.

[0028] In certain embodiments, an antibody or antigen-binding fragment that binds to human TIGIT without competing with CD155 for binding to TIGIT comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or consists of SEQ ID NO: 280, HCDR2 comprises or consists of SEQ ID NO: 281, HCDR3 comprises or consists of SEQ ID NO: 282, and LCDR1 comprises or consists of SEQ ID NO: 292, LCDR2 comprises or consists of SEQ ID NO: 293, and LCDR3 comprises or consists of SEQ ID NO: 294.

[0029] In certain such embodiments, the heavy chain variable domain comprises or consists of the amino acid sequence shown in SEQ ID NO:333 or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto, and the light chain variable domain comprises or consists of the amino acid sequence shown in SEQ ID NO:334 or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0030] In certain preferred embodiments, the antibody that binds to human TIGIT without competing with CD155 for binding to TIGIT comprises a heavy chain variable domain and a light chain variable domain, wherein HCDR1 comprises SEQ ID NO:353, HCDR2 comprises SEQ ID NO:354, HCDR3 comprises SEQ ID NO:355, and LCDR1 comprises SEQ ID NO:356, LCDR2 comprises SEQ ID NO:357, and LCDR3 comprises SEQ ID NO:358.

[0031] In certain such embodiments, the heavy chain variable domain can comprise the amino acid sequence set forth in SEQ ID NO:367, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto, and the light chain variable domain can comprise the amino acid sequence set forth in SEQ ID NO:368, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0032] In another aspect, the present invention provides an isolated anti-TIGIT antibody or antigen-binding fragment thereof that preferentially depletes Treg cells expressing TIGIT, optionally wherein the antibody or antigen-binding fragment is an antibody or antigen-binding fragment according to the first aspect of the present invention, such as the antibodies exemplified herein.

[0033] In another aspect, the invention provides affinity variants of antibodies according to other aspects of the invention (eg, antibodies exemplified herein).

[0034] In another aspect, the invention provides an isolated polynucleotide or combination of isolated polynucleotides encoding an antibody or antigen-binding fragment according to any other aspect of the invention, such as an antibody exemplified herein.

[0035] In another aspect, the present invention provides an isolated polynucleotide encoding the VH and / or VL domain of an anti-TIGIT antibody, wherein the polynucleotide comprises one or more sequences selected from the group consisting of SEQ ID Nos: 241-270, 335-342 and 369-370.

[0036] In another aspect, the present invention provides an expression vector comprising a polynucleotide or combination of polynucleotides according to the present invention operably linked to regulatory sequences that allow the antigen binding polypeptide to be expressed in a host cell or a cell-free expression system.

[0037] In another aspect, the present invention provides a host cell or a cell-free expression system comprising the expression vector according to the present invention.

[0038] In another aspect, the present invention provides a method for producing a recombinant antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell or a cell-free expression system according to the present invention under conditions allowing expression of the antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

[0039] In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment according to the invention (eg, an antibody exemplified herein) and at least one pharmaceutically acceptable carrier or excipient.

[0040] In another aspect, the invention provides an antibody or antigen-binding fragment according to the invention or a pharmaceutical composition according to the invention for use in therapy.

[0041] In another aspect, the invention provides an antibody or antigen-binding fragment according to the invention (eg, an antibody exemplified herein) or a pharmaceutical composition according to the invention for use in a method of treating cancer.

[0042] In another aspect, the invention provides an antibody or antigen-binding fragment according to the invention (eg, an antibody exemplified herein) or a pharmaceutical composition according to the invention for use in a method of treating a viral infection, optionally a CMV infection.

[0043] In another aspect, the invention provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment according to the invention (e.g., an antibody exemplified herein) or a pharmaceutical composition according to the invention, thereby treating the cancer.

[0044] In another aspect, a method for treating a viral infection in a subject is provided, the method comprising administering to the subject an effective amount of an antibody or antigen binding fragment according to the present invention or a pharmaceutical composition according to the present invention, thereby treating the viral infection. In a preferred embodiment, the viral infection is CMV infection.

[0045] In another aspect, a method for promoting T cell activity is provided, the method comprising contacting a T cell population with an antibody or antigen binding fragment according to the present invention. In certain embodiments, the method promotes αβT cell activity. In certain embodiments, the method promotes γδT cell activity. In certain embodiments, the method is performed in vitro. In certain embodiments, the method is performed in vivo, for example, in a human subject.

[0046] In certain embodiments, a method according to the invention, or an antibody or antigen binding fragment or a pharmaceutical composition for use in a method according to the invention is provided, wherein the method further comprises administering one or more additional therapeutic agents. In certain preferred embodiments, the one or more additional agents are selected from: chemotherapeutic agents, anti-PD1 antibodies, anti-PD-L1 antibodies, anti-41BB antibodies, anti-OX40 antibodies, anti-GITR antibodies, and anti-ICOS antibodies.

[0047] In another aspect, a combination is provided, comprising an anti-TIGIT antibody or an antigen-binding fragment thereof, and one or more of a chemotherapeutic agent, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody, an anti-GITR antibody, and an anti-ICOS antibody. In another aspect, a combination according to the present invention is provided for use in therapy. In another aspect, a combination according to the present invention is provided for use in a method for treating cancer or for use in a method for treating a viral infection. In another aspect, a combination according to the present invention is provided for use in a method according to the present invention. In a preferred embodiment, the anti-TIGIT antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment of the present invention.

[0048] In all relevant aspects, it is preferred that any subject to be treated is a human subject.In all relevant aspects, it is preferred that the cell (eg, T cell) contacted with the antibody according to the invention is a human cell (eg, a human T cell).

[0049] Unless technically incompatible or indicated to the contrary, any preferred embodiment described may optionally be used in combination with one or more of all other preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Table providing VH CDR sequences, i.e., heavy chain variable domain (VH) complementarity determining region (CDR) sequences of antibodies of the present invention

[0051] Figure 2 Table of VL CDR sequences, i.e., light chain variable domain (VL) CDR sequences of antibodies of the present invention is provided

[0052] Figure 3 A table providing VH FR sequences, i.e., heavy chain variable domain (VH) framework (FR) sequences of antibodies of the present invention is provided.

[0053] Figure 4 A table providing VL FR sequences, i.e., light chain variable domain (VL) framework (FR) sequences of antibodies of the invention is provided.

[0054] Figure 5 Tables providing variable domain protein sequences, i.e., amino acid sequences of the heavy chain variable domain (VH) and light chain variable domain (VL) of the antibodies of the present invention

[0055] Figure 6 Tables providing variable domain DNA sequences, i.e. polynucleotide sequences encoding the VH and VL domains of antibodies according to the invention

[0056] Figure 7 Graph showing the results of competition assays for hCD155 and anti-TIGIT antibody binding to Jurkat-hTIGIT

[0057] Figure 8 TIGIT expression on different immune populations of PBMC from healthy donors is shown. (A) A graph showing the proportion of TIGIT positive cells in specific T cell populations of PBMC from 7 healthy human donors. (B) A graph showing the proportion of TIGIT positive cells in different immune populations of PBMC from 7 healthy human donors.

[0058] Fig. 9 Graph showing binding to Jurkat-hTIGIT and binding assay results of anti-TIGIT antibodies on Jurkat-hTIGIT

[0059] Fig.10 Shown with human primary CD8 + (A and B) show the binding of primary CD8 T cells from human healthy PBMCs. + Graph showing the results of binding assays of anti-TIGIT antibodies on T cells. (C) shows primary memory CD8 + Graph showing the results of binding assays of anti-TIGIT antibodies on T cells and Tregs

[0060] Fig.11 Shown is the cynomolgus monkey primary CD8 + T cells and primary CD8 + Graph showing the results of binding assay of anti-TIGIT antibodies on T cells

[0061] Fig.12Graph showing the effect of anti-TIGIT antibodies in CHO-TCR-CD155 and Jurkat-hTIGIT bioassays

[0062] Fig.13 The anti-TIGIT antibody was shown to be based on human CD8 + Effect in T cell functional assays Graph showing the effect of anti-TIGIT antibodies on enhancing IFNg secretion in a functional assay of human primary CD8 T cells from healthy donors activated with CHO-TCR-CD155 cells

[0063] Fig.14 The effect of anti-TIGIT antibodies in a human TIL-based functional assay is shown in the presence of human primary CD8 T cells from ovarian ascites activated with CHO-TCR-CD155 cells. + Histogram of the effect of anti-TIGIT antibodies on enhancing IFNg secretion in a functional assay of TILs

[0064] Fig.15 Characterization of mouse surrogate anti-TIGIT antibodies that exhibit functional activity in mice is shown. (A) A graph showing the results of a competition assay between mouse CD155 and anti-TIGIT antibodies for binding to Jurkat-mTIGIT. (B) A graph showing the effect of anti-TIGIT antibodies in enhancing IFNg secretion in a functional assay of mouse OT-1 T cells. (C) A graph showing the effect of anti-TIGIT antibodies in enhancing cytotoxicity in a functional assay of mouse OT-1 T cells.

[0065] Fig.16 The anti-tumor efficacy of anti-TIGIT antagonist antibodies is shown. (A) A graph showing the anti-tumor efficacy of anti-TIGIT antibodies as monotherapy in the CT26 tumor model. (B and C) Graphs showing the anti-tumor efficacy of anti-TIGIT antibodies in combination with anti-PD1 in the CT26 tumor model.

[0066] Fig.17 Isotype-dependent anti-tumor efficacy of anti-TIGIT antagonist antibodies is shown. (A) A graph showing the isotype-dependent anti-tumor efficacy of anti-TIGIT antibodies as monotherapy in the CT26 tumor model. (B) A graph showing the isotype-dependent anti-tumor efficacy of anti-TIGIT antibodies in combination with anti-PD1 in the CT26 tumor model.

[0067] Fig.18 The mechanism of anti-tumor efficacy of anti-TIGIT antagonist antibodies is shown. (A and G) show the expression of total CD4+ T cells in CT26 tumors treated with anti-TIGIT antibodies as monotherapy or in combination with anti-PD1. +Figure 2 shows the regulation of the proportion of Treg cells in the T cell population. (B and H) show the regulation of total CD45 Treg cells in CT26 tumors treated with anti-TIGIT antibodies as monotherapy or in combination with anti-PD1. + CD8 + Figure 2 shows the modulation of CD8 T cell ratio in CT26 tumors treated with anti-TIGIT antibodies as monotherapy or in combination with anti-PD1. + (D and J) show the regulation of IFNg secreting CD4 Treg T cell ratio in CT26 tumors treated with anti-TIGIT antibodies as monotherapy or in combination with anti-PD1. + (E) shows the regulation of IFNg-secreting CD8 T cells in CT26 tumors treated with anti-TIGIT antibodies. + Figure 1 shows the regulation of T cells. (L and F) show the expression of IFNg / IL-10 secreting CD4 T cells in CT26 tumors treated with anti-TIGIT antibodies as monotherapy or in combination with anti-PD1. + (K) shows the ratio of IL-10 secreting CD4 T cells in CT26 tumors treated with anti-TIGIT antibody in combination with anti-PD1 antibody. + Diagram of T cell regulation.

[0068] Fig.19 Figure 2 shows the mechanism of anti-tumor efficacy of anti-TIGIT antagonist antibodies (transcriptional analysis). (A) Volcano plot showing the effect of anti-TIGIT antibody treatment on gene expression in CT26 tumors and measured by NanoString analysis. (B) Box plot showing the modulation of cytotoxicity score in CT26 tumors treated with anti-TIGIT antibodies either as monotherapy or in combination with anti-PD1. (C) Box plot showing the modulation of CD8 in CT26 tumors treated with anti-TIGIT antibodies either as monotherapy or in combination with anti-PD1. + Box plot of modulation of T cell score

[0069] Fig. 20 (A) shows ADCC activity against healthy human PBMC. (B) shows ADCC activity against TIGIT in PBMC from healthy human volunteers. + CD4 + 、CD8 + (B) shows the histogram of the ratio of T cell and Treg populations. (C) shows the expression of anti-TIGIT antibodies on conventional CD4 + 、CD8 + Graph of in vitro cytotoxic effects of T cell and Treg populations.

[0070] Fig.21 Preferential depletion of Treg cells in mouse tumor suspension and the effect of anti-TIGIT antibody on conventional CD4 in CT26 tumors + 、CD8 + Graph of ex vivo cytotoxic effects of T cell and Treg populations.

[0071] Fig. 22 (A) Graph showing the results of binding assays of anti-TIGIT antibody clones on Jurkat-hTIGIT cells. (B) Graph showing the results of binding assays of anti-TIGIT antibody clones on primary CD8 + Figure 2 shows the results of binding assays of anti-TIGIT antibody clones on T cells. (C) shows the results of binding assays of anti-TIGIT antibody clones on primary CD8 T cells from PBMCs of cancer patients. + Graph of binding assay results of anti-TIGIT antibody clones on T cells.

[0072] Fig.23 Graph showing the results of a competition assay of human CD155 and anti-TIGIT antibody clones binding to Jurkat-hTIGIT

[0073] Fig.24 Figures showing functional characterization of antagonist α-TIGIT clones. (A) Graph showing the effect of anti-TIGIT antibodies in a functional assay (luciferase reporter assay) using Jurkat-hTIGIT effector cells. (B) Graph showing the effect of anti-TIGIT antibodies in measuring primary human CD8 + Graph showing the effect of anti-TIGIT antibody clone 31282 in a functional assay for IFNg secretion by T cells. (C) shows the effect of anti-TIGIT antibody clone 31282 in measuring CD3 + (D) Graph showing the effect of anti-TIGIT antibody clone 31282 in a functional assay measuring intracellular cytokine staining in TILs or PBMCs of cancer patients.

[0074] Fig.25 Effect of a-TIGIT clone 31282 on total memory CD4 in PBMCs from cancer patients + or CD8 + Cytotoxic activity of T cells and Treg populations

[0075] Fig.26 Figures showing the characterization of TIGIT expression on immune populations from cancer patients. (A) Frequency of TIGIT expression on immune populations from cancer patient PBMCs and TILs. (B) Absolute quantification of TIGIT expression on immune populations from cancer patient PBMCs and TILs.

[0076] Fig. 27 (A) Structure of the Fab:TIGIT complex shown as a ribbon diagram; (B) Complete binding interface between clone 31282 and TIGIT; (C) Binding interface between clone 31282 and TIGIT, showing contacting residues.

[0077] Fig.28 Competition assay between anti-TIGIT clones 31282 and 32959.

[0078] Fig.29 Measurement of plasma concentrations of anti-TIGIT clone 31282 in cynomolgus monkeys following single intravenous injection of 0.1 mg / kg (top row), 1 mg / kg (middle row), or 10 mg / kg (bottom row). Left column: 31282 IgG1; right column 31282 IgG4.

[0079] Fig.30 The malignant and normal CD4 + Graphs showing characterization of TIGIT expression on T cell populations. (A) Separation of malignant and normal CD4 + Gating strategy for T cells. (B) MFI of TIGIT staining for two different populations.

[0080] Fig.31 Graphs showing the characterization of TIGIT expression on malignant and normal B cell populations from CLL patients. (A) Gating strategy to separate malignant and normal B cells. (B) MFI of TIGIT staining for 2 different populations.

[0081] Fig.32 (A to C) Graphs showing tumor growth curves in mice inoculated with EL4-mTIGIT tumors. (A) Median tumor growth curve. (B) Individual tumor growth curves in mice treated with hIgG1 isotype control antibody. (C) Individual tumor growth curves in mice treated with mouse surrogate antagonist a-TIGIT antibody (hIgG1). (D to F) Graphs showing tumor growth curves in mice inoculated with EL4-GFP tumors. (D) Median tumor growth curve. (E) Individual tumor growth curves in mice treated with hIgG1 isotype control antibody. (F) Individual tumor growth curves in mice treated with surrogate antagonist a-TIGIT (hIgG1).

[0082] Fig.33(A to D) Graphs showing tumor growth curves in mice inoculated with CT26 tumors. (A) Median and individual tumor growth curves for mice treated with anti-TIGIT and anti-4-1BB antibodies. (B) Median and individual tumor growth curves for mice treated with anti-TIGIT and anti-OX-40 antibodies. (C) Median and individual tumor growth curves for mice treated with anti-TIGIT and anti-GITR antibodies. (D) Median and individual tumor growth curves for mice treated with anti-TIGIT and anti-ICOS antibodies.

[0083] Fig.34 Graphs showing the effects of anti-TIGIT antibodies on γδ T cells. (A) Vδ2 from PBMCs of CMV positive and negative human donors - The median proportion of TIGIT-positive cells and TIGIT MFI signal in the γδT cell population. (B) shows the median proportion of TIGIT-positive cells and TIGIT MFI signal in the γδT cell population. + (C) A graph showing the activity of anti-TIGIT Ab in increasing IFNg secretion in a functional assay for γδ T cells. (C) A graph showing the activity of anti-TIGIT Ab in increasing IFNg secretion in a functional assay for total PBMCs. DETAILED DESCRIPTION

[0084] As used herein, the term "immunoglobulin" includes polypeptides having a combination of two heavy chains and two light chains, whether or not they have any relevant specific immunoreactivity. "Antibody" refers to such a combination that has significant known specific immunoreactivity to a target antigen (e.g., TIGIT). The term "TIGIT antibody" or "anti-TIGIT antibody" is used herein to refer to antibodies that exhibit immunological specificity for TIGIT protein. Antibodies and immunoglobulins comprise light and heavy chains, with or without interchain covalent linkages between them. The basic immunoglobulin structure in vertebrate systems has been relatively well understood.

[0085] The general term "immunoglobulin" includes five different classes of antibodies that can be distinguished biochemically. Although all five classes of antibodies are within the scope of the present invention, the following discussion is generally directed to immunoglobulin molecules of the IgG class. With respect to IgG, immunoglobulins contain two identical polypeptide light chains of molecular weight of about 23,000 daltons and two identical heavy chains of molecular weight of 53,000 to 70,000. The four chains are connected by disulfide bonds into a "Y" configuration, wherein the light chains start from the opening of the "Y" and continue through the variable region to bracket the heavy chains.

[0086] The light chains of antibodies are classified as kappa or lambda (κ, λ). Each heavy chain category can be combined with a κ or λ light chain. In general, the light chain and the heavy chain are covalently bound to each other, and when immunoglobulins are produced by B cells or genetically engineered host cells, the "tails" of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will understand that the heavy chain is classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), and there are also some subclasses (such as γ1-γ4). The properties of the chain determine the "class" of the antibody as IgG, IgM, IgA, IgD or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, etc. have been fully characterized and are known to confer functional specialization. Modified forms of each of these classes and isotypes are readily discernible to those skilled in the art in view of the present disclosure, and such modified forms are therefore within the scope of the present invention.

[0087] As mentioned above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and VH domain of an antibody combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementarity determining regions (CDRs) on each of the VH and VL chains.

[0088] As used herein, the terms "TIGIT protein" or "TIGIT antigen" or "TIGIT" are used interchangeably and refer to a human T cell immune receptor (GenBank Accession No.: NM_173799) that binds to the poliovirus receptor (PVR-also known as CD155). TIGIT is also known as VSIG9, VSTM3, or WUCAM. References to TIGIT include native human TIGIT proteins naturally expressed in human hosts and / or on the surface of human cultured cell lines, and recombinant forms and fragments thereof, as well as naturally occurring mutant forms.

[0089] As used herein, the term "binding site" comprises a polypeptide region responsible for selective binding to a target antigen (e.g., TIGIT). A binding domain comprises at least one binding site. Exemplary binding domains include antibody variable domains. Antibody molecules of the present invention may comprise a single binding site or multiple (e.g., two, three, or four) binding sites.

[0090] As used herein, the term "derived from" a specified protein (e.g., a TIGIT antibody or an antigen-binding fragment thereof) refers to the source of a polypeptide. In one embodiment, a polypeptide or amino acid sequence derived from a specific starting polypeptide is a CDR sequence or a sequence associated therewith. In one embodiment, the amino acid sequence derived from a specific starting polypeptide is not continuous. For example, in one embodiment, one, two, three, four, five or six CDRs are derived from a starting antibody. In one embodiment, a polypeptide or amino acid sequence derived from a specific starting polypeptide or amino acid sequence has an amino acid sequence substantially identical to the amino acid sequence of the starting sequence or a portion thereof, or for a person of ordinary skill in the art, it can be otherwise identified as an amino acid sequence derived from the starting sequence, wherein the portion is composed of at least 3 to 5 amino acids, at least 5 to 10 amino acids, at least 10 to 20 amino acids, at least 20 to 30 amino acids, or at least 30 to 50 amino acids. In one embodiment, the one or more CDR sequences derived from the starting antibody are changed to produce a variant CDR sequence (e.g., an affinity variant), wherein the variant CDR sequence retains TIGIT binding activity.

[0091] As used herein, "conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide can be replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0092] As used herein, the term "heavy chain portion" includes an amino acid sequence of a constant domain derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain portion includes at least one of the following: a CH1 domain, a hinge (e.g., upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, the antibody or antigen-binding fragment of the present invention may include the Fc portion of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, the antibody or antigen-binding fragment of the present invention may lack at least a portion of a constant domain (e.g., the entire or a portion of a CH2 domain). In certain embodiments, at least one and preferably all constant domains are derived from a human immunoglobulin heavy chain. For example, in a preferred embodiment, the heavy chain portion includes a fully human hinge domain. In other preferred embodiments, the heavy chain portion includes a fully human Fc portion (e.g., hinge, CH2 and CH3 domain sequences from a human immunoglobulin).

[0093] In certain embodiments, the constituent constant domains of the heavy chain portion are from different immunoglobulin molecules. For example, the heavy chain portion of the polypeptide may include a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domain is a chimeric domain that includes portions of different immunoglobulin molecules. For example, a hinge may include a first portion from an IgG1 molecule and a second portion from an IgG3 or IgG4 molecule. As described above, it will be appreciated by those of ordinary skill in the art that the constant domains of the heavy chain portion may be modified so that they differ from naturally occurring (wild-type) immunoglobulin molecules in amino acid sequence. That is, the polypeptides of the present invention disclosed herein may include changes or modifications to one or more heavy chain constant domains (CH1, hinge, CH2 or CH3) and / or to a light chain constant region domain (CL). Exemplary modifications include addition, deletion or substitution of one or more amino acids in one or more domains.

[0094] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are intended to have equivalent meanings. The term "variable" refers to the fact that certain portions of the variable domains VH and VL differ extensively in sequence between antibodies and are used for the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of an antibody. The variability is concentrated in three segments called "hypervariable loops" in each of the VL and VH domains that form part of the antigen binding site. λThe first, second and third hypervariable loops of the light chain domain are referred to herein as L1 (λ), L2 (λ) and L3 (λ), and can be defined as comprising residues 24-33 (L1 (λ), consisting of 9, 10 or 11 amino acid residues), 49-53 (L2 (λ), consisting of 3 residues) and 90-96 (L3 (λ), consisting of 5 residues) in the VL domain (Morea et al., Methods 20, 267-279, 2000). κ The first, second and third hypervariable loops of the light chain domain are referred to herein as L1 (κ), L2 (κ) and L3 (κ), and can be defined as comprising residues 25-33 (L1 (κ), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2 (κ), consisting of 3 residues) and 90-97 (L3 (κ), consisting of 6 residues) in the VL domain (Morea et al., Methods 20, 267-279, 2000). The first, second and third hypervariable loops of the VH domain are referred to herein as H1, H2 and H3, and can be defined as comprising residues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea et al., Methods 20, 267-279, 2000).

[0095] Unless otherwise indicated, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops of the VL domain, respectively, and encompass the entire VL domain. κ and V λ Hypervariable loops obtained from an isotype. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops, respectively, of a VH domain, and encompass hypervariable loops obtained from any known heavy chain isotype, including gamma, epsilon, delta, alpha, or mu.

[0096] As defined below, hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise a portion of a "complementarity determining region" or "CDR". The terms "hypervariable loop" and "complementarity determining region" are not strictly synonymous, because hypervariable loops (HV) are defined based on structure, while complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991) and the limits of HV and CDRs may be different in some VH and VL domains.

[0097] The CDRs of VL and VH domains can generally be defined as comprising the following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or 31-35b (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991). Thus, HV can be contained within the corresponding CDRs, and unless otherwise indicated, references herein to "hypervariable loops" of VH and VL domains should be interpreted as also covering the corresponding CDRs, and vice versa.

[0098] As defined below, the more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs (FR1, FR2, FR3, and FR4, respectively), mainly in a β-folded configuration connected by three hypervariable loops. The hypervariable loops in each chain are closely held together by the FRs, and together with the hypervariable loops from the other chain, contribute to the formation of the antigen binding site of the antibody. The structural analysis of antibodies reveals the relationship between the sequence and shape of the binding site formed by the complementarity determining region (Chothia et al., J. Mol. Biol. 227, 799-817, 1992; Tramontano et al., J. Mol. Biol, 215, 175-182, 1990). Although it has high sequence variability, five of the six loops only adopt a small amount of main chain conformation, referred to as "canonical structure". These conformations are determined firstly by the length of the loop and secondly by the presence of key residues at certain positions in the loop and framework regions, which dictate the conformation through their ability to pack, hydrogen bond, or assume unusual main-chain conformations.

[0099] As used herein, the term "CDR" or "complementarity determining region" means a non-contiguous antigen binding site found within the variable region of both heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616, 1977; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991; Chothia et al., J. Mol. Biol. 196, 901-917, 1987; and MacCallum et al., J. Mol. Biol. 262, 732-745, 1996, wherein definitions include overlaps or subsets of amino acid residues when compared to each other. The amino acid residues covering the CDRs defined by each reference cited above are shown for comparison. Preferably, the term "CDR" is a CDR defined by Kabat based on sequence comparison.

[0100] Table 1: CDR definitions.

[0101]

[0102]

[0103] 1 The residue numbering follows the nomenclature of Kabat et al. (supra).

[0104] 2 The residue numbering follows the nomenclature of Chothia et al. (supra).

[0105] 3 The residue numbering follows the nomenclature of MacCallum et al. (supra).

[0106] As used herein, the term "framework region" or "FR region" includes amino acid residues that are part of the variable region but not part of the CDR (e.g., using the Kabat definition of CDR). Thus, the variable region framework is about 100 to 120 amino acids in length, but includes only those amino acids outside the CDR. For the specific example of the heavy chain variable domain and the CDR defined by Kabat et al., framework region 1 corresponds to the variable region domain covering amino acids 1-30; framework region 2 corresponds to the variable region domain covering amino acids 36-49; framework region 3 corresponds to the variable region domain covering amino acids 66-94; and framework region 4 corresponds to the variable region domain from amino acid 103 to the end of the variable region. The framework region of the light chain is similarly separated by each light chain variable region CDR. Similarly, using the CDR definitions of Chothia et al. or McCallum et al., the framework region boundaries are separated by the ends of the corresponding CDRs as described above. In some preferred embodiments, the CDRs are as defined by Kabat.

[0107] In naturally occurring antibodies, the six CDRs present in each monomer antibody are short, non-continuous amino acid sequences that are specifically positioned to form an antigen binding site when the antibody presents its three-dimensional configuration in an aqueous environment. The rest of the heavy and light chain variable domains show less intermolecular variability in the amino acid sequence and are called framework regions. The framework region mainly adopts a β-folded configuration, and the CDR forms a loop that connects the β-folded structure and forms a part of the β-folded structure in some cases. Therefore, these framework regions play a role in forming a scaffold that positions the six CDRs in the correct direction by non-covalent interactions between chains. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. The complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. One of ordinary skill in the art can easily identify the position of the CDR.

[0108] As used herein, the term "fragment" refers to a part or portion of an antibody or antibody chain, which contains less amino acid residues than a complete or complete antibody or antibody chain. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with a complete antibody (i.e., a complete antibody from which a polypeptide fragment is derived) for binding to an antigen (i.e., specifically binds to TIGIT). As used herein, the term "fragment" of an antibody molecule includes an antigen-binding fragment of an antibody, for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (scFv), a F(ab')2 fragment, a Fab fragment, a Fd fragment, a Fv fragment, and a single domain antibody fragment (DAb). Fragments can be obtained, for example, by chemical or enzymatic treatment of a complete or complete antibody or antibody chain or by recombinant means.

[0109] As used herein, the term "valency" refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to a target molecule or a specific site on a target molecule. When a polypeptide comprises more than one target binding site, each target binding site can specifically bind to the same or different molecules (for example, different ligands or different antigens, or different epitopes on the same antigen) . The binding molecules of the present invention have at least one binding site that is specific for TIGIT.

[0110] As used herein, the term "specificity" refers to the ability to bind to (e.g., immune response) a given target (e.g., TIGIT). A polypeptide may be monospecific and contain one or more binding sites that specifically bind to a target, or a polypeptide may be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In one embodiment, the antibodies of the present invention are specific for more than one target. For example, in one embodiment, a multispecific binding molecule of the present invention binds to TIGIT and a second target molecule. In this case, the second target molecule is a molecule other than TIGIT.

[0111] As used herein, the term "synthetic" with respect to polypeptides includes polypeptides containing non-naturally occurring amino acid sequences. For example, non-naturally occurring polypeptides are modified forms of naturally occurring polypeptides (e.g., containing mutations such as additions, substitutions, or deletions) or contain a first amino acid sequence (which may or may not be naturally occurring) linked in a linear amino acid sequence to a second amino acid sequence (which may or may not be naturally occurring) that is not naturally linked to the first amino acid sequence in nature.

[0112] As used herein, the term "engineering" includes the manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides, or some combination of these techniques). Preferably, the antibodies of the invention have been engineered to improve one or more properties, such as antigen binding, stability / half-life, or effector function.

[0113] As used herein, the term "modified antibody" includes synthetic forms of antibodies that are altered so that they do not exist naturally, for example, antibodies that include at least two heavy chain portions instead of two complete heavy chains (such as domain-deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that are altered to bind to two or more different antigens or different epitopes on a single antigen; heavy chain molecules connected to scFv molecules, and the like. ScFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. In addition, the term "modified antibody" includes multivalent forms of antibodies (e.g., trivalent, tetravalent antibodies that bind to three or more copies of the same antigen, etc.). In another embodiment, the modified antibody of the present invention is a fusion protein that includes at least one heavy chain portion lacking a CH2 domain and includes a binding domain of a polypeptide containing a binding portion of one member of a receptor ligand pair.

[0114] The term "modified antibody" may also be used herein to refer to amino acid sequence variants of the TIGIT antibodies of the present invention. Those skilled in the art will appreciate that the TIGIT antibodies of the present invention may be modified to produce variant TIGIT antibodies that differ in amino acid sequence from the TIGIT antibodies from which they are derived. For example, nucleotide or amino acid substitutions (e.g., in CDR and / or framework residues) may be made that produce conservative substitutions or changes at "non-essential" amino acid residues. Amino acid substitutions may include replacing one or more amino acids with naturally occurring or non-natural amino acids.

[0115] "Antibody fragments" comprise a portion of a full-length antibody, typically its antigen binding domain or variable domain. Some examples of antigen-binding antibody fragments include Fab, Fab', F(ab')2, bispecific Fab and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments (see Holliger and Hudson, Nature Biotechnol. 23: 1126-1136, 2005, the contents of which are incorporated herein by reference).

[0116] As used herein, the term "affinity variant" refers to a variant antibody that exhibits one or more changes in the amino acid sequence compared to a reference TIGIT antibody of the present invention, wherein the affinity variant exhibits an altered affinity for TIGIT compared to the reference antibody. Preferably, the affinity variant will exhibit improved affinity for TIGIT compared to the reference TIGIT antibody. This improvement can be manifested as a lower KD for TIGIT, or a slower dissociation rate for TIGIT. Compared to the reference TIGIT antibody, affinity variants typically exhibit one or more changes in the amino acid sequence of the CDR. Such substitutions can result in the original amino acid present at a given position in the CDR being replaced by a different amino acid residue, which can be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue. Amino acid substitutions can be conservative or non-conservative.

[0117] As used herein, the term "affinity" or "binding affinity" should be understood based on the usual meaning in the art in the context of antibody binding, and reflects the binding strength and / or stability between an antigen and a binding site on an antibody or antigen-binding fragment thereof.

[0118] The anti-TIGIT antibodies provided herein are characterized by high affinity binding to human TIGIT. Binding affinity to TIGIT can be assessed using standard techniques known to those skilled in the art.

[0119] Binding affinity can also be expressed as the dissociation constant or K for a specific antibody. D . K D The smaller the value, the stronger the binding interaction between the antibody and its target antigen. In one embodiment, the binding interaction between the antibody and its target antigen can be determined by using methods known in the art, such as by ForteBio TM system, by MSD-solution equilibrium titration (SET) or by surface plasmon resonance, for example using Biacore as described in the accompanying examples TM The Fab fragments of the antibodies according to the present invention generally exhibited binding affinity to TIGIT as determined by the ForteBio TM Measured in 1x10 -10 Up to 5x10 -8 M, optional 7x10 -10 Up to 4x10 -8 K in the M range D In this range, K D This can be taken as an indication that the Fab and the corresponding bivalent mAb exhibit high affinity binding to hTIGIT. DThe bivalent mAb of Fab was also found to exhibit high affinity binding to hTIGIT. -11 Up to 5x10 -9 , optionally 2x10 -11 Up to 1x10 -9 MSD K in the range D This can be used as an indication of high affinity binding to hTIGIT. The Fab fragments of the antibodies according to the present invention generally show high affinity binding to TIGIT as measured by Biacore TM Measured in 1x10 -10 M to 1x10 -9 M, optional 1x10 -10 Up to 7x10 10 , optionally 2x10 -10 Up to 7x10 -10 K in the M range D In this range, K D This can be taken as an indication that the Fab and the corresponding bivalent mAb exhibit high affinity binding to hTIGIT.

[0120] Binding affinity to human TIGIT can also be assessed using a cell-based system as described in the accompanying Examples, in which the mAb is tested for binding to mammalian cells (cell lines expressing TIGIT or ex vivo cells), for example, using ELISA or flow cytometry. For example, an EC of no more than 0.5 nM as measured by flow cytometry (e.g., FACS) analysis (such as that described in Example 10) is sufficient to detect the binding affinity of the mAb to human TIGIT. 50 A high affinity for TIGIT may be indicated. In certain embodiments, the antibodies of the invention exhibit a cell-binding EC of no more than 0.5 nM, optionally no more than 0.2 nM. 50 . Expressed as EC 50 Cell-based affinity assays are preferably determined using Jurkat cells expressing hTIGIT or primary CD8 T cells from human peripheral blood mononuclear cells (PBMCs).

[0121] As used herein, "Treg cells" or "Treg" for short refer to regulatory CD4+T cells - that is, T cells that reduce the effector function of conventional T cells (CD8 or CD4 T cells). Tregs can be identified according to methods known in the art, such as using flow cytometry to identify CD4 cells that express high levels of CD25 and low levels of CD127 or the absence of CD127.

[0122] As summarized above, the present invention relates, at least in part, to antibodies and antigen-binding fragments thereof that bind to TIGIT.The properties and characteristics of the TIGIT antibodies and antibody fragments according to the present invention will now be described in more detail.

[0123] Anti-TIGIT antibodies

[0124] In one aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that binds to human TIGIT and comprises a protein selected from Figure 1 The isolated antibody or antigen-binding fragment thereof further comprises a heavy chain variable domain comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3) having the HCDR1, HCDR2 and HCDR3 sequences shown in Figure 2 The light chain variable domains of light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) of the LCDR1, LCDR2 and LCDR3 sequences shown. That is, the present invention provides an isolated antibody or antigen-binding fragment thereof, which binds to human TIGIT and comprises a heavy chain variable domain comprising heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3), wherein:

[0125] (i) HCDR1 is selected from the group consisting of SEQ ID No: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 271, 274 and 277;

[0126] (ii) HCDR2 is selected from the group consisting of SEQ ID No: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 272, 275 and 278;

[0127] (iii) HCDR3 is selected from the group consisting of SEQ ID No: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 273, 276 and 279;

[0128] The isolated antibody or antigen-binding fragment thereof further comprises a light chain variable domain comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3), wherein

[0129] (iv) LCDR1 is selected from the group consisting of SEQ ID Nos: 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 283, 286 and 289;

[0130] (v) LCDR2 is selected from the group consisting of SEQ ID Nos: 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 284, 287 and 290; and

[0131] (vi) LCDR3 is selected from the group consisting of SEQ ID No: 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 285, 288 and 291.

[0132] Any given anti-TIGIT antibody or antigen-binding fragment thereof comprising a VH domain that pairs with a VL domain to form an antigen (human TIGIT) binding site will comprise a combination of the following six CDRs: variable heavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variable light chain CDR2 (LCDR2), and variable light chain CDR1 (LCDR1). Although many different combinations of six CDRs selected from the group of CDR sequences listed above are permissible and within the scope of the invention, certain combinations of the six CDRs are particularly preferred; these are "natural" combinations within a single mAb that exhibit high affinity binding to human TIGIT. In certain embodiments, the antibody or antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the combination is selected from Figure 1 The HCDRs of the antibodies in Figure 2 The groups of combinations formed by the LCDRs of the corresponding antibodies.

[0133] That is, in certain embodiments, the antibody or antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the combination is selected from the group consisting of:

[0134] (i) a HCDR1 comprising SEQ ID NO: 1, a HCDR2 comprising SEQ ID NO: 2, a HCDR3 comprising SEQ ID NO: 3, a LCDR1 comprising SEQ ID NO: 46, a LCDR2 comprising SEQ ID NO: 47, and a LCDR3 comprising SEQ ID NO: 48;

[0135] (ii) HCDR1 comprising SEQ ID NO:4, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:6, LCDR1 comprising SEQ ID NO:49, LCDR2 comprising SEQ ID NO:50, and LCDR3 comprising SEQ ID NO:51;

[0136] (iii) HCDR1 comprising SEQ ID NO:7, HCDR2 comprising SEQ ID NO:8, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:52, LCDR2 comprising SEQ ID NO:53, and LCDR3 comprising SEQ ID NO:54;

[0137] (iv) HCDR1 comprising SEQ ID NO: 10, HCDR2 comprising SEQ ID NO: 11, HCDR3 comprising SEQ ID NO: 12, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 56, and LCDR3 comprising SEQ ID NO: 57;

[0138] (v) HCDR1 comprising SEQ ID NO: 13, HCDR2 comprising SEQ ID NO: 14, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 58, LCDR2 comprising SEQ ID NO: 59, and LCDR3 comprising SEQ ID NO: 60;

[0139] (vi) HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, HCDR3 comprising SEQ ID NO: 18, LCDR1 comprising SEQ ID NO: 61, LCDR2 comprising SEQ ID NO: 62, and LCDR3 comprising SEQ ID NO: 63;

[0140] (vii) HCDR1 comprising SEQ ID NO: 19, HCDR2 comprising SEQ ID NO: 20, HCDR3 comprising SEQ ID NO: 21, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 65, and LCDR3 comprising SEQ ID NO: 66;

[0141] (viii) HCDR1 comprising SEQ ID NO:22, HCDR2 comprising SEQ ID NO:23, HCDR3 comprising SEQ ID NO:24, LCDR1 comprising SEQ ID NO:67, LCDR2 comprising SEQ ID NO:68, and LCDR3 comprising SEQ ID NO:69;

[0142] (ix) a HCDR1 comprising SEQ ID NO:25, a HCDR2 comprising SEQ ID NO:26, a HCDR3 comprising SEQ ID NO:27, a LCDR1 comprising SEQ ID NO:70, a LCDR2 comprising SEQ ID NO:71, and a LCDR3 comprising SEQ ID NO:72;

[0143] (x) a HCDR1 comprising SEQ ID NO:28, a HCDR2 comprising SEQ ID NO:29, a HCDR3 comprising SEQ ID NO:30, a LCDR1 comprising SEQ ID NO:73, a LCDR2 comprising SEQ ID NO:74, and a LCDR3 comprising SEQ ID NO:75;

[0144] (xi) a HCDR1 comprising SEQ ID NO:31, a HCDR2 comprising SEQ ID NO:32, a HCDR3 comprising SEQ ID NO:33, a LCDR1 comprising SEQ ID NO:76, a LCDR2 comprising SEQ ID NO:77, and a LCDR3 comprising SEQ ID NO:78;

[0145] (xii) a HCDR1 comprising SEQ ID NO:34, a HCDR2 comprising SEQ ID NO:35, a HCDR3 comprising SEQ ID NO:36, a LCDR1 comprising SEQ ID NO:79, a LCDR2 comprising SEQ ID NO:80, and a LCDR3 comprising SEQ ID NO:81;

[0146] (xiii) HCDR1 comprising SEQ ID NO:37, HCDR2 comprising SEQ ID NO:38, HCDR3 comprising SEQ ID NO:39, LCDR1 comprising SEQ ID NO:82, LCDR2 comprising SEQ ID NO:83, and LCDR3 comprising SEQ ID NO:84;

[0147] (xiv) a HCDR1 comprising SEQ ID NO:40, a HCDR2 comprising SEQ ID NO:41, a HCDR3 comprising SEQ ID NO:42, a LCDR1 comprising SEQ ID NO:85, a LCDR2 comprising SEQ ID NO:86, and a LCDR3 comprising SEQ ID NO:87;

[0148] (xv) a HCDR1 comprising SEQ ID NO:43, a HCDR2 comprising SEQ ID NO:44, a HCDR3 comprising SEQ ID NO:45, a LCDR1 comprising SEQ ID NO:88, a LCDR2 comprising SEQ ID NO:89, and a LCDR3 comprising SEQ ID NO:90;

[0149] (xvi) a HCDR1 comprising SEQ ID NO: 271, a HCDR2 comprising SEQ ID NO: 272, a HCDR3 comprising SEQ ID NO: 273, a LCDR1 comprising SEQ ID NO: 283, a LCDR2 comprising SEQ ID NO: 284, and a LCDR3 comprising SEQ ID NO: 285;

[0150] (xvii) a HCDR1 comprising SEQ ID NO: 274, a HCDR2 comprising SEQ ID NO: 275, a HCDR3 comprising SEQ ID NO: 276, a LCDR1 comprising SEQ ID NO: 286, a LCDR2 comprising SEQ ID NO: 287, and a LCDR3 comprising SEQ ID NO: 288;

[0151] (xviii) HCDR1 comprising SEQ ID NO:277, HCDR2 comprising SEQ ID NO:278, HCDR3 comprising SEQ ID NO:279, LCDR1 comprising SEQ ID NO:289, LCDR2 comprising SEQ ID NO:290, and LCDR3 comprising SEQ ID NO:291.

[0152] In certain embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable domain having an amino acid sequence selected from the group consisting of: SEQ ID No: 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 327, 329 and 331, and amino acid sequences that exhibit at least 90%, 95%, 97%, 98% or 99% sequence identity thereto; and optionally comprises a light chain variable domain having an amino acid sequence selected from the group consisting of: SEQ ID The amino acid sequences of Nos:212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 328, 330 and 332, and amino acid sequences that exhibit at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0153] While all possible pairings of VH and VL domains selected from the groups of VH and VL domain sequences listed above are permissible and within the scope of the invention, certain VH and VL combinations are particularly preferred; these are the "natural" combinations within a single mAb that exhibit high affinity binding to human TIGIT.

[0154] In certain embodiments, the antibody or antigen-binding fragment comprises a combination of a heavy chain variable domain and a light chain variable domain, wherein the combination is selected from Figure 5 The VH of each antibody in the present invention or an amino acid sequence thereof that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity with the ... Figure 5 In certain embodiments, the antibody or antigen-binding fragment comprises a combination of a heavy chain variable domain and a light chain variable domain, wherein the combination is selected from the group consisting of:

[0155] (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 211 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 212;

[0156] (ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 213 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 214;

[0157] (iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 215 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 216;

[0158] (iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 217 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 218;

[0159] (v) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 219 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 220;

[0160] (vi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 221 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 222;

[0161] (vii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 223 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 224;

[0162] (viii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 225 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 226;

[0163] (ix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 227 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 228;

[0164] (x) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 229 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 230;

[0165] (xi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 231 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 232;

[0166] (xii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 233 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 234;

[0167] (xiii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 235 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 236;

[0168] (xiv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 237 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 238;

[0169] (xv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 239 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 240;

[0170] (xvi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 327, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 328, or an amino acid sequence that is at least 90% identical to said amino acid sequence;

[0171] (xvii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 329, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 330, or an amino acid sequence that is at least 90% identical to said amino acid sequence; and

[0172] (xviii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 331, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 332, or an amino acid sequence that is at least 90% identical to said amino acid sequence.

[0173] For each of the specific VH / VL combinations listed above, it is permissible and within the scope of the invention to combine a VH domain having an amino acid sequence that is at least 90%, 92%, 95%, 97% or 99% identical to the recited VH domain sequence with a VL domain having an amino acid sequence that is at least 90%, 92%, 95%, 97% or 99% identical to the recited VL domain sequence. Nevertheless, embodiments in which the amino acid sequence of the VH domain exhibits less than 100% sequence identity to a given reference VH sequence may comprise heavy chain CDRs that are identical to HCDR1, HCDR2 and HCDR3 of the reference sequence, yet exhibit amino acid sequence variations within the framework regions. Likewise, embodiments in which the amino acid sequence of the VL domain exhibits less than 100% sequence identity to a given reference sequence may comprise light chain CDRs that are identical to LCDR1, LCDR2 and LCDR3 of the reference sequence, yet exhibit amino acid sequence variations within the framework regions.

[0174] In the previous paragraph and elsewhere herein, the structure of the antibody / antigen binding fragment is defined based on the % sequence identity to the described reference sequence (with a given SEQ ID NO). In this case, the % sequence identity between two amino acid sequences can be determined by comparing the two sequences aligned in an optimal manner, and wherein the amino acid sequence to be compared may contain additions or deletions relative to the reference sequence to optimally align the two sequences. The percent identity is calculated by determining the number of identical positions where the amino acid residues are identical between the two sequences, dividing the number of identical positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percent identity between the two sequences. Typically, the comparison window corresponds to the full length of the compared sequence. For example, the BLAST program "BLAST2sequences" (Tatusova et al., "Blast 2sequences-a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250) can be used, which can be obtained at the website http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, and the parameters used are those given by default (particularly the parameters "open gap penalty": 5, and "extended gap penalty": 2; the selected matrix is, for example, the matrix "BLOSUM62" proposed by the program), and the percentage of identity between the two sequences to be compared is calculated directly by the program. Determining the sequence identity of the query sequence to the reference sequence is within the capabilities of the technician, and commercially available analysis software such as BLAST can be used. TM conduct.

[0175] In certain preferred embodiments, the antibody or antigen-binding fragment may comprise a heavy chain variable domain and a light chain variable domain, wherein HCDR1 comprises SEQ ID NO:16, HCDR2 comprises SEQ ID NO:17, HCDR3 comprises SEQ ID NO:18, and LCDR1 comprises SEQ ID NO:61, LCDR2 comprises SEQ ID NO:62 and LCDR3 comprises SEQ ID NO:63.

[0176] In certain such embodiments, the heavy chain variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 221, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto, and the light chain variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 222, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto. In certain such embodiments, the heavy chain variable domain and the light chain variable domain are the VH and VL domains of antibody 31282 provided herein.

[0177] The antibody 31282 provided herein is derived from the antibody 29489. Antibody 31282 is produced by substitution of MT at amino acid 116 in the VH FR4 region of 29489. It is understood that this substitution removes potential oxidation sites of the antibody and thereby improves stability without affecting function. Therefore, antibodies 31282 and 29489 share the same HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences, with only different frameworks.

[0178] Therefore, in certain embodiments of the antibodies or antigen-binding fragments of the present invention, the heavy chain variable domain may comprise the amino acid sequence shown in SEQ ID NO: 219 or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence, and the light chain variable domain may comprise the amino acid sequence shown in SEQ ID NO: 220 and an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence. In certain such embodiments, the heavy chain variable domain and the light chain variable domain are the VH and VL domains of the antibody 29489 provided herein.

[0179] Nevertheless, embodiments in which the amino acid sequence of the VH domain exhibits less than 100% sequence identity to the sequence shown in SEQ ID NO: 221 or 219 may comprise heavy chain CDRs identical to HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 221 and 219 (SEQ ID NO: 16, 17 and 18, respectively), however exhibiting amino acid sequence variation within the framework regions. Similarly, embodiments in which the amino acid sequence of the VL domain exhibits less than 100% sequence identity to the sequence shown in SEQ ID NO: 222 or 220 may comprise light chain CDRs identical to LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 222 and 220 (SEQ ID NO: 61, 62 and 63, respectively), however exhibiting amino acid sequence variation within the framework regions.

[0180] This article describes Figures 1 to 5 The exemplary TIGIT antibodies with the sequences shown in were developed from five parent antibody clones. Table 2 summarizes the repertoire of antibodies described herein. Parents are anti-TIGIT antibodies, and those antibodies that show high functional activity to TIGIT are selected (gray rows, named 26...), and affinity maturation is performed. Then, the selected affinity matured antibodies are expressed in mammalian cells (white rows below each parent, named 29... or 3...). In addition, antibody 31282 is produced by 29489 through MT substitution at amino acid 116 in the VH FR4 region. It is understood that this substitution will remove potential oxidation sites of the antibody, and thus improve stability without affecting function. In addition, antibody 31288 is produced by 29494 through VL substitution at amino acid 2 in the VH FR1 region and MT substitution at amino acid 120 in the VH FR4 region. It is understood that VL substitutions will restore the sequence of the VH4-39 germline, and MT substitutions will remove potential oxidation sites of the antibody and thus improve stability without affecting function.

[0181] Table 2

[0182]

[0183]

[0184] The second generation antibodies exhibited higher affinity than the corresponding parental antibodies.

[0185] In certain embodiments, the present invention provides an anti-TIGIT antibody or an antigen-binding fragment thereof, wherein the VH domain is derived from a human V region germline sequence selected from the group consisting of: VH3-07, VH3-30, VH1-46, VH4-0B, VH4-39, VH1-69, VH3-09, VH3-33, VH3-30. In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain derived from human V region germline VH1-46.

[0186] A VH domain is "derived from" a particular V region germline sequence if the sequence of the heavy chain variable region is more likely to be derived from the given germline than from any other germline.

[0187] TIGIT epitope

[0188] The present invention also provides an antibody or an antigen-binding fragment thereof that binds to human TIGIT at an epitope comprising residues Q56 and I109. In certain embodiments, the antibody or its antigen-binding fragment binds to human TIGIT at least at residues Q56, N58 and I109. In certain embodiments, the antibody or its antigen-binding fragment binds to human TIGIT at an epitope comprising residues Q56, N58 and I109 and optionally one or more of residues E60, I68, L73 and H76. In certain embodiments, the antibody or its antigen-binding fragment binds to human TIGIT at an epitope comprising residues Q56, N58, E60, I68, L73, H76 and I109.

[0189] In certain embodiments, the antibody or antigen-binding fragment thereof binds to human TIGIT at an epitope consisting of TIGIT residues Q56, N58, E60, I68, L73, H76, and 1109. In certain embodiments, the antibody or antigen-binding fragment thereof binds to the same epitope as antibody 31282.

[0190] Where the antibody or antigen-binding fragment binds to a human TIGIT epitope comprising specified TIGIT residues, the antibody binds to each of these residues and optionally to other residues of TIGIT. Where the antibody or antigen-binding fragment binds to a human TIGIT epitope consisting of TIGIT residues Q56, N58, E60, I68, L73, H76, and I109, the antibody binds to each of these residues without binding to other residues of TIGIT.

[0191] If an antibody or antigen-binding fragment contacts a specified TIGIT amino acid residue when bound to TIGIT, it binds to human TIGIT at a given epitope. As used herein, an antibody contacts a TIGIT residue if, in a protein complex formed by antibody-TIGIT binding, the residue meets each of the following criteria: (i) the calculated binding free energy contribution of the residue is greater than 0.3 kcal / mol, (ii) the experimental average B-factor of the residue is lower than the average B-factor of all residues in the X-ray structure, (iii) the residue forms at least 3 pairs of heavy atom interatomic contacts with antibody atoms at a distance of less than or equal to 4.0 angstroms, (iv) the residue does not produce only solvent-exposed hydrogen bonds or ionic interactions, (v) if the residue is a non-aromatic polar residue (Asn, Gln, Ser, Thr, Asp, Glu, Lys or Arg), it produces at least one hydrogen bond or ionic interaction with the antibody. Calculation of binding free energy is within the capabilities of the skilled person. Preferably, an empirical force field is used to calculate the binding free energy, preferably FoldX. FoldX is familiar to the skilled person and can be used in http: / / foldxsuite.crg.eu / Publicly available. Calculation of binding free energy using FoldX is also described in Guerois et al. J. Mol. Biol. 2002; 320(2):369-87, which is incorporated herein by reference. As is familiar to the skilled person, heavy atoms are all non-hydrogen atoms (including C, N, O, S).

[0192] Therefore, the present invention also provides antibodies or antigen-binding fragments thereof that contact human TIGIT at least at residues Q56 and I109. In certain embodiments, the antibody or its antigen-binding fragment contacts human TIGIT at least at residues Q56, N58 and I109. In certain embodiments, the antibody or its antigen-binding fragment contacts human TIGIT at least at residues Q56, N58 and I109 and optionally at one or more of residues E60, I68, L73 and H76. In certain embodiments, the antibody or its antigen-binding fragment contacts human TIGIT at least at residues Q56, N58, E60, I68 L73, H76 and I109.

[0193] In certain such embodiments, the antibody or antigen-binding fragment thereof contacts human TIGIT only at residues Q56, N58, E60, I68, L73, H76, and I109.

[0194] Methods for determining the TIGIT residues contacted by antibodies or antigen-binding fragments are familiar to the skilled artisan and include X-ray crystallography, such as described in Example 23.

[0195] Also provided are isolated antibodies or antigen-binding fragments thereof that bind to the same epitope as the antibodies or antigen-binding fragments described herein.

[0196] Antibody subtype

[0197] TIGIT antibodies can adopt a variety of different embodiments in which both VH domains and VL domains are present. The term "antibody" is used in the broadest sense herein, and covers but is not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), as long as they show appropriate immunological specificity for human TIGIT protein. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homologous antibody population, i.e., except for a small amount of possible naturally occurring mutations, the individual antibodies constituting the population are the same. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different determinants (epitopes) on an antigen, each monoclonal antibody is directed against a single determinant or epitope on an antigen.

[0198] In non-limiting embodiments, the TIGIT antibodies provided herein may include the following CH1 domain and / or CL domain, the amino acid sequence of which is completely or substantially human. If the TIGIT antibody is intended for human therapeutic use, the entire constant region of the antibody or at least a portion thereof generally has an amino acid sequence that is completely or substantially human. Therefore, one or more or any combination of the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may be completely or substantially human in terms of their amino acid sequence. Such antibodies may be of any human isotype, with human IgG4 and IgG1 being particularly preferred.

[0199] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain, if present) may all have completely or substantially human amino acid sequences. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to an amino acid sequence identity of at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% with a human constant region. In this context, the term "human amino acid sequence" refers to an amino acid sequence encoded by a human immunoglobulin gene, including germline, rearranged, and somatically mutated genes. Such antibodies may be any human isotype, with human IgG4 and IgG1 being particularly preferred.

[0200] Also provided are TIGIT antibodies comprising "human" sequence constant domains that are altered relative to the human sequence by one or more amino acid additions, deletions or substitutions.

[0201] The TIGIT antibodies provided herein can be of any isotype. Antibodies intended for human therapeutic use will generally be of IgA, IgD, IgE, IgG, IgM type, often of IgG type, in which case they may belong to any of the four subclasses of IgG1, IgG2a and b, IgG3 or IgG4. In each of these subclasses, one or more amino acid substitutions, insertions or deletions within the Fc portion are permitted, or other structural modifications are performed, for example, to enhance or reduce Fc-dependent functions.

[0202] In certain preferred embodiments, the TIGIT antibodies provided herein are IgG antibodies. In certain embodiments, the antibodies according to the present invention are IgG1 antibodies. In certain alternative embodiments, the antibodies according to the present invention are IgG4 antibodies.

[0203] IgG4 antibodies are known to undergo Fab arm exchange (FAE), which can result in unpredictable pharmacodynamic properties of IgG4 antibodies. FAE has been shown to be prevented by the S228P mutation in the hinge region (Silva et al., J Biol Chem. 2015 Feb 27; 290(9): 5462–5469). Thus, in certain such embodiments in which the antibody according to the invention is an IgG4 antibody, the antibody comprises the mutation S228P—i.e., a mutation of serine to proline at position 228 (according to EU numbering).

[0204] In non-limiting embodiments, it is expected that one or more amino acid substitutions, insertions or deletions may be made in the constant region of the heavy chain and / or light chain, particularly in the Fc region. Amino acid substitutions may result in the substituted amino acid being replaced by a different naturally occurring amino acid or by a non-natural or modified amino acid. Other structural modifications are also allowed, such as changes in glycosylation patterns (e.g., additions or deletions of glycosylation sites linked by N- or O-). Depending on the intended use of the TIGIT antibody, it may be desirable to modify the antibody of the present invention with respect to its binding properties to Fc receptors (e.g., regulating effector functions).

[0205] In certain embodiments, a TIGIT antibody may comprise an Fc region of a given antibody isotype (eg, human IgG1) that is modified to reduce or substantially eliminate one or more antibody effector functions naturally associated with that antibody isotype.

[0206] As demonstrated herein, antibodies with cytolytic effector function can effectively reduce the Treg cell population, but surprisingly, do not adversely affect the conventional effector T cell population. This selectivity may allow for more robust inhibition of the regulatory effects of Tregs while preserving anti-tumor effector T cells.

[0207] Therefore, in certain alternative embodiments, the TIGIT antibody retains one or more antibody effector functions naturally associated with the antibody isotype. For example, the TIGIT antibody of the present invention can be an IgG1 antibody that retains ADCC functionality. In other embodiments, the TIGIT antibody can comprise an Fc region of a given antibody isotype (e.g., human IgG1), and the Fc region is modified to enhance one or more antibody effector functions naturally associated with the antibody isotype. In this context, "antibody effector functions" include one or more or all of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0208] In certain embodiments, the anti-TIGIT antibody is a modified antibody.

[0209] In certain embodiments, a bispecific antibody is provided, which comprises a first arm specific for TIGIT and a second arm specific for a second target. In some preferred embodiments, the second target is an immune checkpoint molecule. In certain embodiments, the second target is OX40. In certain embodiments, the second target is ICOS. In certain embodiments, the second target is GITR. In certain embodiments, the second target is 4-1BB. In certain embodiments, the second target is PD-1. In certain embodiments, the second target is PD-L1. In certain embodiments, the first arm specific for TIGIT comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of an antibody according to the present invention. In certain embodiments, the first arm comprises a heavy chain variable domain and a light chain variable domain, wherein HCDR1 comprises SEQ ID NO: 16, HCDR2 comprises SEQ ID NO: 17, HCDR3 comprises SEQ ID NO: 18, and LCDR1 comprises SEQ ID NO: 61, LCDR2 comprises SEQ ID NO: 62 and LCDR3 comprises SEQ ID NO: 63.

[0210] Monoclonal antibodies or antigen-binding fragments thereof that "cross-compete" with the TIGIT antibodies disclosed herein are those that bind to human TIGIT at sites that are identical or overlapping with sites that bind to the TIGIT antibodies of the present invention. Competitive monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, by antibody competition assays. For example, a sample of purified or partially purified human TIGIT can be bound to a solid support. Then, an antibody compound of the present invention or an antigen-binding fragment thereof and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with this antibody compound of the present invention are added. One of the two molecules is labeled. If the labeled compound and the unlabeled compound bind to separate and discontinuous sites on TIGIT, the labeled compound will have the same level of binding, regardless of whether the suspected competitive compound is present. However, if the interaction sites are identical or overlapping, the unlabeled compound will compete, and the amount of labeled compound bound to the antigen will be reduced. If the unlabeled compound is present in excess, very little (if any) labeled compound will bind.

[0211] For the purposes of the present invention, a competitive monoclonal antibody or its antigen-binding fragment is one that reduces the binding of the antibody compound of the present invention to TIGIT by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or about 99%. The details of the procedures for carrying out such competitive assays are well known in the art and can be found, for example, in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, 567-569, 1988, ISBN 0-87969-314-2. These assays can be quantified using purified antibodies. A standard curve is established by titrating an antibody against itself, i.e., using the same antibody for labeling and competitor. The ability of the unlabeled competitive monoclonal antibody or its antigen-binding fragment to inhibit the binding of the labeled molecule to the plate is titrated. The results are plotted and the concentrations necessary to achieve the desired degree of binding inhibition are compared.

[0212] The antibodies of the present invention exhibit high affinity for TIGIT and compete with CD155

[0213] In certain embodiments, the antibodies or antigen-binding fragments of the present invention exhibit high affinity for human TIGIT. In certain embodiments, the Fab fragments of the antibodies according to the present invention exhibit high affinity for TIGIT by ForteBio TM Measured in 1x10 -10 Up to 5x10 -8 M, optional 7x10 -10 Up to 4x10 -8 K in the M range D In certain embodiments, antibodies according to the present invention exhibit a -11 Up to 5x10 -9 M, optionally 2x10 -11 Up to 1x10 -9 MSD K in the range D In certain embodiments, the Fab fragments of the antibodies according to the present invention exhibit TIGIT activity as measured by Biacore TM Measured in 1x10 -10 M to 1x10 -9 M, optional 1x10 -10 Up to 7x10 -10 M, optionally 2x10 -10 Up to 7x10 -10 K in the M range D .

[0214] Table 3

[0215]

[0216]

[0217] As demonstrated in the Examples, antibody 31282 exhibits surprisingly high affinity for TIGIT expressed on transgenic cells. Thus, in certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments provided herein exhibit binding EC to human TIGIT. 50 In preferred such embodiments, the antibody or antigen-binding fragment exhibits a binding EC of about 0.05 to about 0.4 nM, preferably about 0.05 to about 0.3 nM, preferably about 0.05 to about 0.2 nM, preferably about 0.05 to about 0.15 nM. 50 In certain preferred embodiments, the antibodies or antigen-binding fragments exhibit binding to human TIGIT. 50 is about 0.1 nM. In a preferred embodiment, the antibody comprises the CDRs of antibody 31282. Preferably, Jurkat cells expressing human TIGIT are used to determine EC 50 , as described in Example 18. In certain embodiments, the antibodies or antigen-binding fragments of the invention cross-react with mouse TIGIT and / or cynomolgus monkey TIGIT.

[0218] Since the "29..." second generation antibodies are affinity matured progeny of the highly functional parent antibodies, they are expected to exhibit functional properties at least similar or equivalent to those of the parent antibodies, and vice versa.

[0219] As described herein, in certain embodiments, the antibodies or antigen-binding fragments of the invention have equivalent affinity for TIGIT expressed by CD8 T cells and TIGIT expressed by Treg cells. As used herein, if the affinity for CD8 T cells is in the range of 0.5-1.5 times the affinity for Treg cells, the antibody or antigen-binding fragment has "equivalent affinity" for CD8 T cells and Treg cells. For example, an antibody having equivalent affinity for CD8 T cells and Treg cells that exhibits an affinity of 0.03 nM for Treg cells will exhibit an affinity for CD8 T cells in the range of 0.015-0.045 nM.

[0220] Table 3 provides a summary of the affinity properties of the anti-TIGIT antibodies of the invention, wherein grey cells indicate parental antibody clones, with second and third generation antibodies of each lineage shown directly below the corresponding parent antibody (see also Table 2).

[0221] As demonstrated in the Examples, antibody 31282 exhibits surprisingly high affinity for TIGIT expressed on human primary CD8+ T cells. Thus, in certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments provided herein exhibit binding EC to human TIGIT. 50 In preferred such embodiments, the antibody or antigen-binding fragment exhibits a binding EC of about 0.05 to about 0.4 nM, preferably about 0.1 to about 0.3 nM. 50 In certain preferred embodiments, the antibodies or antigen-binding fragments exhibit binding to human TIGIT. 50 is about 0.2 nM. In a preferred embodiment, the antibody or antigen-binding fragment comprises the CDRs of antibody 31282. Preferably, EC is determined using CD8+ T cells from human PBMCs (preferably from healthy individuals) as described in Example 18. 50 .

[0222] As demonstrated in the accompanying examples, in certain embodiments, the antibodies or antigen-binding fragments of the present invention exhibit high affinity for CD8 T cells expressing TIGIT and high affinity for Treg cells expressing TIGIT. In certain embodiments, the antibodies or antigen-binding fragments of the present invention exhibit high affinity for CD8 T cells expressing TIGIT and Treg cells expressing TIGIT characterized by EC 50 An affinity of less than 0.5 nM, preferably less than 0.3 nM, preferably less than 0.2 nM. In certain embodiments, the antibodies or antigen-binding fragments of the invention exhibit equivalent affinity for CD8 T cells expressing TIGIT and Treg cells expressing TIGIT.

[0223] The antibodies according to the present invention (e.g., antibody 31282) exhibit surprisingly high affinity for CD8+ T cells from cancer patients. This is particularly advantageous because increasing the effector activity of T cells from cancer patients by inhibiting TIGIT signaling can produce more effective tumor control. Therefore, in certain embodiments, the anti-TIGIT antibodies or antigen-binding fragments provided herein exhibit a binding EC of less than 0.5 nM for human TIGIT on human CD8+ T cells from cancer patients. 50 In preferred such embodiments, the antibody or antigen-binding fragment exhibits a binding EC of about 0.05 to about 0.4 nM, preferably about 0.1 to about 0.3 nM. 50 In certain preferred embodiments, the antibody or antigen-binding fragment exhibits an EC of about 0.1 nM to about 0.2 nM for human TIGIT. 50In a preferred embodiment, the antibody or antigen-binding fragment comprises the CDRs of antibody 31282. Preferably, EC is determined using CD8+ T cells from PBMCs obtained from cancer patients. 50 , as described in Example 18.

[0224] As demonstrated in the accompanying examples, in certain embodiments, the antibodies or antigen-binding fragments of the invention compete with CD155 / PVR for binding to TIGIT. In certain embodiments, the antibodies or antigen-binding fragments of the invention exhibit competition with CD155, characterized in that IC 50 is 0.2 nM or less, preferably 0.1 nM or less. In certain embodiments, the antibody or antigen-binding fragment exhibits competition with CD155, characterized in that IC 50 In certain preferred embodiments, the IC 50 Without wishing to be bound by theory, it is expected that antibodies that compete with CD155 for binding to TIGIT will reduce the level of CD155-induced TIGIT-mediated signaling, thereby increasing the level of effector T cell activation.

[0225] The invention also provides "affinity variants" of the antibodies described herein.

[0226] The present invention also provides an isolated antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment described herein for binding to human TIGIT.

[0227] The antibodies of the present invention promote pro-inflammatory T cell activity

[0228] The antibodies according to the invention (e.g., antibody 31282) are surprisingly effective in promoting the pro-inflammatory activity of CD8+ T cells. As demonstrated in the Examples, the antibodies or antigen-binding fragments according to the invention (particularly 31282) are more effective in promoting pro-inflammatory CD8+ T cell activity (indicated by IFNg release) than comparative anti-TIGIT antibodies (see Fig.24 ). This improved efficacy relative to the control antibody was demonstrated in transgenic Jurkat reporter cells expressing TIGIT and primary CD8 T cells. Thus, in certain embodiments, as described in Example 19, the anti-TIGIT antibodies or antigen-binding fragments provided herein exhibit less than 5 nM activation EC against human TIGIT expressed by Jurkat reporter cells. 50 In preferred such embodiments, the antibody or antigen-binding fragment exhibits an EC of about 1 nM to about 4 nM, preferably about 2 nM to about 4 nM. 50 .

[0229] In certain embodiments, as described in Example 19, the anti-TIGIT antibodies or antigen-binding fragments provided herein exhibit an activation EC of less than 0.4 nM on CD8 T cells from healthy individuals. 50 CD8 T cell activity (i.e., pro-inflammatory activity) can be measured by the production of inflammatory cytokines (e.g., IFNg). In preferred such embodiments, the antibody or antigen-binding fragment exhibits an EC of about 0.05 nM to about 0.4 nM, preferably about 0.1 nM to about 0.2 nM. 50 Preferably, EC is determined using CD8+ T cells from PBMCs obtained from healthy individuals. 50 , as described in Example 19.

[0230] In the accompanying examples, it is additionally and surprisingly demonstrated that the anti-TIGIT antibodies provided are effective in increasing the activity of gamma-delta (γδ, or g / d) T cells (i.e., T cells expressing γδTCR subunits that are completely different from conventional αβTCR subunits). Such γδT cells form a unique and important component of the immune system, and the ability of the antibodies provided herein to promote the activity of these cells highlights the utility of the antibodies.

[0231] Therefore, the present invention also provides a method for promoting γδT cell activity, the method comprising contacting a γδT cell population with an anti-TIGIT antibody. In certain embodiments, the method is performed in vitro. In certain embodiments, the method is performed in a human subject. In certain such embodiments, the human subject suffers from cancer. In certain embodiments, the anti-TIGIT antibody or antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of an antibody according to the present invention. In certain embodiments, the anti-TIGIT antibody comprises a heavy chain variable domain and a light chain variable domain, wherein HCDR1 comprises SEQ ID NO: 16, HCDR2 comprises SEQ ID NO: 17, HCDR3 comprises SEQ ID NO: 18, and LCDR1 comprises SEQ ID NO: 61, LCDR2 comprises SEQ ID NO: 62 and LCDR3 comprises SEQ ID NO: 63.

[0232] Selective depletion of T-REG cells

[0233] As demonstrated herein, anti-TIGIT antibodies are able to selectively deplete Treg cells expressing TIGIT. That is, anti-TIGIT antibodies reduce the proportion of Treg cells expressing TIGIT relative to the total T cell population to a greater extent than they reduce the proportion of effector or memory CD4 or CD8 T cells.

[0234] In certain embodiments, the antibody or antigen-binding fragment thereof selectively depletes Treg cells expressing TIGIT.

[0235] Such selective depletion of Treg cells expressing TIGIT can be achieved by selective lysis of Treg cells expressing TIGIT (e.g., by ADCC or CDC (see Fig. 20 , Fig.21 and Fig.25 )) is mediated. It is believed that Tregs expressing TIGIT are stronger regulatory cells than Tregs that do not express TIGIT. Without wishing to be bound by theory, it is expected that selective depletion by lysing Treg cells expressing TIGIT will improve T cell effector function (e.g., T cell-mediated cytotoxicity, proinflammatory cytokine release) by depleting the total number of Treg cells and depleting those Treg cells that exhibit stronger regulatory function. Fig.24 This enhanced T cell effector function was demonstrated in .

[0236] Thus, in certain embodiments, the antibodies or antigen-binding fragments of the invention selectively lyse Treg cells expressing TIGIT.

[0237] The selective depletion of Treg cells expressing TIGIT can also be mediated by inducing TIGIT receptor internalization so that it is no longer expressed on the cell membrane. Without wishing to be bound by theory, by inducing TIGIT internalization, TIGIT+Treg cells become TIGIT-Treg cells, and it is expected that the regulatory function of these cells will become less strong (because TIGIT+Treg is a stronger regulatory cell). Due to receptor internalization and the subsequent decrease in the regulatory efficacy of these Tregs, it is expected that T cell effector function will increase. Therefore, in certain embodiments, the antibody or antigen-binding fragment of the present invention inhibits the inhibitory activity of Treg cells expressing TIGIT, preferably by inducing TIGIT-expressing Treg cells to internalize TIGIT to inhibit.

[0238] It is particularly advantageous that the anti-TIGIT antibodies according to the present invention exhibit high affinity to CD8 T cells and Treg cells, and also exhibit selective depletion of Treg cells, thereby promoting T cell effector functions through two mechanisms. The maintenance of antibody effector functions (e.g., ADCC, CDC) leads to effective depletion of Treg, and the selectivity means that the antibody effector function does not lead to unnecessary depletion of effector T cells. This selectivity is particularly surprising because previous attempts to produce anti-TIGIT antibodies have attempted to eliminate antibody effector functions to avoid the lysis of effector T cells expressing TIGIT. Moreover, since the TIGIT antibodies of the present invention exhibit affinity to effector T cells (e.g., CD8 T cells), TIGIT-mediated signaling in these cells can be inhibited by competing for CD155 binding and / or inducing TIGIT internalization on effector T cells. In combination, these effects of the antibodies of the present invention can lead to a significant upregulation of T cell effector functions.

[0239] Other surprising favorable properties exhibited by antibodies and antigen-binding fragments according to the present invention include improving the T cell effector function (e.g., release of proinflammatory cytokines) of tumor infiltrating lymphocytes (TILs). Exposure to the tumor microenvironment can cause TILs to exhibit anergy or so-called "exhaustion" phenotype, which may be caused by excessive antigen exposure and / or immunosuppressive tumor microenvironment. It is desirable to enhance the effector function of TILs because it is these cells that infiltrate the tumor itself and are therefore located in the loci that are most suitable for reducing tumor size or growth; however, due to the anergy or exhaustion phenotype of many TILs, it is expected to be difficult to enhance their effector function. Therefore, the increase in proinflammatory reactions from TILs after exposure to the antibodies of the present invention is surprising, and indicates that the antibodies can be particularly effective therapeutic agents.

[0240] Other surprising favorable properties exhibited by antibodies and antigen-binding fragments include the ability to enhance the pro-inflammatory activity of gamma-delta (γδ) T cells. The ability of anti-TIGIT antibodies to promote the activity of unconventional T cells such as γδ T cells has not been previously reported, and this ability provides the potential for treating diseases other than cancer in which γδ T cells are known to be important. For example, γδ T cells are reported to be involved in responses to pathogenic infections (bacteria, viruses (e.g., CMV), fungi) and to play a role in preventing autoimmune diseases. In addition, the surprising ability to promote the activity of unconventional T cells provides further efficacy for the anti-tumor effects of the antibodies.

[0241] In another aspect, a method for selectively depleting Treg cells from a T cell population is provided, the method comprising contacting a T cell population with an anti-TIGIT antibody or an antigen-binding fragment thereof, whereby the anti-TIGIT antibody selectively depletes a Treg cell population. In certain embodiments, the method is performed in vitro. In certain embodiments, the method is performed in vivo in a human subject. In certain such embodiments, the human subject suffers from cancer. In certain embodiments, the anti-TIGIT antibody or antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody according to the present invention. In certain embodiments, the anti-TIGIT antibody comprises a heavy chain variable domain and a light chain variable domain, wherein HCDR1 comprises SEQ ID NO: 16, HCDR2 comprises SEQ ID NO: 17, HCDR3 comprises SEQ ID NO: 18, and LCDR1 comprises SEQ ID NO: 61, LCDR2 comprises SEQ ID NO: 62, and LCDR3 comprises SEQ ID NO: 63.

[0242] As demonstrated in the accompanying examples, the present invention also provides anti-TIGIT antibodies that do not compete with CD155 / PVR for binding to TIGIT. Thus, in another aspect, the present invention provides human TIGIT antibodies or antigen-binding fragments thereof that do not compete with CD155 / PVR for binding to human TIGIT. In certain such embodiments, the Fab fragments of the CD155 non-competitive anti-TIGIT antibodies according to the present invention exhibit binding to TIGIT by ForteBio TM Measured in 5x10 -9 Up to 5x10 -8 M, optional 1x10 -8 Up to 3x10 -8 K in the M range D .

[0243] In certain preferred embodiments, the antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein HCDR1 comprises SEQ ID NO: 280, HCDR2 comprises SEQ ID NO: 281, HCDR3 comprises SEQ ID NO: 282, and LCDR1 comprises SEQ ID NO: 292, LCDR2 comprises SEQ ID NO: 293, and LCDR3 comprises SEQ ID NO: 294. In certain such embodiments, the heavy chain variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 333, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto, and the light chain variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 334, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0244] Nevertheless, embodiments in which the amino acid sequence of the VH domain exhibits less than 100% sequence identity to the sequence set forth in SEQ ID NO: 333 may comprise heavy chain CDRs identical to HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 333 (SEQ ID NOs: 280, 281, and 282, respectively), however exhibiting amino acid sequence variation within the framework regions. Similarly, embodiments in which the amino acid sequence of the VL domain exhibits less than 100% sequence identity to the sequence set forth in SEQ ID NO: 334 may comprise light chain CDRs identical to LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 334 (SEQ ID NOs: 292, 293, and 294, respectively), however exhibiting amino acid sequence variation within the framework regions.

[0245] In certain preferred embodiments, the antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein HCDR1 comprises SEQ ID NO: 353, HCDR2 comprises SEQ ID NO: 354, HCDR3 comprises SEQ ID NO: 355, and LCDR1 comprises SEQ ID NO: 356, LCDR2 comprises SEQ ID NO: 357, and LCDR3 comprises SEQ ID NO: 358. In certain such embodiments, the heavy chain variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 367, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto, and the light chain variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 368, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0246] Nevertheless, embodiments in which the amino acid sequence of the VH domain exhibits less than 100% sequence identity with the sequence set forth in SEQ ID NO: 367 may comprise heavy chain CDRs identical to HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 367 (SEQ ID NOs: 353, 354, and 355, respectively), yet exhibit amino acid sequence variation within the framework regions. Similarly, embodiments in which the amino acid sequence of the VL domain exhibits less than 100% sequence identity with the sequence set forth in SEQ ID NO: 368 may comprise light chain CDRs identical to LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 368 (SEQ ID NOs: 356, 357, and 358, respectively), yet exhibit amino acid sequence variation within the framework regions.

[0247] Polynucleotides, vectors and expression systems

[0248] The present invention also provides a polynucleotide molecule encoding the TIGIT antibody of the present invention, an expression vector containing a nucleotide sequence encoding the TIGIT antibody of the present invention operably linked to a regulatory sequence allowing expression of the antigen-binding polypeptide in a host cell or a cell-free expression system, and a host cell or a cell-free expression system containing the expression vector.

[0249] Polynucleotide molecules encoding TIGIT antibodies of the present invention include, for example, recombinant DNA molecules. As used herein, the terms "nucleic acid," "polynucleotide," or "polynucleotide molecule" are interchangeable and refer to any single-stranded or double-stranded DNA or RNA molecule, and if single-stranded, also refer to molecules of its complementary sequence. When discussing nucleic acid molecules, the sequence or structure of a specific nucleic acid molecule may be described herein according to the conventional practice of providing sequences in the 5' to 3' direction. In some embodiments of the present invention, nucleic acids or polynucleotides are "isolated." When applied to nucleic acid molecules, the term refers to a nucleic acid molecule separated from a sequence that is closely adjacent to it in the naturally occurring genome of an organism from which it is derived. For example, an "isolated nucleic acid" may include a DNA molecule that is inserted into a vector (such as a plasmid or viral vector), or integrated into the genomic DNA of a prokaryotic cell or eukaryotic cell or a non-human host organism. When applied to RNA, the term "isolated polynucleotide" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been purified / separated from other nucleic acids with which it is associated in its natural state (i.e., in a cell or tissue). An isolated polynucleotide (DNA or RNA) may also refer to a molecule that has been directly produced by biological or synthetic means and separated from other components present during its production.

[0250] In order to recombinantly produce a TIGIT antibody according to the present invention, a recombinant polynucleotide encoding the antibody can be prepared (using standard molecular biology techniques) and inserted into a replicable vector for expression in a selected host cell or a cell-free expression system. Suitable host cells can be prokaryotes, yeast or higher eukaryotic cells, particularly mammalian cells. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59-74, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980; or CHO-derived clones such as CHO-K1, ATCC CCL-61, Kao and Puck, Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells, Proc. Natl. Acad. Sci. 60: 1275-1281, 1968); mouse Sertoli cells (TM4; Mather, Biol. Reprod. 23: 243-252, 1980); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581; ATCC CRL8287) or NS0 (HPA culture collection number 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383: 44-68, 1982); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2), and DSM's PERC-6 cell line. Expression vectors suitable for use in each of these host cells are also generally known in the art.

[0251] It should be noted that the term "host cell" generally refers to a cultured cell line. An intact human into which an expression vector encoding an antigen-binding polypeptide according to the invention has been introduced is specifically excluded from the definition of "host cell".

[0252] In an important aspect, the present invention also provides a method for producing the TIGIT antibody of the present invention, the method comprising culturing a host cell (or a cell-free expression system) containing a polynucleotide (e.g., an expression vector) encoding the TIGIT antibody under conditions that allow expression of the TIGIT antibody, and recovering the expressed TIGIT antibody. This recombinant expression method can be used for large-scale production of TIGIT antibodies according to the present invention, including monoclonal antibodies intended for human therapeutic use. Suitable vectors, cell lines, and production methods for large-scale production of recombinant antibodies suitable for in vivo therapeutic use are generally available in the art and are well known to those skilled in the art.

[0253] Thus, according to the present invention, there is provided an isolated polynucleotide or a combination of isolated polynucleotides encoding an antibody or antigen-binding fragment comprising a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the combination is selected from the group consisting of:

[0254] (i) a HCDR1 comprising SEQ ID NO: 16, a HCDR2 comprising SEQ ID NO: 17, a HCDR3 comprising SEQ ID NO: 18, a LCDR1 comprising SEQ ID NO: 61, a LCDR2 comprising SEQ ID NO: 62, and a LCDR3 comprising SEQ ID NO: 63;

[0255] (ii) HCDR1 comprising SEQ ID NO:4, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:6, LCDR1 comprising SEQ ID NO:49, LCDR2 comprising SEQ ID NO:50, and LCDR3 comprising SEQ ID NO:51;

[0256] (iii) HCDR1 comprising SEQ ID NO:7, HCDR2 comprising SEQ ID NO:8, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:52, LCDR2 comprising SEQ ID NO:53, and LCDR3 comprising SEQ ID NO:54;

[0257] (iv) HCDR1 comprising SEQ ID NO: 10, HCDR2 comprising SEQ ID NO: 11, HCDR3 comprising SEQ ID NO: 12, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 56, and LCDR3 comprising SEQ ID NO: 57;

[0258] (v) HCDR1 comprising SEQ ID NO: 13, HCDR2 comprising SEQ ID NO: 14, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 58, LCDR2 comprising SEQ ID NO: 59, and LCDR3 comprising SEQ ID NO: 60;

[0259] (vi) HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 3, LCDR1 comprising SEQ ID NO: 46, LCDR2 comprising SEQ ID NO: 47, and LCDR3 comprising SEQ ID NO: 48;

[0260] (vii) HCDR1 comprising SEQ ID NO: 19, HCDR2 comprising SEQ ID NO: 20, HCDR3 comprising SEQ ID NO: 21, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 65, and LCDR3 comprising SEQ ID NO: 66;

[0261] (viii) HCDR1 comprising SEQ ID NO:22, HCDR2 comprising SEQ ID NO:23, HCDR3 comprising SEQ ID NO:24, LCDR1 comprising SEQ ID NO:67, LCDR2 comprising SEQ ID NO:68, and LCDR3 comprising SEQ ID NO:69;

[0262] (ix) a HCDR1 comprising SEQ ID NO:25, a HCDR2 comprising SEQ ID NO:26, a HCDR3 comprising SEQ ID NO:27, a LCDR1 comprising SEQ ID NO:70, a LCDR2 comprising SEQ ID NO:71, and a LCDR3 comprising SEQ ID NO:72;

[0263] (x) a HCDR1 comprising SEQ ID NO:28, a HCDR2 comprising SEQ ID NO:29, a HCDR3 comprising SEQ ID NO:30, a LCDR1 comprising SEQ ID NO:73, a LCDR2 comprising SEQ ID NO:74, and a LCDR3 comprising SEQ ID NO:75;

[0264] (xi) a HCDR1 comprising SEQ ID NO:31, a HCDR2 comprising SEQ ID NO:32, a HCDR3 comprising SEQ ID NO:33, a LCDR1 comprising SEQ ID NO:76, a LCDR2 comprising SEQ ID NO:77, and a LCDR3 comprising SEQ ID NO:78;

[0265] (xii) a HCDR1 comprising SEQ ID NO:34, a HCDR2 comprising SEQ ID NO:35, a HCDR3 comprising SEQ ID NO:36, a LCDR1 comprising SEQ ID NO:79, a LCDR2 comprising SEQ ID NO:80, and a LCDR3 comprising SEQ ID NO:81;

[0266] (xiii) HCDR1 comprising SEQ ID NO:37, HCDR2 comprising SEQ ID NO:38, HCDR3 comprising SEQ ID NO:39, LCDR1 comprising SEQ ID NO:82, LCDR2 comprising SEQ ID NO:83, and LCDR3 comprising SEQ ID NO:84;

[0267] (xiv) a HCDR1 comprising SEQ ID NO:40, a HCDR2 comprising SEQ ID NO:41, a HCDR3 comprising SEQ ID NO:42, a LCDR1 comprising SEQ ID NO:85, a LCDR2 comprising SEQ ID NO:86, and a LCDR3 comprising SEQ ID NO:87;

[0268] (xv) a HCDR1 comprising SEQ ID NO:43, a HCDR2 comprising SEQ ID NO:44, a HCDR3 comprising SEQ ID NO:45, a LCDR1 comprising SEQ ID NO:88, a LCDR2 comprising SEQ ID NO:89, and a LCDR3 comprising SEQ ID NO:90;

[0269] (xvi) a HCDR1 comprising SEQ ID NO: 271, a HCDR2 comprising SEQ ID NO: 272, a HCDR3 comprising SEQ ID NO: 273, a LCDR1 comprising SEQ ID NO: 283, a LCDR2 comprising SEQ ID NO: 284, and a LCDR3 comprising SEQ ID NO: 285;

[0270] (xvii) a HCDR1 comprising SEQ ID NO: 274, a HCDR2 comprising SEQ ID NO: 275, a HCDR3 comprising SEQ ID NO: 276, a LCDR1 comprising SEQ ID NO: 286, a LCDR2 comprising SEQ ID NO: 287, and a LCDR3 comprising SEQ ID NO: 288;

[0271] (xviii) HCDR1 comprising SEQ ID NO:277, HCDR2 comprising SEQ ID NO:278, HCDR3 comprising SEQ ID NO:279, LCDR1 comprising SEQ ID NO:289, LCDR2 comprising SEQ ID NO:290, and LCDR3 comprising SEQ ID NO:291.

[0272] In certain embodiments, an isolated polynucleotide or a combination of isolated polynucleotides is provided that encodes an antibody or antigen-binding fragment comprising a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein:

[0273] (i) HCDR1 comprises or consists of SEQ ID NO: 16, HCDR2 comprises or consists of SEQ ID NO: 17, HCDR3 comprises or consists of SEQ ID NO: 18, LCDR1 comprises or consists of SEQ ID NO: 61, LCDR2 comprises or consists of SEQ ID NO: 62, and LCDR3 comprises or consists of SEQ ID NO: 63.

[0274] In addition, according to the present invention, there is provided an isolated polynucleotide or a combination of isolated polynucleotides encoding the antibodies or antigen-binding fragments described herein. In certain embodiments, there is provided an isolated polynucleotide encoding the antibody 31282 or antigen-binding fragment thereof provided herein.

[0275] In addition, according to the present invention, an isolated polynucleotide encoding the VH and / or VL domain of an anti-TIGIT antibody is provided, wherein the polynucleotide comprises one or more sequences selected from the group consisting of SEQ ID Nos: 241-270, 335-342, and 369-370. In certain embodiments, the isolated polynucleotide comprises a sequence according to SEQ ID NO: 251 and / or a sequence according to SEQ ID NO: 252. In certain embodiments in which the polynucleotide comprises a sequence according to SEQ ID NO: 251 and a sequence according to SEQ ID NO: 252, the sequences are contiguous. In certain embodiments in which the polynucleotide comprises a sequence according to SEQ ID NO: 251 and a sequence according to SEQ ID NO: 252, the sequences are not contiguous.

[0276] Furthermore, according to the present invention, an expression vector is provided, which comprises a polynucleotide according to the present invention operably linked to a regulatory sequence which allows the antigen-binding polypeptide to be expressed in a host cell or a cell-free expression system.

[0277] Furthermore, according to the present invention, a host cell or a cell-free expression system containing the expression vector according to the present invention is provided.

[0278] In addition, according to the present invention, a method for producing a recombinant antibody or an antigen-binding fragment thereof is provided, the method comprising culturing a host cell or a cell-free expression system according to the present invention under conditions allowing expression of the antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

[0279] Pharmaceutical composition

[0280] Also provided herein is a pharmaceutical composition comprising an antibody or antigen-binding fragment according to the present invention formulated with one or more pharmaceutically acceptable carriers or excipients. Such compositions may comprise a TIGIT antibody or a combination of (e.g., two or more different) TIGIT antibodies. The techniques for preparing antibodies for human therapeutic use are well known in the art and are reviewed in, for example, Wang et al., Journal of Pharmaceutical Sciences, Vol. 96, pp. 1-26, 2007.

[0281] The TIGIT antibodies and pharmaceutical compositions provided herein can be used in therapy, particularly therapeutic treatment of diseases, particularly disorders that benefit from inhibition of TIGIT function.

[0282] Combination Products

[0283] As demonstrated herein, the antibodies of the present invention or their antigen-binding fragments are particularly effective when administered in combination with immune checkpoint inhibitors, particularly anti-ICOS antagonist antibodies or anti-PD-1 antibodies (i.e., antagonist antibodies specific for human immunomodulatory molecule PD-1). Compared to any single antibody, the administration of anti-TIGIT antibodies in combination with anti-ICOS or anti-PD-1 antibodies results in a synergistic reduction in tumor growth. It is expected that similar effects will be observed using a combination of anti-TIGIT antibodies and anti-PD-L1 antibodies according to the present invention.

[0284] It is further demonstrated herein that the antibodies of the present invention or their antigen-binding fragments are particularly effective when administered in combination with agonist antibodies specific for immune checkpoint co-stimulatory molecules, particularly anti-4-1BB, anti-OX40 or anti-GITR agonist antibodies. Compared to any single antibody, the administration of anti-TIGIT antibodies in combination with anti-4-1BB, anti-OX40 or anti-GITR agonist antibodies results in a synergistic reduction in tumor growth.

[0285] In another aspect, a combination product is provided, which comprises an anti-TIGIT antibody or an antigen-binding fragment thereof, and one or more of a chemotherapeutic agent, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody, an anti-GITR antibody, and an anti-ICOS antibody. In certain preferred embodiments, the anti-TIGIT antibody or antigen-binding fragment is an antibody or antigen-binding fragment provided according to the present invention. In a most preferred embodiment, the anti-TIGIT antibody or antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein:

[0286] HCDR1 comprises SEQ ID NO: 16 (YTFTSYYMH),

[0287] HCDR2 comprises SEQ ID NO: 17 (VIGPSGASTSYAQKFQG),

[0288] HCDR3 comprises SEQ ID NO: 18 (ARDHSDYWSGIMEV),

[0289] LCDR1 comprises SEQ ID NO:61 (RASQSVRSSYLA),

[0290] LCDR2 comprises SEQ ID NO:62 (GASSRAT), and

[0291] LCDR3 comprises SEQ ID NO:63 (QQYFSPPWT).

[0292] Also provided is a combination as provided herein for use in a method of treating cancer or a viral infection, optionally wherein the viral infection is a CMV infection.Also provided is a combination as provided herein for use in a method provided herein.

[0293] As used herein, where two or more active agents are provided as a "combination," "therapeutic combination," or "combination therapy" (these terms are used interchangeably), this does not require or preclude the active agents from being formulated into a single composition. Combination therapy is given a conventional explanation: two or more active agents are administered so that the patient can obtain benefit from each agent. "Combination therapy" does not necessitate co-formulation, co-administration, simultaneous administration, or fixed dose formulation.

[0294] Treatment

[0295] The TIGIT antibodies or antigen-binding fragments thereof and pharmaceutical compositions provided herein can be used to inhibit the growth of cancerous tumor cells in vivo and are therefore useful in treating tumors.

[0296] Therefore, other aspects of the invention relate to methods of inhibiting tumor cell growth in human patients, and methods of treating or preventing cancer, comprising administering to a patient in need thereof an effective amount of a TIGIT antibody or antigen-binding fragment as described herein, a pharmaceutical composition as described herein, or a combination as described herein.

[0297] Another aspect of the present invention provides a TIGIT antibody or antigen binding fragment as described herein for use in inhibiting tumor cell growth in a human patient. Yet another aspect of the present invention provides a TIGIT antibody or antigen binding fragment as described herein for use in treating or preventing cancer in a human patient.

[0298] On the other hand, the present invention provides a method for selectively depleting Treg cells in cancer patients, the method comprising administering an anti-TIGIT antibody or an antigen-binding fragment thereof to the patient. In certain embodiments, the anti-TIGIT antibody binds to an epitope on human TIGIT, the epitope comprising residues Q56, N58, E60, I68 L73, H76 and I109, preferably consisting of residues Q56, N58, E60, I68 L73, H76 and I109. In certain embodiments, the anti-TIGIT antibody is an anti-TIGIT antibody provided herein.

[0299] In certain embodiments, an anti-TIGIT antibody comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: HCDR1 comprises or consists of SEQ ID NO:16 (YTFTSYYMH), HCDR2 comprises or consists of SEQ ID NO:17 (VIGPSGASTSYAQKFQG), HCDR3 comprises or consists of SEQ ID NO:18 (ARDHSDYWSGIMEV), LCDR1 comprises or consists of SEQ ID NO:61 (RASQSVRSSYLA), LCDR2 comprises or consists of SEQ ID NO:62 (GASSRAT), and LCDR3 comprises or consists of SEQ ID NO:63 (QQYFSPPWT).

[0300] In certain preferred embodiments, the patient to be treated has a cancer selected from the group consisting of kidney cancer (e.g., renal cell carcinoma), breast cancer, brain tumor, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, B-cell lymphoma (e.g., CLL), T-cell lymphoma (e.g., Sezary syndrome)), nasopharyngeal carcinoma, melanoma (e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), skin cancer, cutaneous or intraocular malignant melanoma In some embodiments, the inhibited cancer is lung cancer, bladder cancer, breast cancer, kidney cancer (e.g., renal cancer), head and neck cancer (e.g., HNSCC), or colon cancer (e.g., colon adenocarcinoma). In some embodiments, the cancer is colon cancer (e.g., colon adenocarcinoma) or lung cancer. In some embodiments, the cancer is blood cancer. In some such embodiments, the cancer is lymphoma. In certain embodiments, the cancer is T-cell lymphoma or B-cell lymphoma.

[0301] In certain embodiments, the methods of treating cancer further comprise administering an additional therapeutic agent, such as a chemotherapeutic agent.

[0302] As demonstrated herein, the antibodies of the present invention or their antigen-binding fragments are particularly effective when administered in combination with immune checkpoint inhibitors, particularly anti-ICOS antagonist antibodies or anti-PD-1 antibodies (i.e., antagonist antibodies specific to human immunomodulatory molecule PD-1). Compared to any single antibody, the administration of anti-TIGIT antibodies in combination with anti-ICOS or anti-PD-1 antibodies results in a synergistic reduction in tumor growth. It is expected that similar effects will be observed using a combination of anti-TIGIT antibodies and anti-PD-L1 antibodies according to the present invention.

[0303] It is also demonstrated herein that the antibodies of the present invention or their antigen-binding fragments are particularly effective when administered in combination with agonist antibodies specific for immune checkpoint co-stimulatory molecules, particularly anti-4-1BB, anti-OX40 or anti-GITR agonist antibodies. Compared to any single antibody, the administration of anti-TIGIT antibodies in combination with anti-4-1BB, anti-OX40 or anti-GITR agonist antibodies results in a synergistic reduction in tumor growth.

[0304] Therefore, the present invention also provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of an anti-TIGIT antibody or an antigen-binding fragment thereof according to the present invention, and also administering an effective amount of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody and an anti-GITR antibody, or an anti-ICOS antibody.

[0305] In addition, the data provided herein demonstrating that anti-TIGIT antibodies can increase the activity of γδ cells as well as conventional T cells indicate that anti-TIGIT antibodies can be used to treat diseases other than cancer. Specifically, γδT cells are known to be important in responding to infections (e.g., bacterial, fungal, or viral infections). As shown in Example 29, when contacted with anti-TIGIT antibodies, γδT cells from CMV seropositive subjects exhibited significantly increased activation, characterized by increased IFNg secretion. The ability to promote γδT cell activation in CMV patients in this way indicates that administration of anti-TIGIT antibodies will promote the antiviral activity of γδT cells.

[0306] Therefore, provided herein is a method for treating a viral infection in a subject, the method comprising administering an effective amount of an anti-TIGIT antibody or an antigen-binding fragment thereof. Also provided is a method for treating a viral infection in a subject, the method comprising administering to the subject an effective amount of an anti-TIGIT antibody or antigen-binding fragment or a pharmaceutical composition provided herein, thereby treating the viral infection. In a preferred embodiment, the viral infection is a CMV infection.

[0307] In certain embodiments, the method further comprises administering one or more additional therapeutic agents. In certain embodiments, the one or more therapeutic agents are selected from: anti-PD1 antibodies, anti-PD-L1 antibodies, anti-41BB antibodies, anti-OX40 antibodies, anti-GITR antibodies, and anti-ICOS antibodies.

[0308] As demonstrated in the Examples, the anti-TIGIT antibodies disclosed herein effectively promote T cell activity, particularly pro-inflammatory T cell activity. T cell activity can be measured by methods familiar to those skilled in the art, such as by measuring the production of IFNg as described in the Examples.

[0309] Thus, also provided herein is a method of promoting T cell activity, the method comprising contacting a population of T cells with an antibody or antigen-binding fragment described herein.

[0310] In certain embodiments, the method for promoting T cell activity is performed in vitro. In certain embodiments, the method for promoting T cell activity is performed in vivo in a human subject. In certain such embodiments, the human subject suffers from cancer. In certain embodiments, the human subject suffers from a viral infection, such as CMV infection.

[0311] In certain embodiments, the method promotes conventional αβ T cell activity. In certain embodiments, the method promotes CD4 T cell activity. In certain embodiments, the method promotes CD8 T cell activity. In certain embodiments, the method promotes γδ (gamma-delta) T cell activity.

[0312] It is also demonstrated in the examples that when used in combination with anti-PD1 antibodies, anti-PD-L1 antibodies, anti-41BB antibodies, anti-OX40 antibodies, anti-GITR antibodies or anti-ICOS antibodies, the anti-TIGIT antibodies disclosed herein will be particularly effective in promoting T cell activity. Significantly, the combination provides a synergistic (i.e., higher than addition) increase in T cell activity.

[0313] Therefore, in certain embodiments, the method of promoting T cell activity further comprises contacting the T cell population with one or more of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody, an anti-GITR antibody, and an anti-ICOS antibody.

[0314] Variations and equivalents of the embodiments of the invention described herein that do not depart from the spirit and scope of the invention will be familiar to the skilled artisan.The invention will be further understood with reference to the following non-limiting examples.

[0315] Example

[0316] Example 1: Selection of TIGIT antigen binding protein

[0317] The TIGIT ABP is selected from a synthetic library of human antibodies expressed and presented on the surface of yeast cells in IgG format, generally as described, for example, in WO2009036379; WO2010105256; WO2012009568; and Xu et al., Protein Eng Des Sel., Vol. 26(10), pp. 663-670 (2013), and more specifically as provided below. Figures 1 to 6 The sequences and characteristics of ABPs isolated from recombinant libraries are provided in.

[0318] As previously described, eight 9 The original human synthetic yeast library of diversity was propagated (see, e.g., Xu et al., 2013; WO2009036379; WO2010105256 and WO2012009568). For the first two rounds of selection, magnetic bead sorting technology using the Miltenyi MACS system was performed as described (see, e.g., Siegel et al., 2004). Briefly, yeast cells (about 10 10 cells / library) were incubated with biotinylated TIGIT-Fc antigen (Creative Biomart) in FACS wash buffer (phosphate buffered saline (PBS) / 0.1% bovine serum albumin (BSA)). After washing once with 50 ml of ice-cold wash buffer, the cell pellet was resuspended in 40 mL of wash buffer, and Streptavidin MicroBeads (500 μl) were added to the yeast and incubated at 4°C for 15 minutes. Next, the yeast was precipitated, resuspended in 5 mL of wash buffer, and loaded onto a Miltenyi LS column. After loading 5 mL, the column was washed 3 times with 3 ml of FACS wash buffer. The column was then removed from the magnetic field, the yeast was eluted with 5 mL of growth medium, and then grown overnight. The following rounds of sorting were performed using flow cytometry. Approximately 1×10 8Yeast pellets were washed three times with wash buffer and incubated with biotinylated TIGIT-Fc fusion antigen (10 nM) at room temperature under equilibrium conditions. The yeast were then washed twice and stained with LC-FITC (1:100 dilution) and SA-633 (1:500 dilution) or EA-PE (1:50 dilution) secondary reagents for 15 minutes at 4°C. After washing twice with ice-cold wash buffer, the cell pellet was resuspended in 0.4 mL wash buffer and transferred to a sorting tube covered with a filter. Sorting was performed using a FACS ARIA sorter (BD Biosciences) and sorting gates were assigned to select specific binders relative to background controls. Subsequent rounds of selection were used to reduce the number of nonspecific binders using soluble membrane proteins from CHO cells (see, e.g., WO2014179363 and Xu et al., Protein Eng DesSel, Vol. 26(10), pp. 663-670 (2013)), and binders with improved affinity for TIGIT were identified using the TIGIT-Fc antigen. After the final round of sorting, the yeast were plated and individual colonies were picked for characterization and designation of clones for affinity maturation. 63 clones were screened for functional activity. Based on the screening, clones 26518, 26452, 26486, 26521, and 26493 had the best functional activity and were selected for further optimization.

[0319] Example 2: Antibody Optimization

[0320] Optimization of primary clones was performed using three maturation strategies: light chain diversification; diversification of HCDR1 and HCDR2; and HCDR3 diversification within selected HCDR1 and HCDR2 diversity pools.

[0321] Light chain diversification: Heavy chain variable regions were extracted from the original output (described above) and converted to a diversity of 1x10 6 Selection was performed as described above, with one round of MACS sorting and three rounds of FACS sorting, using 10 nM or 1 nM biotinylated TIGIT-HIS antigen (Creative Biomart) for each round.

[0322] HCDR1 and HCDR2 selection: HCDR3 of clones selected from the light chain diversification program were recombined to have a diversity of 1x10 8 Premade libraries of HCDR1 and HCDR2 variants of the same protein were selected using monomeric HIS-TIGIT antigen. Affinity pressure was applied by using decreasing concentrations of biotinylated HIS-TIGIT antigen (100 to 1 nM) under equilibrium conditions at room temperature.

[0323] HCDR3 / HCDR1 / HCDR2 selection: Oligonucleotides were ordered from IDT, which contained HCDR3 and homologous flanking regions on each side of HCDR3. The amino acid positions in HCDR3 were variegated by NNK diversity at two positions per oligonucleotide throughout the HCHR3. The HCDR3 oligonucleotides were double-stranded using primers that annealed to the flanking regions of HCDR3. The remaining FWR1 to FWR3 of the heavy chain variable region were amplified from an antibody library with improved affinity, which was isolated from the HCDR1 and HCDR2 diversity selected above. The library was then generated by transforming the double-stranded HCDR3 oligonucleotides, the FWR1 to FWR3 merged fragments, and the heavy chain expression vector into yeast that already contained the light chain of the original parent. Four rounds of selection were performed as during the previous cycle using FACS sorting. For each round of FACS, the library was evaluated for PSR binding, species cross-reactivity, and affinity pressure, and sorted to obtain a population with the desired characteristics. Affinity pressure for these selections was performed as described above in HCDR1 and HCDR2 selections.

[0324] Example 3: Antibody Production and Purification

[0325] A. Produced in yeast

[0326] In order to produce enough optimized and unoptimized selected antibodies for further characterization, yeast clones were grown to saturation and then induced at 30°C under shaking for 48 hours. After induction, yeast cells were precipitated and harvested supernatant for purification. Protein A column was used to purify IgG and eluted with acetic acid at pH 2.0. Fab fragments were produced by papain digestion and purified in a two-step process of protein A (GE LifeSciences) and KappaSelect (GE Healthcare LifeSciences).

[0327] B. Produced in mammalian cells

[0328] In order to produce sufficient amounts of optimized and unoptimized selected antibodies for further characterization, DNA vectors encoding specific antibody clones were generated and transduced into HEK cells. Human codon-optimized synthetic DNA fragments of the antibody variable domains were ordered at Geneart. The variable domain sequences were seamlessly linked to the pUPE expression vector containing the mouse Igκ signal sequence and the constant region of the corresponding antibody class. The expression vector was verified by restriction analysis and DNA sequencing. For transient transfection, endotoxin-free DNA maxiprep (Sigma) was produced, and the heavy and light chain vectors were co-transfected into HEK293EBNA1 cells in Freestyle medium (ThermoFisherScientific) according to established protocols. Primatone (0.55% final volume) was added 24 hours after transfection. Conditioned medium was harvested 6 days after transfection. Antibodies were purified in batches by Mabselect sureLX (GE Healthcare) affinity chromatography. The bound antibodies were washed in two steps with PBS containing 1M NaCl and PBS. Antibodies were eluted with 20 mM citrate 150 mM NaCl pH 3 and neutralized to approximately pH 7 with 1 / 6 volume of 1 M K2HPO4 / KH2PO4 pH 8.

[0329] Next, the antibody was further purified by gel filtration using a Superdex200 column equilibrated in PBS. The fractions were analyzed by NuPAGE and the fractions containing the antibody were pooled. The final product was sterilized through a 0,22 μM syringe filter. The product was analyzed by NuPAGE and the endotoxin level was measured by LAL assay.

[0330] Example 4: Affinity determination of anti-TIGIT antibodies binding to recombinant human TIGIT protein

[0331] A. ForteBio K D Measurement

[0332] ForteBio affinity measurements of selected antibodies were generally performed as previously described (see, e.g., Estep et al., Mabs, Vol. 5(2), pp. 270-278 (2013)). Briefly, ForteBio affinity measurements were performed by loading IgG onto the AHQ sensor online. The sensor was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds for baseline establishment. The sensors with loaded IgG were exposed to 100 nM antigen (human TIGIT-Fc, human TIGIT-His, or cynomolgus monkey TIGIT-Fc) for 5 minutes and then transferred to assay buffer for 5 minutes for dissociation rate measurements. Kinetics were analyzed using a 1:1 binding model. The affinity of more than 90 antibodies was tested by ForteBio, and Table 3 provides data for 15 selected anti-TIGIT antibodies, demonstrating strong binding to recombinant TIGIT protein.

[0333] B.MSD-SET K D Measurement

[0334] Equilibrium affinity measurements of selected antibodies were generally performed as previously described (Estep et al., Mabs, Vol. 5(2), pp. 270-278 (2013)). Briefly, solution equilibrium titrations (SET) were performed in PBS + 0.1% IgG-free BSA (PBSF), where the antigen (TIGIT-His monomer) was kept constant at 10 to 100 pM and incubated with 3- to 5-fold serial dilutions of Fab or mAb (starting from 10 pM to 10 nM). Antibodies (20 nM in PBS) were applied to standard binding MSD-ECL plates at 4°C overnight or at room temperature for 30 minutes. The plates were then blocked with BSA for 30 minutes at 700 rpm shaking and then washed three times with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied and incubated on the plates for 150 seconds at 700 rpm shaking and then washed once. Antigen captured on the plate was detected with 250 ng / mL sulfotag-labeled streptavidin in PBSF by incubating the plate for 3 minutes. The plate was washed three times with wash buffer and then read on an MSD Sector Imager 2400 instrument using 1x Read Buffer T with surfactant. The percentage of free antigen was plotted in Prism against the variation of titrated antibody and a quadratic equation was fitted to extract K D To increase throughput, a liquid handling robot was used throughout the MSD-SET experiment, including SET sample preparation. The affinity of the selected antibodies was tested by MSD, and Table 4 provides data for seven anti-TIGIT clones demonstrating strong binding to recombinant TIGIT protein.

[0335] Table 4: MSD analysis of the affinity of selected anti-TIGIT antibodies

[0336]

[0337] C. Biacore measurement

[0338] Biosensor analysis was performed using a Biacore 8K optical biosensor (GE Healthcare, Marlboro, MA) docked to a CM5 sensor chip at 25°C in a HBS-EP buffer system (10 mM HEPES pH 7.3, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). The sample hotel was kept at 8°C. Goat anti-human IgG capture antibodies (specific Fcγ fragments, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 109-005-098) were fixed (11700 + / - 200 RU) to two flow cells of the sensor chip using standard amine coupling chemistry. This surface type provides a form in which fresh analytical antibodies can be repeatedly captured after each regeneration step. Flow cell 2 was used to analyze captured antibodies (60-90 RU), while flow cell 1 was used as a reference flow cell. Antigen concentrations of 30 to 0.123 nM (3-fold dilution) were prepared in running buffer. Each antigen sample concentration was run as a single repeat. Two blank (buffer) injections were also run and used to evaluate and subtract system artifacts (artefacts). The association (300 seconds) and dissociation (600 seconds) stages of all antigen concentrations were carried out at a flow rate of 30uL / min. The surface was regenerated by three consecutive injections (15 seconds, 15 seconds and 60 seconds) of 10mM glycine (pH 1.5) at a flow rate of 30uL / min. Data were compared, double referenced and fitted to a 1:1 binding model using Biacore 8K evaluation software version 1.0. The affinity of the selected antibodies was tested by Biacore, and Table 5 provides the data of 5 anti-TIGIT clones, demonstrating strong binding to recombinant TIGIT protein.

[0339] Table 5: Biacore analysis of affinity of selected anti-TIGIT antibodies

[0340]

[0341] Example 5: Competition assay between anti-TIGIT antagonist antibodies and TIGIT natural ligands

[0342] A. Octet Red384 Epitope Binning / Ligand Blocking

[0343] The epitope reclassification / ligand blocking of the selected antibodies was performed using a standard sandwich format cross-blocking assay. The control anti-target IgG was loaded onto the AHQ sensor, and the unoccupied Fc binding sites on the sensor were blocked with an unrelated human IgG1 antibody. The sensor was then exposed to 100nM target antigen (hTIGIT, Creative Biomart), followed by exposure to a second anti-target antibody or ligand (anti-TIGIT antibody and CD155 or CD113 or CD112). The data was processed using ForteBio's data analysis software 7.0. The additional binding of the second antibody or ligand after antigen association indicates an unoccupied epitope (non-competitor), while the absence of binding indicates epitope blocking (competitor or ligand blocking). The competition of the parent antibody (before optimization) with the natural ligand was tested, and Table 6 summarizes the data obtained for the competition with CD155, CD112 and CD113. It was found that the parent clone 26432 did not compete with CD155 for binding to TIGIT. All other selected anti-TIGIT antibodies competed with the natural ligand for binding to recombinant human TIGIT protein.

[0344] Table 6: Re-ranking analysis of non-optimized anti-TIGIT antibodies against TIGIT natural ligands

[0345]

[0346]

[0347] B. Competition of anti-TIGIT antagonist antibodies with CD155 on Jurkat-hTIGIT

[0348] Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT) were collected and cultured at 10 5 Cells were dispensed into wells and incubated for 45 min at 37°C in complete medium with anti-human TIGIT antibodies at the following concentrations: 166,6; 53,24; 17,01; 5,43; 1,73; 0,55; 0,17; 0,05; 0,01; 5,78x10 -3 ;1,85x10 -3 ; 5,9x10 -3nM. Excess antibody was washed off, and the cells were then incubated with 5 μg / ml CD155-His (Creative Biomart, PVR-3141H) at 37°C for 45 minutes. Bound CD155-His was then detected using anti-His tag-PE (Biolegend, 362603, 2 μl per test) and incubated at 4°C for 30 minutes. Cells were analyzed by FACS using BD LSRFortessa, and the half concentration (IC) that prevented CD155 binding was calculated based on the geometric mean fluorescence. 50 ).

[0349] The results were as follows: 0,101 nM for clone 29489; 0,07 nM for clone 29494; 0,102 nM for clone 29520 and 0,078 nM for clone 29527. Figure 7 The values ​​for the other antibodies tested are summarized in Table 7 below. In summary, the results demonstrate that the antagonist anti-TIGIT antibodies tested strongly compete with CD155 for binding to membrane expressed TIGIT.

[0350] Table 7: ICs of CD155 competing with TIGIT 50 data

[0351]

[0352] Example 6: Characterization of Hydrophobic Interaction Chromatography (MAbs. 2015 May-June; 7(3):553–561)

[0353] The anti-TIGIT IgG1 antibody sample was buffer exchanged to 1M ammonium sulfate and 0.1M sodium phosphate at pH 6.5 using a Zeba 40kDa 0.5mL centrifugal column (Thermo Pierce, catalog number 87766). A salt gradient was established on a Dionex ProPac HIC-10 column from 1.8M ammonium sulfate, 0.1M sodium phosphate (pH 6.5) to the same conditions without ammonium sulfate. The gradient was run for 17 minutes at a flow rate of 0.75ml / min. An acetonitrile wash step was added at the end of the run to remove any residual protein, and the column was rebalanced with 7 column volumes before the next injection cycle. The peak retention time was monitored at A280 absorbance, and the ammonium sulfate concentration during elution was calculated based on the gradient and flow rate. Table 8 summarizes the results obtained for 15 selected anti-TIGIT antibodies.

[0354] Table 8: Hydrophobic interaction chromatography analysis of selected anti-TIGIT antibodies

[0355]

[0356] Example 7: Characterization of Multispecific Agent PSR Formulations

[0357] A. Preparation of multispecific reagents:

[0358] According to Xu et al., mAbs 2013, a multispecific reagent (PSR) was prepared. Briefly, 2.5 liters of CHO-S cells were used as raw materials. The cells were precipitated at 2,400 x g for 5 minutes in a 500 mL centrifuge bottle filled to 400 mL. The cell pellets were combined and then resuspended in 25 ml of buffer B and precipitated at 2,400 x g for 3 minutes. The buffer was decanted and the wash was repeated once. The cell pellet was resuspended in 3 x precipitation volumes of buffer B containing 1 x protease inhibitor (Roche, cOmplete, EDTA-free) using a polytron homogenizer, and the cells were kept on ice. The homogenate was then centrifuged at 2,400 x g for 5 minutes, the supernatant was retained and precipitated once more (2,400 x g / 5 minutes) to ensure that unbroken cells, cell debris and nuclei were removed; the resulting supernatant was a total protein preparation. The supernatant was then transferred to two Nalgene Oak Ridge 45mL centrifuge tubes and precipitated at 40,000xg for 40 minutes at 4°C. The supernatant containing the separated cytosolic proteins (SCP) was then transferred to a clean Oak Ridge tube and centrifuged again at 40,000xg. In parallel, the precipitate containing the membrane fraction (EMF) was retained and centrifuged at 40,000 for 20 minutes to remove the residual supernatant. The EMF precipitate was then rinsed with buffer B. 8mL of buffer B was then added to the membrane precipitate to remove the precipitate and transferred to a Dounce homogenizer. After the precipitates were homogenized, they were transferred to 50mL conical tubes and represented the final EMF preparation.

[0359] ~10 6 –10 71 billion mammalian cells (e.g., CHO, HEK293, Sf9) of 1 cell / mL were transferred to 4x250mL conical tubes from a tissue culture environment and precipitated at 550x g for 3 minutes. All subsequent steps were performed at 4°C or on ice with ice-cold buffer. The cells were washed with 100mL PBSF (1xPBS+1mg / mL BSA) and merged into a conical tube. After removing the supernatant, the cell pellet was then resuspended in 30mL buffer B (50mM HEPES, 0.15M NaCl, 2mM CaCl2, 5mM KCl, 5mM MgCl2, 10% glycerol, pH 7.2) and precipitated at 550x g for 3 minutes. The buffer B supernatant was decanted and the cells were resuspended in 3x precipitation volumes of buffer B plus 2.5x protease inhibitors (Roche, cOmplete, EDTA-free). Protease inhibitors in buffer B were included from this point on. The cells were homogenized 4 times with 30 second pulses each time (Polyton homogenizer, PT1200E), and the membrane fraction was precipitated at 40,000xg for 1 hour at 4C. The precipitate was rinsed with 1 mL of buffer B; the supernatant was retained and represented by s. The precipitate was transferred to a Dounce homogenizer with 3 mL of buffer B and resuspended by slowly moving the pestle up and down 30-35 times. The enriched membrane fraction (EMF) was moved to a new collection tube and the pestle was rinsed to collect all possible proteins. The protein concentration of the purified EMF was determined using the Dc-protein assay kit (BioRad). To dissolve the EMF, it was transferred to a solubilization buffer (50 mM HEPES, 0.15 M NaCl, 2 mM CaCl2, 5 mM KCl, 5 mM MgCl2, 1% n-dodecyl-bD-maltopyranoside (DDM), 1x protease inhibitor, pH 7.2) to a final concentration of 1 mg / mL. The mixture was rotated overnight at 4°C and then centrifuged at 40,000 xg for 1 hour in a 50 mL Oak Ridge tube (Fisher Scientific, 050529-ID).The supernatant representing soluble membrane protein (SMP) was collected and protein yield was quantified as described above.

[0360] For biotinylation, NHS-LC-biotin stock solution was prepared according to the manufacturer's protocol (Pierce, Thermo Fisher). Briefly, 20ul biotin reagent was added to each 1mg EMF sample and incubated at 4°C for 3 hours under gentle stirring. The volume was adjusted to 25mL with buffer B and transferred to an Oak Ridge centrifuge tube. Biotinylated EMF (b-EMF) was precipitated at 40,000xg for 1 hour and rinsed twice with 3mL buffer C (buffer B minus glycerol) without disturbing the precipitation. Residual solution was removed. As previously described, the precipitation was resuspended in 3mL buffer C with a Dounce homogenizer. The resuspended precipitation now represents biotinylated EMF (b-EMF) and is dissolved as described above to prepare b-SMP.

[0361] B. PSR binding analysis

[0362] PSR analysis is usually performed as described in WO2014 / 179363. Briefly, in order to characterize the PSR spectrum of monoclonal antibodies presented on yeast, 2 million IgG-presenting yeasts are transferred to a 96-well assay plate and precipitated at 3000 x g for 3 minutes to remove the supernatant. The precipitate is resuspended in a 1:10 dilution of 50ul freshly prepared b-PSR stock solution and incubated on ice for 20 minutes. The cells are washed twice with 200ul cold PBSF, and the precipitate is resuspended in a secondary labeling mixture (Extravidin-R-PE, anti-human LC-FITC and propidium iodide) of 50ul. The mixture is incubated on ice for 20 minutes, then washed twice with 200ul ice-cold PBSF. The cells are resuspended in 100ul ice-cold PBSF, and the plate is run on FACS Canto (BD Biosciences) using an HTS injector. The mean fluorescence intensity in the R-PE channel of the flow cytometry data was analyzed and normalized to appropriate controls to assess non-specific binding. Table 9 summarizes the results of multi-specific reagent binding obtained for the 15 selected anti-TIGIT antibodies, which confirms that most clones scored low.

[0363] Table 9: Analysis of multispecific reagents

[0364]

[0365]

[0366] Example 8: Characterization of TIGIT expression on immune populations from healthy human PBMCs

[0367] TIGIT expression profile on AT cell subsets

[0368] Flow cytometry analysis was performed to evaluate TIGIT expression on immune cell subsets in freshly isolated PBMCs from healthy individuals. Conjugated antibodies were purchased from Ebioscience / Thermo Fisher Scientific, BioLegend or BD Biosciences. Cells were stained using filtered FACS buffer (PBS+2mM EDTA+0.1% BSA) and Brilliant Stain buffer (BD#563794) according to the manufacturer's instructions. Cells were blocked with appropriate human FcBlock (BD#564220) before staining and fixed using IC fixation buffer (eBioscience#00-8222-49) before acquisition. Acquisition was performed on a FACS Fortessa (BD Biosciences) and analyzed using FlowJo software (FlowJo, LLC). Live cells were gated at forward and side scatter. The following various immune cell subsets were gated: CD19 + (B cells), CD3 - CD19 - CD14 + (monocytes), CD3 + TCRab - (TCRgd T cells), CD3 + TCRab + (TCRab T cells), CD3 - CD19 - CD14 - HLA-DR - CD56 低 / 高 (NK cells), CD3 - CD19 - CD14 - HLA-DR + (dendritic cells), CD3 + TCRab + CD4 + CD127 低 CD25 + (regulatory T cells), CD3 + TCRab + CD4 + or CD8 + CD45RO - CCR7 + (CD4 or CD8 naive T cells), CD3 + TCRab + CD4 + or CD8 + CD45RO +(memory T cells) and CD45RO - CD62L - (Effector T cells).

[0369] like Figure 8 A and Figure 8 As shown in Figure 2, TIGIT is preferentially expressed on NK cells, regulatory T cells, and CD8 memory T cells. It is less present on other T cell subsets, with a low proportion of naive T cells showing TIGIT expression. In addition, TIGIT is not expressed on monocytes, dendritic cells, and B cells ( Figure 8 B). This set of data is consistent with published data (Yu et al. NI 2008 and Wang et al. EJI 2015).

[0370] Example 9: Cell Binding of Anti-TIGIT Antagonistic Antibodies

[0371] A. Binding of anti-TIGIT antibodies to Jurkat-hTIGIT and Jurkat-mTIGIT

[0372] The affinity of human anti-TIGIT antibodies has been measured using Jurkat E6.1 cells transduced with human TIGIT (Jurkat hTIGIT) or Jurkat E6.1 cells transduced with mouse TIGIT (Jurkat-mTIGIT). To analyze the affinity of the selected antibodies for hTIGIT or mTIGIT, 10 5 cells and treated with a single dose of 100 nM anti-TIGIT antibody (Table 3) or with decreasing concentrations (166,6; 53,24; 17,01; 5,43; 1,73; 0,55; 0,17; 0,05; 0,01; 5,78x10 -3 ;1,85x10 -3 ; 5,9x10 -3 nM) of the selected antibody ( Fig. 9 ). The antibodies were incubated with cells at 4 ° C in FACS buffer for 20 minutes. After washing, the cells were incubated with anti-human Ig (Fcγ specific)-PE (eBioscience, 12-4998-82, 2.5 μg / ml) on ice for 20 minutes and washed twice. The geometric mean fluorescence intensity was analyzed using LSR BD Fortessa. For each antibody, cell binding was recorded as the median fluorescence intensity of PE on the transfected cell line compared to the untransfected cell line (Table 3). In order to calculate the EC 50 Binding, the half-maximal binding concentration (EC) for hTIGIT-Jurkat was calculated using a four-variable curve fitting equation in Prism. 50), and the values ​​obtained were as follows: 0,082 nM for clone 29489; 0,07 nM for clone 29494; 0,119 nM for clone 29520 and 0,05 nM for clone 29527, data at Fig. 9 The results demonstrate that the tested anti-TIGIT antibodies strongly bind to membrane-expressed human TIGIT.

[0373] B. Binding of anti-TIGIT antagonist antibodies to primary human T cells

[0374] Isolated human PBMCs from healthy volunteers were analyzed for binding to antagonist anti-TIGIT antibodies. 5x10 5 Cells were incubated with anti-CD16 (clone 3G8, BioLegend 302002), CD32 (clone FLI8.26, BD Bioscience 557333), and CD64 (BD Bioscience 555525) for 10 min at room temperature, and the indicated anti-human TIGIT antibodies were added directly at the following final concentrations in FACS buffer: 12,65; 4; 1,26; 0,40; 0,126; 0,040; 0,12 and 4x10 -3 nM and incubated at 4°C for 20 minutes. After washing, the cells were incubated with anti-human Ig (Fcγ specific)-PE (eBioscience, 12-4998-82, 2.5 μg / ml) at 4°C for 20 minutes. Fig.10 A and Fig.10 B, cells were washed and incubated with the following antibody and LVD mixture: anti-CD4-PercP-Cy5.5 (clone A161A1, BioLegend 357414); anti-CD8-BV510 (clone SK1, BD Bioscience 563919) and LVD efluor 520 (eBioscience 65-0867-14). Fig.10C, cells were washed and incubated with the following antibodies and LVD mixture: LVD efluor 520 (eBioscience 65-0867-14), anti-TCRab-PercP-Cy5.5 (clone IP26, Biolegend 306723), anti-CD4-BV510 (clone SK3, BD Horizon 562970), anti-CD8-APC-Cy7 (clone SK1, Biolegend 344714), anti-CD25-BV605 (clone 2A3, Biolegend 562660), anti-CD127-APC (A019D5, Biolegend 351316), anti-CCR7-BV421 (clone G043H7, Biolegend 353207), and anti-CD45RO-PE-Cy7 (clone UCHL1, Biolegend 304229).

[0375] Using a gated LVD - CD8 + The percentage of T cells positive for TIGIT staining was calculated with CD8 + Human primary T cells bind EC 50 value( Fig.10 A and Fig.10 B) Using a gated LVD - TCRab + CD45RO + CD8 + T cells (for memory CD8 + T cells) or gated LVD - TCRab + CD127 lo CD25 hi CD4 + Calculation of the percentage of positive TIGIT-stained cells on T cells (for Tregs) and human memory CD8 + or Treg primary T cells bound to EC 50 value, and is shown in Fig.10 C.

[0376] like Fig.10 As shown in A, compared with total human CD8 + T cell-bound EC 50 The values ​​were as follows: 0,123 nM for clone 29489; 0,181 nM for clone 29520, and 0,253 nM for clone 29527. A direct comparison between 29489 and 31282 (a mutant of 29489 with an M to T mutation at residue 116) was performed, and the EC 50The values ​​were 0.057 nM and 0.086 nM, respectively, demonstrating that these two clones have strong affinity for human primary CD8 + T cells have strong and similar binding efficacy ( Fig.10 B) Acquired and memory CD8 + T cell and Treg-bound EC 50 The values ​​were 0.039 nM and 0.03 nM, respectively, demonstrating strong and similar affinities for both populations ( Fig.10 C).

[0377] C. Binding of anti-TIGIT antagonist antibodies to cynomolgus monkey primary T cells

[0378] Isolated PBMCs from cynomolgus monkeys (Macaca fascicularis) were obtained from BioPRIM. Cells were thawed and stimulated using the T cell activation / expansion kit for non-human primates (Miltenyi Biotec) according to the manufacturer's instructions at a 1:2 (bead: cell ratio). The next day, cells were collected, counted, and plated at 5×10 4 Cells were incubated with anti-CD16 (clone 3G8, BioLegend 302002), CD32 (clone FLI8.26, BD Bioscience 557333), and CD64 (BD Bioscience 555525) for 10 min at room temperature, and the selected anti-human TIGIT antibodies were added directly at the following final concentrations in FACS buffer: 12,65, 4, 1,26, 0,40, 0,126, 0,040, 0,12, and 4x10 -3 nM and incubated at 4°C for 20 minutes. After washing, the cells were incubated with anti-human Ig (Fcγ specific)-PE (eBioscience, 12-4998-82, 2.5 μg / ml) at 4°C for 20 minutes. Fig.11 A and Fig.11 Data shown in B, cells were washed and incubated with the following antibody and LVD mixture: anti-CD4-PercP-Cy5.5 (clone A161A1, BioLegend 357414); anti-CD8-BV510 (clone SK1, BD Bioscience 563919), CD69-APC-Cy7 (clone FN50, BioLegend, 310914) and LVDefluor 520 (eBioscience 65-0867-14). The stained cells were analyzed by FACS using BD LSR Fortessa. LVD - CD69 + CD8 +The percentage of positive TIGIT-stained cells gated on T cells was used to calculate the EC of binding 50 Value. Fig.11 As shown, the cynomolgus monkey CD8 + T cell-bound EC 50 The values ​​were as follows: 0,487 nM for clone 29489, 1,73 nM for clone 29520 and 0,378 nM for clone 29527. A comparison was also made between clones 29489 and 31282 (a mutant of 29489 with an M to T mutation at residue 116), EC 50 The values ​​are 0.25nM and 0.26nM respectively. Fig.11 As shown in B, these two clones were shown to be effective for primary CD8 + T cells have a similar and strong affinity.

[0379] Example 10: In vitro functional characterization of antagonistic anti-TIGIT activity

[0380] A. TIGIT bioassay for CHO-TCR-CD155 and Jurkat-hTIGIT co-cultures

[0381] To characterize the functional consequences of blocking the human TIGIT receptor, we co-cultured Jurkat cells expressing hTIGIT and a luciferase reporter protein activated upon TCR engagement (TIGIT effector cells from Promega, used as thaw) and a CHO-K1 cell line engineered to express human CD155 and TCR activator (CD155 aAPC / CHO-K1 from Promega, used as thaw). Activation of Jurkat cells overexpressing TIGIT can be induced by contact with CHO-K1 cells expressing CD155 upon TCR engagement on Jurkat cells and can be increased in the presence of antagonistic anti-TIGIT antibodies. To compare the potency of different antibodies to increase Jurkat cell activation, experiments were performed in the presence of increasing antibody concentrations and the EC was calculated. 50 value.

[0382] Thawed CD155 aAPC / CHO-K1 (Promega, CS198811) cells were plated and incubated at 37°C in a 5% CO2 incubator O / N according to the manufacturer's recommendations. The next day, thawed TIGIT effector cells (Promega, CS198811) were added to the CD155 aAPC / CHO-K1 cell plate containing fresh complete medium with 133 nM anti-TIGIT antibody ( Fig.12A) or increasing concentrations (0, 22, 0, 54, 1, 36, 3, 41, 8, 53, 21, 3, 53, 3, 133, 33, and 333 nM) of anti-TIGIT antibody ( Fig.12 B).

[0383] After 6 h of incubation, activation of TIGIT effector cells was assessed by measuring luciferase activity using the Bio-Glo™ Luciferase Assay System (Promega, G7941).

[0384] Fig.12 A shows the effect of adding selected clones on luciferase signal compared to isotype control. The data demonstrate the antagonistic activity of antibodies that lead to stronger activation of Jurkat-hTIGIT cells. Table 10 summarizes the fold change induction of luciferase expression obtained for different anti-TIGIT antibodies relative to the isotype control clone (03847).

[0385] Table 10: Fold change induction relative to isotype control

[0386]

[0387] like Fig.12 As shown in B, Jurkat-hTIGIT cell activation was assessed with anti-TIGIT antibody at 0.22 nM to 333 nM, giving the following EC 50 Values: 3.0 nM for clone 29489; 4.4 nM for clone 29494; 2.3 nM for clone 29520 and 32 nM for clone 29527; 2.7 nM for clone 32919 and 3.2 nM for clone 32931, demonstrating a strong functional activity of continuous blockade of TIGIT inhibitory signaling. Clones 29489 and 31282 (a mutant of 29489 with a mutation from M to T at residue 116) were also compared, EC 50 The values ​​are 4.3nM and 8.1nM, respectively. Fig.12 Shown in C, similar functional activities of these two clones were demonstrated.

[0388] B. Based on human primary CD8 + T cell functional assay

[0389] To characterize the functional consequences of blocking the human TIGIT receptor, we isolated primary human CD8 +T cells were co-cultured with a CHO-K1 cell line engineered to express human CD155 and activated human T cells. We observed that blocking hTIGIT with an anti-TIGIT antagonist antibody could increase CD8 + IFNg release by T cells. To compare the potency of these antibodies in increasing IFNg release, experiments were performed in the presence of increasing antibody concentrations and the EC 50 value.

[0390] Thawed CD155 aAPC / CHO-K1 (Promega, CS198811) cells were seeded in U-bottom 96-well plates according to the manufacturer's recommendations and incubated at 37°C in a 5% CO2 incubator O / N. The next day, CD8 + T cells. The purified CD8 T cells were then incubated with increasing concentrations (0,11nM, 0,33nM, 1,06nM, 3,3nM, 10,6nM, 33,3nM, 105,5nM and 333nM) of antibodies (100,000 CD8T cells / 100ul complete medium containing antibodies) for 1 hour. Afterwards, the antibody-CD8 mixture was added to a CD155aAPC / CHO-K1 cell plate containing 50μl of fresh complete medium and incubated at 37°C, 5% CO2 for 5 days. Finally, the IFNg concentration in the cell supernatant was assessed using an ELISA assay (Affymetrix eBioscience, 88-7316-86) performed according to the manufacturer's recommendations.

[0391] like Fig.13 As shown in A, all anti-TIGIT antibodies increased IFNg secretion compared to isotype control. The highest increase was observed for clone 29489 (6.4-fold), followed by 29494 (5.8-fold), 29520 (5.4-fold), 29499 (5.2-fold), 29527 (4.5-fold), and 29513 (3.2-fold).

[0392] A dose-ranging study (0,22nM to 333nM of anti-TIGIT antibody) was also performed to evaluate the EC effect of increased IFNg secretion by human primary CD8 T cells. 50 Value. Fig.13 As shown in B, anti-TIGIT antibody 29489 showed the best activity, EC 50 3,5nM, followed by clone 29527EC50 =5,1 nM), clone 29494 (EC 50 =6,1 nM) and clone 29520 (EC 50 =11,1 nM). Finally, clone 29489 and its mutant 31282 were tested in parallel and demonstrated similar activities, EC 50 The values ​​were 0.49nM and 0.50nM ( Fig.13 C). Taken together, these data demonstrate that antagonistic anti-TIGIT antibodies block CD8 + TIGIT in human T cells has potent functional activity that inhibits signaling and enhances effector function, as characterized by a robust increase in IFNg production.

[0393] C. Human TIL Function Assay

[0394] To characterize the functional consequences of blocking the human TIGIT receptor on tumor infiltrating lymphocytes (TILs) from cancer patients, we injected primary human CD8 + T cells were co-cultured with a CHO-K1 cell line engineered to express human CD155 and activated human T cells. We observed that blocking hTIGIT with an anti-TIGIT antagonist antibody could increase CD8 + IFNg release by T cells.

[0395] Thawed CD155 aAPC / CHO-K1 (Promega, CS198811) cells were seeded in U-bottom 96-well plates according to the manufacturer's recommendations and incubated at 37°C in a 5% CO2 incubator O / N. The next day, CD8 T cells were purified from frozen human TILs (Immunehealth) isolated from ovarian ascites by using a negative selection kit (Stemcell Technologies, 17953) according to the manufacturer's recommendations. The purified CD8 + T cells were transfected with anti-TIGIT antibody clone 26452 (unoptimized parent of clones 29489 and 31282) (100,000 CD8 + After that, the antibody-CD8 mixture was added to the CD155 aAPC / CHO-K1 cell plate containing 50ul of fresh complete medium and incubated at 37°C, 5% CO2 for 5 days.

[0396] Finally, IFNg concentrations in cell supernatants were assessed using an ELISA assay (Affymetrix eBioscience, 88-7316-86) run according to the manufacturer's recommendations. Fig.14 As seen in Figure 2, when anti-TIGIT antibodies were added to the co-culture, IFNg secretion increased almost 2-fold. These data demonstrate that antagonistic anti-TIGIT antibodies block CD8 + TIGIT in human TILs has potent functional activity that inhibits signaling and enhances effector functions of T cells in the tumor environment.

[0397] Example 11: Characterization of anti-TIGIT antagonist antibodies that are functionally active in mice

[0398] A. Mouse CD155 competition assay using alternative anti-TIGIT antagonist antibodies

[0399] For this assay, Jurkat cells (clone E6-1, ATCC TIB-152) engineered to overexpress mouse TIGIT (Jurkat-mTIGIT) were used. Anti-TIGIT antibody 26493 was used as a surrogate because the antibody showed cross-reactivity to mouse TIGIT and binding to human TIGIT. Cells were pre-incubated with different concentrations of anti-TIGIT antibody clone 26493 (0.03 to 10 μg / ml) in 25 μl complete medium (RPMI+10% FBS) at 37°C for 45 minutes. The cells were washed once and incubated in an incubator with 4 μg / ml mouse CD155-His-Fc tag protein (Thermo Fisher, 50259M03H50) in 50 μl complete medium for 45 minutes. The cells were washed once and stained with PE-anti-His antibody (Biolegend, 362603) at 4°C for 30 minutes. The median fluorescence intensity (MFI) measured by FACS was used as a measure of CD155 binding to Jurkat-mTIGIT. Fig.15 A shows the dose-response curve of anti-TIGIT clone 26493 competition with CD155 to determine the IC 50 The expression of 1:1 isotype was 2.3 nM (the upper dashed line represents the signal from the isotype, the lower dashed line represents the signal from cells without CD155). These results demonstrate that anti-TIGIT antibodies compete with CD155 ligand for the functional efficacy of mouse TIGIT.

[0400] B. Mouse functional in vitro assay: antigen-specific cytotoxicity (OT-I)

[0401] To assess OT-I CD8 +Antigen-specific cytotoxic activity of T cells against OVA-pulsed target cells and the role of anti-TIGIT antibodies in this assay were performed by mechanical dissociation from C57BL / 6-Tg (TcraTcrb) OT1 cells were isolated from the spleens of 1100 Mjb / Crl mice (Charles River) and then analyzed using EasySep TM Mouse T cells were negatively selected using a mouse T cell isolation kit (Stemcell, catalog number 19851). As antigen presenting cells, PanO2 cancer cells naturally expressing CD155 were treated with mitomycin C (25 μg / ml) and subsequently pulsed with OVA-peptide (S7951-1MG, Sigma Aldrich, 1 μg / ml, 1 hour at 37°C). CD8 + T cells and PanO2 were co-cultured for 3 days in the presence of 133 nM anti-TIGIT clone 26493 or isotype control. On day 3, supernatants were collected for detection of IFNg( Fig.15 B) and T cells for cytotoxicity assays ( Fig.15 C). OVA-pulsed PanO2 cells were used as target cells. CFSE (C1157, ThermoFisher) and CellTrace TM Far Red Cell Proliferation Kit (C34564, ThermoFisher) was used to label target cells and unpulsed Pan02 cells (non-target internal control) (1×10 6 These cells were mixed (1:1 ratio) and plated at 2x10 4 Cells were plated at 1x10 5 cells / well (effector cells) were added with stimulated OT-1CD8 + T cells were injected with 10:1 effector to target ratio in the presence of 133 nM anti-TIGIT clone 26493 or isotype control. After 24 hours, cells were washed with PBS and lifted by trypsin digestion. Cells were then stained with a live / dead fixable purple dead cell staining kit (Molecular Probes, L34955). Cytotoxic killing of target cells was then measured by flow cytometry monitoring the change in the ratio of live target cells to non-target cells.

[0402] Fig.15 B shows that anti-TIGIT antibody increased IFNg production by almost 2-fold, while Fig.15 C shows mouse OT-I CD8 + The cytotoxic activity of T cells increased by approximately 60%. In summary, these results confirm that anti-TIGIT antibodies enhance the expression of CD8 + Functional activity of T cell effector functions.

[0403] Example 12: Anti-tumor activity of anti-TIGIT antagonist antibodies as monotherapy and in combination with anti-PD1 antibodies in mouse models

[0404] A. In vivo antitumor activity of anti-TIGIT antagonist antibodies as monotherapy

[0405] For this experiment, anti-TIGIT clone 26493 was generated on the mouse IgG2a isotype in mammalian cells. Female Balb / c mice aged 8 weeks were inoculated subcutaneously with 500.000 CT26 colon cancer cells ( CRL-2638 TM On day 9 after inoculation, when the average tumor volume was 45 mm 3 Around 9 days, mice were randomly divided into treatment groups with equal tumor volumes (n=8 per group). On days 9, 12, and 15, mice were treated with 200 μg anti-TIGIT or isotype control (mIgG2a, BioXcell), or 200 μg anti-PD-1 (RMP1-14, BioXcell) and 200 μg isotype control (mIgG2a, BioXcell), or 200 μg anti-PD-1 (RMP1-14, BioXcell) and different concentrations of anti-TIGIT (200 μg, 60 μg, 20 μg) by intraperitoneal injection. Tumor growth was monitored and tumor volume was measured with an electronic caliper three times a week from day 9 to day 36. When the tumor volume exceeded 2000 mm 3 Mice were sacrificed at 4 pm. Tumor growth curves were statistically analyzed by linear mixed models. Differences between treatment groups were evaluated by testing the interaction of time*treatment group. To test the synergy between anti-TIGIT and anti-PD-1, treatment groups were recorded by the combination of two variables; anti-TIGIT (yes / no) and anti-PD-1 (yes / no). In addition to the additive effect of each treatment (anti-TIGIT*time and anti-PD-1*time), synergy was also evaluated by testing the interaction term of anti-TIGIT*anti-PD-1*time.

[0406] Fig.16 A shows the median tumor growth curve for each group as well as the individual growth curves for mice treated with anti-TIGIT antibodies as monotherapy. While no mice in the control group had tumor regression, 2 / 8 mice treated with anti-TIGIT had a complete response. In the remaining mice, there was a significant delay in tumor growth. In the control group, no mice survived more than 30 days, while in the treated group, 7 / 8 mice survived more than 30 days.

[0407] Fig.16B shows the median tumor growth curve for each group and the individual growth curves of mice treated with anti-PD1 as a monotherapy or in combination with anti-TIGIT. Tumor growth in mice treated with anti-TIGIT + anti-PD-1 was significantly inhibited compared with anti-PD-1 monotherapy (p < 0.0001). The combination of anti-TIGIT + anti-PD-1 achieved a synergistic anti-tumor efficacy that exceeded the additive effect of the two monotherapy treatments (p = 0.02). The combination of anti-TIGIT (200ug) and anti-PD1 antibodies resulted in 7 / 8 mice showing a complete response. The combination of anti-PD1 and lower doses of anti-TIGIT maintained anti-tumor efficacy, with 8 / 8 mice achieving a complete response when the anti-TIGIT antibody was reduced to 60μg, and 5 / 8 mice achieving a complete response when the anti-TIGIT antibody was further reduced to 20μg ( Fig.16 C). These data demonstrate significant anti-tumor efficacy of anti-TIGIT therapy for the treatment of pre-established tumors, either as monotherapy (p<0.0001) or in combination with anti-PD1 antibody (p<0.0001).

[0408] Example 13: Isotype-dependent anti-tumor activity of anti-TIGIT antagonist antibodies as monotherapy and in combination with anti-PD1 antibodies in mouse models.

[0409] For this experiment, anti-TIGIT clone 26493 was generated on mouse IgG2a and mouse IgG1 isotypes in mammalian cells. Female Balb / c mice aged 8 weeks were inoculated subcutaneously with 500.000 CT26 colon cancer cells ( CRL-2638 TM On the 10th day after inoculation, when the tumor volume averaged 100 mm 3 Around 10 days, mice were randomly divided into treatment groups with equal tumor volumes (n=10 per group). To evaluate monotherapy, mice were treated with 200 μg anti-TIGIT or isotype control (mIgG2a, BioXcell) by intraperitoneal injection on days 10, 13, and 16. In order to evaluate the combination with anti-PD-1, mice were treated with 200 μg anti-PD-1 (RMP1-14, BioXcell) and 200 μg isotype control (mIgG2a, BioXcell) or a combination of 200 μg anti-PD-1 (RMP1-14, BioXcell) and 200 μg anti-TIGIT by intraperitoneal injection on days 10, 13, and 16. Tumor growth was monitored, and tumor volume was measured with an electronic caliper three times a week from day 10 to day 33. When the tumor volume exceeded 2000 mm 3 When , the mice were killed.

[0410] Fig.17A shows the median tumor growth curves for each group as well as the individual growth curves for monotherapy with anti-TIGIT antibodies, Fig.17 B shows the individual growth curves for the combination therapy of anti-TIGIT and anti-PD1 antibodies. In monotherapy and in combination with anti-PD-1, treatment with anti-TIGIT antibodies produced significant anti-tumor efficacy when administered as a mouse IgG2a isotype (p = 0.0001 and p = 0.009, respectively). However, no anti-tumor efficacy was observed with anti-TIGIT as a mouse IgG1 isotype, indicating that the interaction of Fc receptors with mIgG2a is important for the anti-tumor activity of anti-TIGIT antagonist antibodies in the murine CT26 model. These data demonstrate the isotype-dependent anti-tumor efficacy of monotherapy or combined anti-TIGIT therapy for the treatment of pre-established tumors.

[0411] Example 14: Characterization of the mechanism of action of anti-TIGIT antagonist antibodies in vivo anti-tumor activity

[0412] A. Flow cytometric analysis of spleen and tumors

[0413] In order to study the in vivo mode of action of antagonistic anti-TIGIT antibodies, after treatment with monotherapy and anti-TIGIT antibody 26493 (IgG2a) in combination with anti-PD-1, the immune cell infiltration of the tumor was analyzed by flow cytometry. Mice were inoculated and treated as described in Example 12. Two days after the second treatment, mice (8 mice per group) were killed and tumors were harvested. Tumors were dissociated with a tumor dissociation kit (Miltenyi Biotec). For direct ex vivo staining, after staining with a viability dye (MolecularProbes, L34955) and Fc-block, cells were stained with anti-CD45, anti-CD4, anti-CD8 and anti-FoxP3 (all from eBioscience). For ex vivo stimulation, cells were incubated with a cell stimulation mixture (eBioscience) and a protein transport inhibitor (eBioscience) for 3 hours. After this, anti-CD4 and anti-CD8 antibodies and Fc-block were stained. After fixation and permeabilization with commercial buffers (IC fixation buffer and permeabilization buffer), cells were stained with anti-IL-10 and anti-IFNg (both from eBioscience). In all figures, the percentage change compared to the relevant control group (isotype control for monotherapy, anti-PD-1 in combination) is shown, where negative values ​​represent a decrease compared to the control group, and positive values ​​represent an increase.

[0414] Fig.18 A shows that in vivo treatment of tumors with anti-TIGIT mIgG2a antibodies resulted in CD4 +The proportion of regulatory T cells within the TIL population decreased by 28% compared with the control group, which was not observed after treatment with anti-TIGIT mIgG1. This suggests that the presence of TIGIT + Depletion of Treg cells may thus explain the differential efficacy of the two isotypes as discussed in Example 14. Fig.18 B shows no CD8 + Instead of depletion of TILs, a slight increase was observed for both isotypes (17% increase for mIgG1 and 16% increase for mIgG2a compared to control). Together, these findings suggest that the CD8 / Treg ratio increased by more than 50% in tumors treated with anti-TIGIT mIgG2a ( Fig.18 C). For the group treated with anti-TIGIT mIgG2a antibody, the function of intratumoral T cells was also improved, with CD4 + ( Fig.18 D) and CD8 + TIL( Fig.18 E) Both strongly increased IFNg production. This resulted in a + TIL / CD8 + The ratio of IFN-g-producing cells to IL-10-producing cells in the population increased strongly after ex vivo stimulation ( Fig.18 F).

[0415] Fig.18 G shows that combining anti-TIGIT mIgG2a with anti-PD-1 resulted in a 33% reduction in regulatory T cells compared with anti-PD-1 monotherapy. Similarly, for CD8 + The opposite was true for T cells, where CD8 + T cell infiltration increased by 22% and 28%, respectively ( Fig.18 H). In summary, for the combination with anti-TIGITmIgG2a, this resulted in a decrease in CD8 + The ratio of TIL to Treg increased more than twofold ( Fig.18 I). In addition, the combination of anti-TIGIT antibody mIgG2a and anti-PD-1 showed that intratumoral CD4 + The phenotype of T cells changed from Th1 to Th2, and the number of CD4 cells producing IFNg increased significantly ( Fig.18 J), while the number of CD4 cells producing IL-10 decreased ( Fig.18 K). This resulted in a decrease in CD4 + The number of cells producing IFNg / IL-10 in the TIL population increased strongly after ex vivo stimulation ( Fig.18 L).

[0416] Table 11: Differentially expressed genes between anti-TIGIT mIgG2a and vehicle treated mice

[0417]

[0418]

[0419] Table 12: Differentially expressed genes between anti-TIGIT mIgG2a+anti-PD-1 and anti-PD1 treated mice

[0420]

[0421]

[0422] B. Transcriptomic analysis of tumors by NanoString

[0423] To study the in vivo mode of action of anti-TIGIT antibodies, immune cell infiltration of tumors treated with monotherapy and anti-TIGIT in combination with anti-PD-1 was analyzed by transcriptomic analysis (Nanostring). Mice were inoculated and treated as described in Example 12. Two days after the third treatment with anti-TIGIT and / or anti-PD1 antibodies, mice were sacrificed and tumors were harvested. RNA was extracted and the expression of 770 genes involved in cancer immunology was directly quantified using nCounter technology (PanCancer Immunoassay Panel, Nanostring; performed by VIB NucleomicsCore). Data were analyzed using nSolver software (Nanostring).

[0424] Fig.19 A shows a volcano plot of differentially regulated genes between vehicle-treated mice and anti-TIGIT mIgG2a-treated mice. Highly statistically significant genes fall at the top of the graph, and highly differentially expressed genes fall to either side (left: downregulated in anti-TIGIT-treated mice, right: upregulated in anti-TIGIT-treated mice). Examples of highly upregulated genes include perforin, granzyme B, and CTLA-4. The solid line indicates an uncorrected p-value of 0.01, and the dotted line indicates a corrected p-value of 0.05 (Benjamini-Hochberg correction). Tables 11 and 12 show genes that are significantly differentially expressed in anti-TIGIT mIgG2a compared to vehicle and aPD-1+ anti-TIGIT mIgG2a compared to anti-PD1, respectively. When multiple genes are summarized by score for functional subsets of immune cells, the most significant findings are cytotoxic cells and CD8 + Increased T cell score ( Fig.19B). The same changes were present in mice treated with anti-PD-1 + anti-TIGIT mIgG2a compared to anti-PD-1 alone. No changes were observed in mice treated with anti-TIGIT mIgG1 as monotherapy or in combination with anti-PD-1.

[0425] Taken together, these results demonstrate that the antitumor efficacy observed after in vivo treatment with anti-TIGIT antibodies is driven by a reduction in Treg infiltration in tumors and a decrease in CD8 + The increase in effector T cell populations is also mediated by + and CD8 + Effector function of TILs, as indicated by a higher proportion of IFNg-producing cells, a shift toward a Th1 response and increased expression of genes important for T cell cytotoxic function.

[0426] Example 15: Antibody-dependent cellular cytotoxicity (ADCC) activity induced by anti-TIGIT antagonist antibodies

[0427] A. In vitro ADCC on human PBMCs from healthy donors

[0428] Isolated PBMCs from healthy human donors were resuspended in complete RPMI medium (supplemented with 10% heat-inactivated FBS + 50U penicillin + 50U streptomycin and supplemented with 200IU IL-2 / ml). 2,5x10 5 Human PBMCs. Anti-human TIGIT antibody clone 26452 or IgG1 isotype control (Biolegend, 403102) produced in mammalian cells was added to each corresponding well at final concentrations of 66, 6, 0, 66 and 0,006 nM. Cells were incubated at 37°C, 5% CO2 for 20 hours. Cells were then collected and stained with the following antibody panel: LVD efluor 520 (eBioscience 65-0867-14), anti-TCRab-PercP-Cy5.5 (clone IP26, Biolegend 306723), anti-CD4-BV510 (clone SK3, BD Horizon 562970), anti-CD8-APC-Cy7 (clone SK1, Biolegend 344714), anti-CD25-BV605 (clone 2A3, Biolegend 562660), anti-CD127-APC (A019D5, Biolegend 351316), anti-CCR7-BV421 (clone G043H7, Biolegend 353207) and anti-CD45RO-PE-Cy7 (clone UCHL1, Biolegend 304229). Results on gated live cells are presented. CD45+ CD4 + or CD45 + CD8 + Represents total CD4 + or CD8 + T cells. CD45 + RO + CD4 + or CD45 + RO + CD8 + Cells represent memory CD4 + or CD8 + T cells, and CD25 hi CD127 低 CD4 + Represents Treg cells. Fig. 20 As shown in A, gating Treg on TIGIT + The proportion of cells was higher than that in gated memory CD8 + T cells and CD4 + The proportion of T cells.

[0429] Absolute quantification was performed using AccuCheck counting beads (Life technologies) according to the manufacturer's instructions. After calculating the absolute number of cells per μl, the % of specific lysis was calculated using the following formula = (1-(absolute number of cells per μl on 26452 TIGIT antibody-treated samples / average of triplicates without antibody treatment)) × 100. Fig. 20 As shown in B, the specific lysis of Treg triggered by anti-TIGIT 26452hIgG1 antibody (62.22%) was higher than that of total CD8 + T cells (12.2%) or total CD4 + T cells (16.36%).

[0430] B. Ex vivo ADCC on mouse tumors

[0431] To confirm that anti-TIGIT mouse IgG2a antibodies can deplete TIGIT + An ex vivo ADCC assay was established for regulatory T cells. Female Balb / c mice aged 8 weeks were inoculated subcutaneously with 500.000 CT26 colon cancer cells ( CRL-2638 TM). Three weeks after inoculation, tumors were harvested and dissociated using a tumor dissociation kit (Miltenyi Biotec). Single cell suspensions were incubated with 133 nM anti-TIGIT antibody 26493 (mIgG1 or mIgG2a isotype) for 20 hours (1 million cells / 200 μl in RPMI+10% FBS). After 20 hours, cells were stained with anti-CD4, anti-TIGIT, anti-CD8, and anti-FoxP3 antibodies (all from eBioscience) after staining with a viability dye (Molecular Probes, L34955) and Fc-block.

[0432] Fig.21 Display for different TIGIT + Immune subsets, absolute TIGIT compared to isotype control treatment + The strongest reduction after treatment with anti-TIGIT mIgG2a antibody was evident in regulatory T cells (around 40% reduction), suggesting that these cells are more potent than conventional CD4 + or CD8 + T cells are more susceptible to ADCC.

[0433] Taken together, these results demonstrate that anti-TIGIT hIgG1 or mIgG2a depletes TIGIT + The efficacy of immune cells showed stronger activity in the Treg population.

[0434] Example 16: Immunogenicity prediction using computer analysis

[0435] The immunogenic potential of clones 29494 and 29489 and their variants 31282 was evaluated by computer prediction using EpiMatrix protein scoring (De Groot et al., (2009) Clinical Immunol. 131: 189). To complete the analysis, the input sequence was parsed into overlapping 9-mer frameworks, and each framework was evaluated for a set of eight common class II HLA alleles. These alleles are "supertypes". Each is functionally equivalent or nearly equivalent to many other allele "family members". In general, these eight supertype alleles and their respective family members "cover" more than 95% of the population (Southwood et al., (1998) J. Immunol 160: 3363). The "evaluation" of each framework allele by allele states the predicted HLA binding affinity. The EpiMatrix evaluation score range is about -3 to +3, and is normally distributed. An EpiMatrix assessment score higher than 1.64 was defined as a “hit”; that is, potentially immunogenic and worthy of further consideration.

[0436] All other factors being equal, the more HLA ligands (i.e., EpiMatrix hits) contained in a given protein, the greater the likelihood that the protein will induce an immune response. The EpiMatrix protein score is the difference between the number of predicted T cell epitopes expected to be found in a protein of a given size and the number of putative epitopes predicted by the EpiMatrix system. The EpiMatrix protein score is correlated with the observed immunogenicity. EpiMatrix protein scores are "normalized" and can be plotted according to a standardized scale. The EpiMatrix protein score for an "average" protein is zero. EpiMatrix protein scores above zero indicate the presence of an excess of MHC ligands and indicate a higher immunogenic potential, while scores below zero indicate the presence of fewer potential MHC ligands than expected and a lower immunogenic potential. Proteins with scores above +20 are considered to have significant immunogenic potential.

[0437] Regulation of the presence of regulatory T cell epitopes

[0438] Antibodies are unique proteins in that the amino acid sequences of their variable domains, particularly their complementarity determining regions (CDRs), can vary to a great extent. It is this variability that allows antibodies to recognize a wide variety of antigens. However, the recombination and mutation events that control antibody maturation can also generate new or new T-cell epitopes. These new epitopes appear to be "foreign" to circulating T cells. The presence of new epitopes in the antibody sequence can lead to the formation of a human anti-human antibody response; also known as a HAHA response or ADA (anti-drug antibodies).

[0439] Regulatory T cells play an important role in suppressing immune responses to peripheral fully human proteins, including those containing mutations and / or highly variable sequences such as antibody CDRs. Regulatory T cells engage and activate through regulatory T cell epitopes. The inherent risks associated with the presence of new epitopes in antibody sequences appear to be balanced by the presence of naturally occurring regulatory T cell epitopes.

[0440] By screening the sequences of many human antibody isolates, EpiVax has identified several highly conserved HLA ligands that are thought to have regulatory potential. Experimental evidence suggests that many of these peptides are actually actively tolerogenic in most subjects. These highly conserved, regulatory, and promiscuous T cell epitopes are currently referred to as Tregitopes (De Groot et al. (2008) Blood 112:3303).

[0441] In many cases, the immunogenic potential of new epitopes contained in humanized antibodies can be effectively controlled in the presence of a large amount of Tregitope. For the purpose of antibody immunogenicity analysis, EpiVax has developed an EpiMatrix score regulated by Tregitope and a corresponding prediction of anti-therapeutic antibody response. In order to calculate the EpiMatrix score regulated by Tregitope, the score of Tregitope is deducted from the EpiMatrix protein score. It has been confirmed that the score regulated by Tregitope has a good correlation with the observed clinical immune response of a group of 23 commercial antibodies (De Groot et al., (2009) Clinical Immunol. 131: 189).

[0442] Clone 29489, 29494 and 31282 antibody sequence scores are at the lower end of the EpiMatrix scale, indicating limited potential for immunogenicity. For antibody clones 29489 and 31282, regression analysis of licensed monoclonal antibodies predicts ADA responses in ~0% of exposed patients. For clone 29494, the analysis predicts ADA responses in 2.78% of exposed patients for baseline VH sequences and 2.88% for variant VH sequences. The data are summarized in Table 13 below.

[0443] Table 13: EpiMatrix scores and Tregitope-adjusted EpiMatrix scores

[0444]

[0445] Example 17: Affinity determination of anti-TIGIT clones binding to recombinant human TIGIT protein

[0446] Antibody 31282 was compared with anti-TIGIT antibody clones described in other patent applications. Specifically, 31282 was compared with: 4.1D3.Q1E (also known as 4.1D3, from WO2017 / 053748); 22G2 (from WO2016106302); 31C6 (from WO2016 / 028656); 313M2 (from WO2016 / 191643); and TIG1 (from WO2017 / 152088). The references and sequences of the compared antibody clones are shown in Table 14 below:

[0447] Table 14: Comparison of VH and VL domain sequences of anti-TIGIT antibodies

[0448]

[0449]

[0450]

[0451] A. Produced in mammalian cells

[0452] In order to generate sufficient amounts of selected α-TIGIT clones for further characterization, DNA vectors encoding specific antibody clones (clones 31282_up, 4.1D3, 22G2, 31C6, 313M32 and TIG1) were generated and transduced into HEK cells for the production of human IgG1 isotypes. Human codon-optimized synthetic DNA fragments of the antibody variable domains were ordered at Geneart. The variable domain sequences were seamlessly linked to the pUPE expression vector containing the mouse Igκ signal sequence and the constant region of the corresponding antibody class. The expression vector was verified by restriction analysis and DNA sequencing. For transient transfection, endotoxin-free DNA maxipreps (Sigma) were produced, and the heavy and light chain vectors were co-transfected into HEK293EBNA1 cells in Freestyle medium (ThermoFisherScientific) according to established protocols. Primatone (0.55% final volume) was added 24 hours after transfection. Conditioned medium was harvested 6 days after transfection. Antibodies were purified in batches by Mabselect sureLX (GE Healthcare) affinity chromatography. The bound antibody was washed in two steps with PBS containing 1M NaCl and PBS. The antibody was eluted with 20mM citrate 150mM NaCl pH 3 and neutralized to about pH 7 with 1 / 6 volume of 1M K2HPO4 / KH2PO4 pH 8.

[0453] Next, the antibody was further purified by gel filtration using a Superdex200 column equilibrated in PBS. The fractions were analyzed by NuPAGE and the fractions containing the antibody were pooled. The final product was sterilized through a 0,22 μM syringe filter. The product was analyzed by NuPAGE and the endotoxin level was measured by LAL assay.

[0454] In addition, also in CHO-K1 cells, clone 31282 is produced on IgG1 or IgG4 isotype as follows (clone 31282_wu).Construct the DNA vector encoding the antibody and transfect it into CHO-K1 cells.Synthesize the CHO codon optimized DNA fragment for the antibody variable domain, and connect it to the expression vector containing the constant region of the signal sequence and the corresponding antibody class.Expression vector is verified by restriction analysis and DNA sequencing.According to the established scheme, heavy chain and light chain vector are cotransfected into CHO-K1 cells by electroporation (Bio-rad).The transfected culture is increased in proportion and inoculated into the fed-batch culture.Batch fed-batch culture is harvested in the conditioned medium after 14 days.

[0455] First, the cell culture of harvest was clarified by two depth filtration stages with DOHC and A1HC (Millipore) connected in series. Then, the harvest clarified by affinity chromatography was first purified with MabSelect SuRe (GE Healthcare). The bound antibody was washed in two steps with 50mM NaAc-HAc (pH 5.5) and 50mM NaAc-HAc (pH 5.5) containing 1M NaCl. The antibody was then eluted with 50mM NaAc-HAc (pH 3.5) and neutralized to about pH 5.5 with 1M Tris-HCl (pH 9.0).

[0456] Next, the neutralized intermediate was further refined (polished) by anion exchange chromatography (AEX) in flow-through mode using POROS HQ50 (Life Tech). Before loading, the column was balanced with 50mM NaAc-HAc (pH 5.5). The AEX flowthrough collected during loading and recovery steps was further refined by cation exchange chromatography (CEX) in bind-elute mode using POROS XS (Life Tech.). The CEX column was balanced in 50mM NaAc-HAc (pH 5.5), and the antibody was eluted by linear gradient elution (LGE) to reach 10CV of 50mM NaAc-HAc (pH 5.5) containing 0.5M NaCl. Pellicon 3, ultracel 30kD, A type (Millipore) was used for final ultrafiltration and diafiltration (UF / DF) to concentrate CEX eluate and exchange buffer into 20mM His-HCl (pH 5.5). Afterwards, polysorbate 80 (PS80) and sucrose were added to the diafiltered sample to obtain a final product at a concentration of about 20 g / L in a buffer of 20 mM His-HCl, 0.01% (w / w) PS 80 and 9% (w / v) sucrose (pH 5.5). The product has passed all PQA tests. SEC purity, endotoxin levels and other standards are all in compliance.

[0457] B. Biacore measurement

[0458] Biosensor analysis was performed at 25°C in a HBS-EP buffer system (10mM HEPES pH 7.3, 150mM NaCl, 3mM EDTA, 0.05% Tween 20) using Biacore T200 technology, CM5 sensor chips (run at Novalix, France). The sample set was kept at 8°C. Goat anti-human IgG capture antibodies (Fcγ fragment specific, Jackson ImmunoResearch Laboratories) were fixed (10000RU) to two flow cells of the sensor chip using standard amine coupling chemistry. This surface type provides a form of repeatable capture of fresh analytical antibodies after each regeneration step. Flow cell 2 was used to analyze the captured antibodies, while flow cell 1 was used as a reference flow cell. Six different antigen concentrations from 30 to 0.123nM were prepared in running buffer. Except for the 3.33nM duplicate run, each antigen sample concentration was run as a single replicate. Two blank (buffer) injections were also run and used to evaluate and subtract system artifacts. The association (300 seconds) and dissociation (600 seconds) phases of all antigen concentrations were performed at a flow rate of 30uL / min. The surface was regenerated by three consecutive injections (15 seconds, 15 seconds and 60 seconds) of 10mM glycine-HCl (pH 1.5). The sensorgram obtained was globally fitted to a 1:1 model (assuming that all concentrations applied had the same kinetic values). Affinity was also determined by the steady state of clone 313M32, because the 1:1 kinetic model fit was not very reliable, showing equilibrium with human TIGIT at the end of the association time. The results obtained by different α-TIGIT clones are reported in Table 15.

[0459] Table 15: Kinetic and affinity evaluation

[0460]

[0461] Example 18: Cell Binding of Anti-TIGIT Antagonistic Antibodies

[0462] A. Binding of anti-TIGIT clones to Jurkat-hTIGIT

[0463] The affinity of human anti-TIGIT antibodies has been measured using Jurkat E6.1 cells transduced with human TIGIT (Jurkat hTIGIT). To analyze the affinity of the selected antibodies for hTIGIT, 10 5 cells and compared with decreasing concentrations (8, 4, 2, 1, 0,5, 0,25, 0,125, 0,062, 0,031, 0,016, 8x10 -3 and 4x10 -3 nM) of various anti-TIGIT antagonist antibody clones were incubated together ( Figure 2 ). The antibodies were incubated with cells at 4°C in FACS buffer for 20 minutes. After washing, the cells were incubated with anti-human Ig (Fcγ specific)-PE (eBioscience, 12-4998-82, 2.5 μg / ml) on ice for 20 minutes and washed twice. Fluorescence intensity was analyzed using LSR BD Fortessa, and cell binding was recorded as the median fluorescence intensity of PE in cells expressing TIGIT on their surface.

[0464] The half-maximal concentration (EC) for binding to Jurkat-hTIGIT was calculated using a four-variable curve-fitting equation in Prism. 50 ). The result is Fig. 22 A, and the values ​​are summarized in Table 16 below. For clone 31282, the EC binding to Jurkat-hTIGIT 50 The values ​​were very close, with no significant differences between antibodies produced in HEK cells (31282_up, 0.13 nM) or CHO-K1 cells (31282-wu, 0.10 nM). Clones 31C6 and TIG1 also showed EC values ​​below 0.2 nM. 50 The affinity of other clones (4.1D3, 22G2 and 313M32) was lower, and the results showed that EC 50 The values ​​ranged from 0.267 to 0.445 nM. The results demonstrated that for anti-TIGIT clones 31282, 31C6, and TIG1, there was strong binding to membrane-expressed human TIGIT in the engineered system, while the other clones had lower affinity.

[0465] Table 16: EC binding of different α-TIGIT clones to Jurkat-hTIGIT 50 Data and comparisons

[0466]

[0467] B. Anti-TIGIT clones and primary CD8 + T cell binding

[0468] Isolated human PBMCs from healthy volunteers were analyzed for binding to antagonist anti-TIGIT antibodies. 5Cells were incubated with anti-CD16 (clone 3G8, BioLegend 302002), CD32 (clone FLI8.26, BD Bioscience 557333), and CD64 (BD Bioscience 555525) for 10 min at room temperature and the indicated anti-human TIGIT antibody clones were added directly at final concentrations of 8, 4, 2, 1, 0,5, 0,25, 0,125, 0,062, 0,031, 0,016, 8x10 -3 and 4x10 -3 nM, and incubated at 4°C for 20 minutes. After washing, the cells were incubated with anti-human Ig (Fcγ specific)-PE (eBioscience, 12-4998-82, 2.5 μg / ml) at 4°C for 20 minutes. Then, the cells were washed and incubated with the following antibody and LVD mixture: anti-CD4-PercP-Cy5.5 (clone A161A1, BioLegend 357414); anti-CD8-BV510 (clone SK1, BD Bioscience 563919) and LVD efluor 660 (eBioscience 65-0864-18).

[0469] Using Live TIGIT + CD8 + MFI signal calculation on T cells and CD8 + Human primary T cells bind EC 50 The result is Fig. 22 As shown in B, EC 50 The concentrations are summarized in Table 17 below. For clone 31282, binding to human primary CD8 + T cell EC 50 The values ​​were very close, with no significant differences between antibodies produced in HEK cells (31282_up, 0.21 nM) or CHO-K1 cells (31282-wu, 0.19 nM). Comparison between different clones of antagonistic α-TIGIT antibodies showed that clone 31282_wu (0.19 nM) and clone 31282_up (0.21 nM) were highly specific for human primary CD8 + T cell binding has optimal EC 50 clones 31C6 and TIG1 showed EC 50 The difference between clones 22G2, 313M32, and 4.1D3 was 6.1 to 9.7 fold. + TIGIT expressed on the membrane of T cells showed the best binding.

[0470] Table 17: Different a-TIGIT clones and human primary CD8 + T cell-bound EC 50 Data and comparisons

[0471]

[0472] C. Anti-TIGIT clones and primary CD8 + T cell binding

[0473] Isolated human PBMCs from cancer patients were analyzed for binding to different antagonist anti-TIGIT antibody clones. 5 Cells were distributed per cell. Cells were incubated with anti-CD16 (clone 3G8, BioLegend 302002), CD32 (clone FLI8.26, BD Bioscience 557333) and CD64 (BD Bioscience 555525) at room temperature for 10 minutes, and the specified anti-human TIGIT antibodies were added directly at final concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062 and 0.031 nM in FACS buffer and incubated at 4°C for 20 minutes. After washing, cells were incubated with anti-human Ig (Fcγ specific)-PE (eBioscience, 12-4998-82, 2.5 μg / ml) at 4°C for 20 minutes. Then, the cells were washed and incubated with the following antibody and life viability dye (LVD) mixture: anti-CD4-PercP-Cy5.5 (clone A161A1, BioLegend 357414); anti-CD8-BV510 (clone SK1, BD Bioscience 563919) and LVD efluor 520 (eBioscience 65-0867-14). The cells were washed and fixed, and the surface staining was quantified using BD LSR Fortessa. Flow cytometry data were analyzed using FlowJo V10.1. The gated LVD - TIGIT + CD8 + TIGIT MFI on cells was used to calculate EC 50 The nonlinear regression curve is shown in Fig. 22 C, and the values ​​are summarized in Table 18 below.

[0474] Clones 31282_wu and 31282_up for CD8 + Binding of T cells showed very close proximity to EC 50The remaining clones showed lower affinities, with clones 31C6, TIG1, and 22G2 showing 1.5, 2.7, and 3.1-fold lower affinities, respectively. The EC of clone 313M32 was 0.14 and 0.12 nM, respectively, compared to clone 31282_up. 50 The measured value was 8.3-fold lower. Clone 4.1D3 showed the lowest affinity, with a 9.5-fold difference in binding from the best clone tested.

[0475] Table 18: Different α-TIGIT clones and human primary CD8 from cancer patients + T cell-bound EC 50 Data and comparisons

[0476]

[0477] Example 19: Competition assay between anti-TIGIT antagonist antibody clones and TIGIT natural ligand (CD155)

[0478] Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT) were collected and cultured according to 5.10 4 Cells were dispensed into wells and incubated in complete medium at 37°C for 45 min with anti-human TIGIT antibody at the following concentrations: 133,33,42,20,13,33,4,22,1,33,0,422,0,133,0,042,0,0133,4,2x10 -3 , 1,3x10 -3 ,4,2x10 -4 , 1,3x10 -4 ,4,2x10 -5 nM. Excess antibody was washed, and the cells were then incubated with 15 μg / ml CD155-His (Creative Biomart, PVR-3141H) at 37°C for 45 minutes. Then, bound CD155-His was detected using anti-His tag-PE (Biolegend, 362603, 2 μl per test) and incubated at 4°C for 30 minutes. Cells were analyzed by FACS using BD LSRFortessa, and the half concentration (IC) that prevented CD155 binding was calculated based on the median fluorescence intensity of PE in total cells. 50 ).

[0479] The results are Fig.23 The values ​​are shown in Table 19 below. Anti-TIGIT clones 31282_wu and 31282_up showed the best IC for CD155 competition on Jurkat cells engineered to express hTIGIT 50The IC values ​​of other clones (4.1D3, 22G2, 31C6, TIG1) were 0.05 and 0.04 nM, respectively. 50 The values ​​were 0.07 to 0.09 nM, while clone 313M32 competed with CD155 for binding to TIGIT much less efficiently (0.65 nM).

[0480] Table 19: IC of different a-TIGIT clones competing for CD155 on human TIGIT 50 Data and comparisons

[0481]

[0482] Example 20: Functional characterization of antagonistic anti-TIGIT clones

[0483] A. TIGIT functional assay using Jurkat-hTIGIT cells

[0484] To characterize the functional consequences of blocking the human TIGIT receptor, we co-cultured Jurkat cells expressing hTIGIT and a luciferase reporter gene activated upon TCR engagement (TIGIT effector cells from Promega, ready-to-use, thawed) with a CHO-K1 cell line engineered to express human PVR / CD155 and TCR activators (CD155aAPC / CHO-K1 from Promega, ready-to-use, thawed). Activation of Jurkat cells overexpressing TIGIT can be induced by contact with CHO-K1 cells expressing CD155 upon TCR engagement on Jurkat cells and can be increased in the presence of antagonistic anti-TIGIT antibodies. To compare the potency of different a-TIGIT clones to increase Jurkat cell activation, experiments were performed in the presence of increasing antibody concentrations and the EC was calculated. 50 value.

[0485] CD155 aAPC / CHO-K1 (Promega, CS198811) cells were seeded according to the manufacturer's recommendations and incubated at 37°C in a 5% CO2 incubator O / N. The next day, TIGIT effector cells (Promega, CS198811) were added to CD155 aAPC / CHO-K1 cell plates containing fresh complete medium with increasing concentrations of anti-TIGIT antibodies (0,03, 0,11, 0,33, 1,06, 3,34, 10,56, 33,38, 105,49; and 333 nM) according to the manufacturer's recommendations and incubated for 6 hours at 37°C, 5% CO2. After 6 hours of incubation, the activation of TIGIT effector cells was assessed by measuring luciferase activity using the Bio-GloTM Luciferase Assay System (Promega, G7941).

[0486] like Fig.24 As shown in A and summarized in Table 20, in the assay, anti-TIGIT antibody 31282 50 The activity observed for clones generated in HEK (31282_up) or CHO-K1 (31282_wu) cells was comparable, with a maximum luciferase signal 8-fold higher than the control isotype (Bioexcell, BE0297) and an EC of 1. 50 The concentrations were 3.3 nM and 3.5 nM, respectively. By comparison, clones 4.1D3, 22G2, and 31C6 had maximal activities 5.3 to 6.7 times that of the isotype control, which is consistent with an EC of 5 to 10 nM. 50 EC values ​​for clones 313M32 and TIG1 could not be determined due to low activity at the concentrations tested and poor curve fit. 50 value( Fig.24 A).

[0487] Table 20: EC values ​​of different α-TIGIT clones for functional activity in Jurkat-hTIGIT cells 50 Data and comparisons

[0488]

[0489] PF: Poor Fit

[0490] B. Primary human CD8 from healthy volunteers + TIGIT function assay for T cells

[0491] To characterize the functional consequences of blocking the human TIGIT receptor, we isolated primary human CD8 + T cells were co-cultured with a CHO-K1 cell line engineered to express human PVR / CD155 and activated human T cells. We observed that blocking hTIGIT with an anti-TIGIT antagonist antibody could abrogate the activation of CD8 + To compare the potency of these antibodies in increasing IFNg release, experiments were performed in the presence of increasing antibody concentrations and the EC 50 value.

[0492] CD155 aAPC / CHO-K1 (Promega, CS198811) cells were seeded in U-bottom 96-well plates according to the manufacturer's recommendations and incubated at 37°C in a 5% CO2 incubator O / N. The next day, CD8 + T cells. Purified CD8 T cells and increasing concentrations (0,011nM, 0,033nM, 0,11nM, 0,33nM, 1,06nM, 3,3nM, 10,6nM, 33,3nM and 105,5nM) of antibodies were then added to CD155aAPC / CHO-K1 (100,000 CD8 T cells / 100ul complete medium containing antibodies) and incubated for 5 days at 37°C, 5% CO2. Finally, IFNg concentrations in cell supernatants were assessed using an ELISA assay (Affymetrix eBioscience, 88-7316-86) run according to the manufacturer's recommendations.

[0493] like Fig.24 As shown in B and summarized in Table 21, a-TIGIT clones 31282 and 4.1D3 showed the best induction of IFNg secretion, with increases of 2.7 and 2.9 fold, respectively, relative to the isotype control antibody. 50 In terms of concentration, clone 31282 had the best efficacy in inducing IFNg production. Clone 31C6 showed a 2.3-fold difference in EC compared to clone 31282. 50 The value of clone 313M32 could not be determined due to low activity at the tested concentrations and poor curve fit ( Fig.24 B).

[0494] Table 21: Effects of different α-TIGIT clones on primary human CD8 + Functional activity of T cells in EC 50 Data and comparisons

[0495]

[0496] C. Human primary CD3 from cancer patients + TIGIT function assay for T cells

[0497] To characterize the functional consequences of blocking the human TIGIT receptor on T cells from cancer patients, human primary CD3 +T cells were co-cultured with a CHO-K1 cell line engineered to express human PVR / CD155 and activated human T cells (CHO-TCR-CD155). We observed that blocking hTIGIT with the anti-TIGIT antagonist antibody 31282 increased CD3 + IFNg release by T cells.

[0498] CD155 aAPC / CHO-K1 (Promega, CS198811) cells were seeded in U-bottom 96-well plates according to the manufacturer's recommendations and incubated at 37°C in a 5% CO2 incubator O / N. The next day, CD3 was purified from fresh human peripheral blood mononuclear cells using a negative selection kit (Stemcell Technologies, 17951) according to the manufacturer's recommendations. + T cells, the fresh human peripheral blood mononuclear cells were isolated from whole blood of cancer patients (HNSCC) collected 24 hours ago (Biopartners). The purified CD3 + T cells were added to CD155 aAPC / CHO-K1 (100,000 CD3 T cells / 100ul complete medium containing antibody) with 66,7nM antibody and incubated for 5 days at 37°C, 5% CO2. Finally, IFNg concentrations in cell supernatants were assessed using an ELISA assay (Affymetrix eBioscience, 88-7316-86) run according to the manufacturer's recommendations.

[0499] like Fig.24 As shown in C, antibody 31282 induced strong functional activity to increase IFNg secretion, demonstrating the potential of this α-TIGIT antibody to reactivate PBMC T cells from cancer patients.

[0500] Da-TIGIT clone 31282 increases intracellular cytokine production in T cells from PBMCs of cancer patients as well as dissociated tumor cells (DTCs)

[0501] In this embodiment, intracellular flow cytometry staining is performed to assess the T cell cytokine production of lymphocytes infiltrated by tumors in matching PBMC and dissociated tumor cells (DTC) from freshly separated patients with renal cancer. For DTC, the tumor is mechanically broken, then incubated under rotation in a gentleMACS dissociator with a tumor dissociation kit (MiltenyiBiotech #130-095-929) according to the manufacturer's instructions for specific tumor types. Before intracellular staining, cells are stimulated for 16 hours with a mixture of T cell-stimulating beads (Dynabeads, Thermo Fisher). During the last 3 hours of stimulation, a mixture of protein transport inhibitors (eBioscience) and a cell stimulation mixture (eBioscience) are added to cells. Conjugated antibodies are purchased from Ebioscience / Thermo Fisher Scientific, BioLegend or BD Biosciences. Surface staining was performed using filtered FACS buffer (PBS + 2mM EDTA + 0,1% BSA) and Brilliant Stain buffer (BD#563794) according to the manufacturer's instructions. Prior to surface staining, cells were blocked with appropriate human FcBlock (BD#564220). For intracellular staining, cells were fixed and permeabilized using BD Cytofix / cytoperm solution (BD Biosciences). Cells were stained with the following antibody panel: anti-CD45-BB515 (clone HI30, BD Horizon 564585), anti-CD73-BV421 (clone AD2, BD Horizon 562430), anti-CD8a-BV510 (clone SK1, BD Horizon 563919), anti-CD3-BV650 (clone SK7, BD Horizon 563999), anti-IFNγ-BV711 (clone 4S.B3, BD Horizon 564793), anti-IL-2-APC (MQ1-17H12, eBioscience 17-7029-82), anti-CD4-APC-R700 (clone RPA-T4, BD Horizon 564975), LVD efluor 780 (eBioscience 65-0865-14), anti-TIGIT-PE (clone MBSA43, eBioscience E13456-108), anti-CD39-PE-Dazzle594 (clone A1, Biolegend 328224), and TNFα-PE-cy7 (clone Mab11, eBioscience 25-7349-82).Acquisition was performed on a FACSFortessa (BD Biosciences) and analysis was performed using FlowJo software (FlowJo, LLC). Live cells were gated at forward and side scatter. T cell subsets were gated as follows: CD45 of PBMC. + CD3 + and CD45 of DTC + CD3 + CD4 + and CD45 + CD3 + CD8 + Unstained and unstimulated controls were used to gate cytokine-secreting T cells.

[0502] Fig.24 D shows that the intracellular levels of IL2, IFNg, and TNFa all increased after activation in the presence of a-TIGIT clone 31282. Fig.24 The data shown in C are from PBMC CD3 + This increase was observed in T cells but not in CD4 T cells from dissociated tumor cells. + and CD8 + This increase was also observed in TILs. This demonstrates the potential of α-TIGIT clone 31282 to increase activation of PBMC and TIL populations from cancer patient T cells.

[0503] Example 21: a-TIGIT Clone 31282 Induces Preferential Cytotoxicity of Tregs in PBMCs from Cancer Patients

[0504] In this example, isolated PBMCs from lung cancer patients were resuspended in complete RPMI medium (supplemented with 10% heat-inactivated FBS + 50U penicillin + 50U streptomycin). 2.5x10 5Human PBMCs. Anti-human TIGIT antibody clone 31282, human IgG1 isotype control (BioXcell BE0297) or Rituximab (InvivoGen hcd20-mab1) was added to each corresponding well at a final concentration of 6.6 nM. Cells were incubated at 37°C, 5% CO2 for 20 hours. Cells were then collected and stained with the following antibody panel: LVD efluor 520 (eBioscience 65-0867-14), anti-TCRab-PercP-Cy5.5 (clone IP26, Biolegend 306723), anti-CD4-BV510 (clone SK3, BD Horizon 562970), anti-CD8-APC-Cy7 (clone SK1, Biolegend 344714), anti-CD25-BV605 (clone 2A3, Biolegend 562660), anti-CD127-APC (A019D5, Biolegend 351316), anti-CCR7-BV421 (clone G043H7, Biolegend 353207) and anti-CD45RO-PE-Cy7 (clone UCHL1, Biolegend 304229). Results on gated live cells are presented. Absolute quantification was performed using AccuCheck counting beads (Life technologies) according to the manufacturer's instructions. After calculating the absolute number of cells per μl, the percentage of specific lysis was calculated using the following formula = (1-(the absolute number of cells per μl on the sample treated with 31282TIGIT antibody / the average of the triplicate samples treated with the control isotype)) × 100. The results are expressed as the average % of specific lysis of the triplicates + / - SD. By measuring the gated CD19 after incubation with rituximab + The cytotoxic activity of ADCC / ADCP effector cells was assessed by the % of specific cell lysis on the cells.

[0505] like Fig.25 As shown, anti-TIGIT clone 31282 triggered more specific lysis on Treg cells (30.1 + / - 3%) than on CD45RO + CCR7 + / - CD8 + T cells (total memory CD8 + T cells) (-1.48 + / - 6%) or CD45RO + CCR7 + / - CD4 + T(Total Memory CD4 + T cells) (0.64 + / - 3%). Rituximab positive control was gated on CD19 +The total data showed that the specific lysis of the cells was 77.9% (+ / - 6.8%). + and CD8 + Treg cells were preferentially depleted from PBMCs of cancer patients compared to T cell populations. Similar preferential depletion of Treg cells was observed using cells from patients with colon adenocarcinoma.

[0506] Example 22: Characterization of TIGIT expression on immune populations from cancer patient PBMCs and dissociated tumor cells

[0507] Flow cytometry analysis was performed to evaluate TIGIT expression on immune cell subsets of tumor-infiltrating lymphocytes in freshly isolated matched PBMCs and dissociated tumor cells (DTCs) from cancer patients. Samples from different indications were obtained: ovarian cancer, renal cancer, HNSSC, skin cancer, melanoma, and lung cancer. For DTCs, the tumors were mechanically disrupted and then incubated under rotation in a gentleMACS dissociator using a tumor dissociation kit (Miltenyi Biotech #130-095-929) according to the manufacturer's instructions for the specific tumor type. PBMCs were isolated from whole blood on a density gradient medium (Lymphoprep Axis-Shield #1115758). Phenotypic data were compared with frozen PBMCs isolated from healthy individuals (n=10).

[0508] Cells were stained using filtered FACS buffer (PBS + 2mM EDTA + 0.1% BSA) and Brilliant Stain buffer (BD#563794) according to the manufacturer's instructions. Cells were blocked with appropriate human FcBlock (BD#564220) prior to staining and fixed using IC Fixation Buffer (eBioscience #00-8222-49) prior to acquisition. DTCs were stained with the following antibody panel: anti-CD45-BB515 (clone HI30, BD Horizon 564585), anti-CD73-BV421 (clone AD2, BD Horizon 562430), anti-CD8a-BV510 (clone SK1, BD Horizon 563919), anti-CD3-BV650 (clone SK7, BD Horizon 563999), anti-CD56-BV711 (clone 5.1H11, Biolegend 362542), anti-CD279-BV785 (clone EH12.2H7, Biolegend 329930), anti-CD127-APC (clone A019D5, Biolegend 351316), anti-CD4-APC-R700 (clone RPA-T4, BD Horizon 564975), LVD efluorescence staining. 780 (eBioscience 65-0865-14), anti-TIGIT-PE (clone MBSA43, eBioscience E13456-108), anti-CD39-PE-Dazzle594 (clone A1, Biolegend 328224), and CD25-PE-cy7 (clone BC96, Biolegend 302612).PBMCs were stained with the following antibody panel: anti-CD45RO-BB515 (clone UCHL1, BD Horizon 564529), anti-CD73-BV421 (clone AD2, BD Horizon 562430), anti-CD8a-BV510 (clone SK1, BD Horizon 563919), anti-CD3-BV650 (clone SK7, BD Horizon 563999), anti-CD56-BV711 (clone 5.1H11, Biolegend 362542), anti-CD197-BV786 (clone 3D12, BD Horizon 563710), anti-CD127-APC (clone A019D5, Biolegend 351316), anti-CD4-APC-R700 (clone RPA-T4, BD Horizon 564975), LVD efluor 780 (eBioscience 65-0865-14), anti-TIGIT-PE (clone MBSA43, eBioscience E13456-108), anti-CD39-PE-Dazzle594 (clone A1, Biolegend 328224) and CD25-PE-cy7 (clone BC96, Biolegend 302612). Acquisition was performed on a FACS Fortessa (BD Biosciences) and analyzed using FlowJo software (FlowJo, LLC). Live cells were gated at forward and side scatter. The following immune cell subsets were gated: CD3. + CD4 + CD127 + CD25 - (CD3 + CD4 + non-Treg cells), CD3 + CD4 + CD127 低 CD25 + (regulatory T cells), CD3 + CD8 + (CD3 + CD8 + T cells), CD3 - CD56 + (NK cells), CD3 + CD56 + (NKT cells), CD3 - CD56 -(Non-T / NK cells). Quantibrite PE beads (BD #340495) were run under the same instrument settings and used to convert the fluorescence data into the number of antibodies bound per cell.

[0509] Percentiles were calculated using Tukey's method and box-and-whisker plots were used. Fig.26 A shows the frequency of TIGIT expression in different immune groups. Fig.26 The TIGIT density of each subpopulation is shown in B.

[0510] The data showed that TIGIT frequencies on T cell subsets were higher on PBMCs from cancer patients compared to PBMCs from healthy donors. This frequency was further increased on DTC TILS ( Fig.26 A) Although checking CD3 + CD4 + Non-Treg cells and CD4 + The same observations were obtained for TIGIT density on the surface of Treg cells, but for CD3 + CD8 + T cells, the number of TIGIT molecules per cell on DTCTILS was reduced ( Fig.26 B).

[0511] Example 23: Structural and functional epitope mapping of TIGIT and clone 31282

[0512] To further characterize and understand the interaction between the anti-TIGIT mAb clone 31282 and TIGIT recombinant protein, the crystal structure of 31282 in complex with TIGIT was determined by X-ray diffraction.

[0513] A. Expression, purification and crystallization of TIGIT and Fab

[0514] Human TIGIT residues 23-128 were produced by Proteros Biostructures GmbH. TIGIT (23-128) with an N-terminal HIS-tag (thrombin cleavable) was cloned into pET15b and expressed in inclusion bodies in LB medium at 37°C in Bl21 (DE3). The inclusion bodies (IB) were washed with a buffer containing Tris / HCl pH 7.4 and Tris / HCl pH 7.4, 0.05% Brij-35. The IB was denatured with 6M Gdm / HCl, 50mM Tris pH 8.5 and 10mM DTT. Refolding was performed in 50mM Tris / HCl pH 8, 1mM GSH, 0.5mM GSSG, 150mM NaCl. The refolded protein was purified on a HIS-trap. The N-terminal HIS-tag was removed by thrombin cleavage and further purified on Superdex-75 equilibrated in 50 mM Tris / HCl pH 7.5, 200 mM NaCl.

[0515] To express Fab fragments, HEK293F cells were grown in Freestyle F17 containing 1% penicillin / streptomycin, 2mM L-glutamine and 0.1% Pluronic. The expansion culture for transfection was cultured in a 3L Erlenmeyer flask (Corning, 2L cell culture working volume, 37°C, 8% v / v CO2, 80-120rpm, 50mm amplitude). The culture was diluted one day before transfection and the cell number was adjusted to 1x10 6 Cells / ml. The volume of the expression culture was 6 L. Transient transfection was performed with plasmids of the light and heavy chains of the Fab. A MasterMix of DNA / FectoPro (FectoPro, PolyPlus) was prepared in pure F17 medium and incubated for 10 minutes (according to the PolyPlus protocol). The transfection mixture was added dropwise to the cell suspension and Booster was immediately added. 18 hours after transfection, 3 g / L glucose was supplied to the culture.

[0516] To purify the Fab fragment, 6 L of HEK293 cell culture supernatant was harvested by centrifugation 6 days after transfection and applied to a 30 ml KappaSelect column. KappaSelect was washed with PBS pH 7.4, eluted with sodium citrate pH 3, and the fraction containing Fab was neutralized with Tris buffer. Fab was further purified on a Superdex S-200 column (the column was balanced in 20 mM Tris pH 8, 100 mM NaCl) and stored at -80°C until further use.

[0517] To form the Fab-TIGIT complex, purified TIGIT was mixed with purified Fab at a ratio of 1.5: 1, and the complex was purified on Superdex-200, which was balanced in 20mM Tris pH 8, 100mM NaCl. The Fab-TIGIT complex was concentrated to 35mg / ml for crystallization. Using the vapor diffusion method, 0.1μl of protein solution (35.3mg / ml, in 20mM TRIS pH8.0; 100mM NaCl) was mixed with a reservoir solution (0.10M sodium cacodylate pH 6.00; 15% (w / v) PEG4000) at a ratio of 1: 1, and the Fab-TIGIT complex was crystallized at 277K. The crystals were cryoprotected by immersing them in a reservoir solution supplemented with 25% glycerol.

[0518] B. Data Collection and Processing

[0519] The cryogenic scheme was established using the Proteros Biostructures GmbH standard scheme. The crystals were rapidly frozen and measured at a temperature of 100K. X-ray diffraction data were collected from Fab:TIGIT composite crystals using cryogenic conditions at SWISS LIGHT SOURCE (SLS, Villigen, Switzerland). The crystals belong to space group P1. Data were processed using programs XDS and XSCALE. Data collection and processing statistics can be found in Table 22.

[0520] Table 22: Data collection and processing statistics

[0521]

[0522] 1 SWISS light source (SLS, Villigen, Switzerland)

[0523] 2 The values ​​in brackets refer to the highest resolution bin.

[0524] 3 Calculated from independent reflections

[0525] C. Structural Modeling and Refinement

[0526] Obtain the necessary phase information of determining and analyzing structure by molecular replacement.Previously resolved Fab structure is used as search model.According to standard scheme, software package CCP4 and COOT are used to perform subsequent model building and refinement.For the calculation of free R factor (a measure of the correctness of cross-validation final model), about 2.5% of measured reflection value is excluded from refinement program (see Table 23).

[0527] TLS refinement has been performed (using REFMAC5, CCP4), which resulted in lower R factors and higher quality of the electron density maps. Automatically generated local NCS restraints have been applied (keyword "ncsr local" in newer REFMAC5 versions). Ligand parameterization and generation of corresponding library files were performed with CHEMSKETCH and LIBCHECK (CCP4), respectively.

[0528] The water model was established using the COOT "Find waters" algorithm by placing water molecules at the peak of the Fo-Fc spectrum with a wavy profile at 3.0, followed by refinement using REFMAC5 and checking all waters using the COOT validation tool. The criteria for the suspected water list were: B factor greater than The 2Fo-Fc map is less than 1.2σ, ​​and the distance to the nearest contact is less than or greater than Manually check suspected water molecules and those water molecules in the ligand binding site (with a distance less than ). The final complex structure was refined with PHENIX. We selected refinement parameters, including XYZ coordinates, real space, individual B factors and group B factors. X-ray / stereochemical weights were optimized and NCS restrictions were also selected for refinement. The Ramachandran Plot of the final model showed 95.39% of all residues in the preferred region and 3.95% in the allowed region. The statistics of the final structure and refinement process are listed in Table 23.

[0529] Table 23: Detailed statistics 1

[0530]

[0531]

[0532] 1 Values ​​measured in PHENIX

[0533] 2 D. Overall structure calculated using COOT

[0534] The heavy and light chains of human Fab antibody fragments show the typical folding of human antibodies ( Fig. 27A). There are two heterotrimers in the asymmetric unit with essentially identical overall conformations. The model contains residues 23 to 128 of TIGIT, residues 1 to 224 of the heavy chain of clone 31282, and residues 1 to 214 of the light chain of clone 31282. A short loop region of the heavy chain is not fully defined by the electron density and has therefore not been included in the model.

[0535] The diffraction images were analyzed using the FoldX program to estimate the energy contributions of the residues and define the hot spots of the interactions. The amino acid residues that form the binding interface were clearly defined in the electron density map. The interpreted X-ray diffraction data clearly showed the interaction between Fab and TIGIT ( Fig. 27 B and 27C). Clone 31282 light chain CDRs interact with two regions of TIGIT, where CDR L1 Arg30 and Tyr33 contact TIGIT residues Asn58 and Glu60; CDR L1 Arg30 and CDR L3 Phe93 contact TIGIT residue Ile109. CDR L2 does not contact TIGIT (Table 24). Clone 31282 heavy chain interacts with different regions of TIGIT, where CDR H1 Tyr33 contacts TIGIT at residue Leu73; CDR H2 Val50, Ser54 and Ser57 contact TIGIT at residue Leu73; CDR H3 Asp102, Tyr103 and Trp104 contact TIGIT at residues Gln56, Ile68, Leu73 and His76.

[0536] Based on this crystal structure of the α-TIGIT clone 31282 / TIGIT complex, the TIGIT residues that contact clone 31282 (TIGIT epitope residues bound by clone 31282) and the residues of clone 31282 that contact TIGIT (TIGIT-bound paratope residues of clone 31282) were determined. Tables 24 and 25 and Fig. 27 C shows TIGIT residues that contact light chain (Table 24) or heavy chain (Table 25) residues of clone 31282. A contacting residue is defined as each amino acid that meets each of the following criteria: (i) its calculated binding free energy contribution is greater than 0.3 kcal / mol, (ii) its experimental average B-factor is lower than the average B-factor of all residues in the X-ray structure, (iii) it forms at least 3 pairs of heavy atom interatomic contacts with antibody atoms at a distance of less than or equal to 4.0 angstroms, (iv) it does not make only solvent exposed hydrogen bonds or ionic interactions, (v) if it is a non-aromatic polar residue (Asn, Gln, Ser, Thr, Asp, Glu, Lys or Arg), it makes at least one hydrogen bond or ionic interaction with the antibody.

[0537] Table 24: Summary of epitope residues of TIGIT and corresponding paratope residues on the light chain of clone 31282

[0538]

[0539] Table 25: Summary of epitope residues of TIGIT and corresponding paratope residues on the heavy chain of clone 31282

[0540]

[0541] Example 24: Competition assay between a-TIGIT clones 31282 and 32959.

[0542] Anti-TIGIT antibody clone 32959 of human IgG1 isotype was produced in HEK cells and purified as described in Example 17 above.

[0543] Jurkat cells overexpressing human TIGIT (Jurkat-hTIGIT) were collected and cultured at 5.10 4 Cells / well were dispensed and incubated with the following concentrations of antagonist α-TIGIT clone 31282: 0 nM (no antibody), 0.08 nM, 0.16 nM, 0.8 nM and 8 nM, representing a concentration range of 0 to 100 times the Kd of the clone. Excess antibody was washed and cells were incubated with decreasing concentrations (8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062, 0.031, 0.016, 0.008 and 0.004 nM) of directly conjugated (AF647) anti-TIGIT clone 32959 at 4°C for 30 minutes. The geometric mean fluorescence intensity was analyzed using LSR BD Fortessa. Cell binding was recorded as the median fluorescence intensity of AF647. In order to calculate the EC of clone 32959 50 Binding, the half-maximal concentration (EC) for hTIGIT-Jurkat binding was calculated using a four-variable curve fitting equation in Prism. 50 ), the obtained values ​​are shown in Table 26 and in Fig.28 The results show strong binding of α-TIGIT clone 32959, independent of the concentration of clone 31282, demonstrating the absence of competition with antagonistic α-TIGIT antibodies.

[0544] Table 26: EC values ​​for binding of a-TIGIT clone 32959 to Jurkat-hTIGIT in the presence of increasing concentrations of antagonist a-TIGIT clone 31282 50 concentration

[0545]

[0546] Example 25: Pharmacokinetic properties of clone 31282 after a single intravenous injection in cynomolgus monkeys

[0547] Cynomolgus monkeys received α-TIGIT clone 31282 IgG1 or IgG4 by intravenous push. The antibody was administered to 2 animals (1 male and 1 female) at 3 different concentrations (0.1 mg / kg, 1 mg / kg, 10 mg / kg). Blood was collected 504 hours after administration on the first day. The blood samples were processed into plasma and analyzed using ELISA method for the concentration of α-TIGIT clone 31282 IgG1 or IgG4. In Phoenix WinNonlin (version 6.3, Pharsight, a Certara Company, Princeton, NJ), an intravascular model was used to calculate the toxicological kinetic parameter values ​​of α-TIGIT clone 31282 IgG1 and IgG4 after intravenous administration using plasma concentration-time data from individual animals.

[0548] Following intravenous bolus administration of 0.1, 1, and 10 mg / kg of a-TIGIT clone 31282 IgG1 and IgG4, plasma concentrations of IgG1 could be quantified in male and female monkeys at 240, 336, and 504 hours post-dose, respectively, and IgG4 could be quantified at 168, 240, and 504 hours post-dose, respectively ( Fig.29 and Table 27). Following intravenous bolus administration of α-TIGIT clone 31282 IgG1 or IgG4, there were no apparent sex-related differences in systemic exposure (Cmax and AUClast) for IgG1 and IgG4, with ratios (male / female) ranging from 0.855 to 1.16.

[0549] Following intravenous bolus administration of α-TIGIT clone 31282 IgG1 to male and female monkeys, plasma IgG1 concentrations declined biphasically at all dose levels, with mean terminal half-lives (t1 / 2) ranging from 84.7 to 174 hours ( Fig.29 ). Systemic clearance (CL) was consistent across the dose range studied, ranging from 0.280 to 0.392 mL / hour / kg. The apparent volume of distribution at steady state (Vss) was consistent between the dose levels tested, with values ​​ranging from 53.7 to 66.5 mL / kg. A 10-fold increase in the dose of a-TIGIT clone 31282 IgG1 over the 0.1 to 1 mg / kg and 1 to 10 mg / kg range resulted in a roughly proportional increase in exposure (9.57 to 14.5-fold increase).

[0550] Following intravenous administration of α-TIGIT clone 31282 IgG4 to male and female monkeys, plasma IgG4 concentrations declined biphasically at all dose levels tested, with t1 / 2 ranging from 148 to 334 hours ( Fig.29 and Table 28). CL was consistent across the dose levels tested, ranging from 0.160 to 0.219 mL / hour / kg. Mean Vss ranged from 41.2 to 70.7 mL / kg. A 10-fold increase in the α-TIGIT clone 31282 IgG4 dose over the 0.1 to 1 mg / kg and 1 to 10 mg / kg range resulted in a roughly proportional increase in IgG4 exposure (9.32 to 12.5-fold increase).

[0551] Table 27: Summary of mean toxicokinetic parameters of a-TIGIT clone 31282 human IgG1 following intravenous bolus injection in cynomolgus monkeys

[0552]

[0553] Table 28: Summary of mean toxicokinetic parameters of a-TIGIT clone 31282 human IgG4 following intravenous bolus injection in cynomolgus monkeys

[0554]

[0555] Example 26: Characterization of TIGIT expression on human tumor cell populations

[0556] Flow cytometric analysis was performed to evaluate TIGIT expression on normal and tumor T or B cells in blood samples from cancer patients with different blood cancer indications.

[0557] Testing of samples from patients with Sjögren's syndrome to compare malignant and normal CD4 +TIGIT expression on T cell populations. To isolate these populations, a Beckman Coulter TCR-Vb lineage kit (#IM3497) was used to pre-determine the malignant clone TCR-Vb rearrangement. Once the malignant clone was identified, the following commercial reagents were used to analyze TIGIT expression on immune cells from patients with S.S. syndrome: anti-CD3Krome Orange (#B00068), anti-CD4-PE (#A07751), anti-CD8-PC7 (#737661), anti-CD56-PC5 (#A07789), anti-CD45-Pacific Blue (#A74763), anti-CD19-AF750 (#A94681) and anti-Vb8-FITC (#IM1233) (all from Beckman-Coulter) and anti-TIGIT-APC (clone MBSA43, ebiosciences #17-9500-42). Flow cytometric analysis of SSRI patient samples was performed on a CytoFlex apparatus (Beckman-Coulter). Data were analyzed using FloJo software (FlowJo, LLC).

[0558] Fig.30 A representative example is shown in A. Fig.30 The gating strategy for this donor with a malignant TCR-Vb8 clone is shown in A, where the malignant cells are CD45 + CD3 + CD4 + Vb8 + , and normal CD4+ T cells are CD45 + CD3 + CD4 + Vb8 - . Compared with normal CD4 + Compared with malignant CD4 T cells, + Strong expression of TIGIT was observed on T cells (MFI 999 and 4987, respectively) ( Fig.30 B).

[0559] Similarly, flow cytometry analysis was performed to evaluate TIGIT expression on normal and malignant B cells in bone marrow samples from CLL patients. Samples were stained with the following antibody panel: LVD efluor 780 (eBioscience 65-0865-14), anti-CD45-BB515 (clone HI30, BD Horizon 564585), anti-CD5-BV510 (clone UCHT2, Biolegend363381), anti-CD19-BV711 (clone SJ25C1, BD Horizon 563036) and anti-TIGIT-PE (clone MBSA43, eBioscience E13456-108). Collection was performed on FACS Fortessa (BD Biosciences) and analyzed with FlowJo software (FlowJo, LLC). Live cells were gated at forward and side scatter. The following various cell subsets were gated: CD45 + CD19 + CD5 - (normal B cells) and CD45 + CD19 + CD5 + (Malignant B-CLL).

[0560] Fig.31 A representative example is shown in FIG, where the gating strategy is as follows: Fig.31 A. A high proportion of malignant B-CLL cells were positive for TIGIT (75%) compared with normal B cells (1%) (MFI of 1440 and 810, respectively) ( Fig.31 B).

[0561] In summary, the data obtained indicate that tumor cells express TIGIT in specific blood cancer indications.

[0562] Example 27: Anti-tumor activity of anti-TIGIT antagonist antibodies as monotherapy in a mouse T-cell lymphoma model.

[0563] For this experiment, EL4 T cell lymphoma cells ( TIB-39 TM) were engineered to stably express mouse TIGIT (EL4-mTIGIT). EL4 cells transduced with a similar vector encoding GFP were used as a control (EL4-GFP). The cell pool was subcloned to obtain clones of EL4-mTIGIT and EL4-GFP. The anti-TIGIT antibody used was a modified form of antibody 29527 (modified so that residue 27 of VH FR3 was mutated from L to V and residue 6 of VH FR4 was mutated from M to T) and was produced on the human IgG1 isotype. Female Balb / c mice aged 8 weeks were subcutaneously inoculated with 1.000.000 EL4-mTIGIT cells or 200.000 EL4-GFP cells. On day 7 after inoculation, when the tumor volume averaged 110 mm 3 Around 24 hours after tumor inoculation, mice were randomly divided into treatment groups with equal tumor volumes (n=15 per group for EL4-mTIGIT and n=10 per group for EL4-GFP). Mice were treated with 200 μg of anti-TIGIT or isotype control antibody (hIgG1, BioXcell) by intraperitoneal injection on days 7, 10, 13, and 16 after tumor inoculation. Tumor growth was monitored and tumor volume was measured three times a week with an electronic caliper from day 7 to day 26. When the tumor volume exceeded 2000 mm 3 Mice were sacrificed at 4 hr. Tumor growth curves were statistically analyzed by linear mixed models. Differences between treatment groups were assessed by testing the interaction of time*treatment group.

[0564] Fig.32 Tumor growth curves of mice inoculated with EL4-mTIGIT (AC) or EL4-GFP (DF) are shown. Median tumor growth curves (A and D) and individual tumor growth curves of mice treated with hIgG1 isotype control (B and E) or antagonist a-TIGIT Ab (C and F) are shown. In mice inoculated with EL4-mTIGIT cells, tumor growth was significantly inhibited when treated with anti-TIGIT Ab compared to the isotype control treated group (p < 0.001). However, in the group treated with isotype control antibody, 3 of 15 mice showed tumor growth control at the end of the model, with a volume of less than 700mm 3 In the group treated with antagonist anti-TIGIT antibodies, this number increased to 8 out of 15 mice. When antagonist α-TIGIT treatment was compared to isotype control antibodies, no antitumor efficacy or complete response could be observed in mice bearing EL4-GFP tumors. In summary, these data demonstrate that antagonist α-TIGIT antibodies (hIgG1) have significant antitumor efficacy in models with tumor cells expressing TIGIT.

[0565] Example 28: Anti-tumor activity of anti-TIGIT antagonist antibodies in combination with immune checkpoint antibodies in the CT26 colon cancer mouse model

[0566] In addition to the combination of anti-TIGIT Ab and anti-PD1 antibody (Examples 12, 13 and 14), the anti-tumor efficacy of anti-TIGIT antibody in combination with agonist antibodies specific for co-stimulatory molecules 4-1BB, OX40 and GITR and antagonist antibodies specific for the checkpoint inhibitory molecule ICOS was also evaluated.

[0567] CT26 tumor cell line was purchased from (CRL-2638 TM ). Eight-week-old female BALB / c mice were inoculated subcutaneously with 500,000 cells on the right flank. On day 9 after inoculation, when the tumor volume averaged 75 mm 3 Around 1:10 p.m., mice were randomly divided into treatment groups with equal tumor volumes (n=10 mice per group). All antibodies were given intraperitoneally every 3 days starting from the day of randomization, for a total of 3 injections. The anti-TIGIT antibody used was a modified form of antibody 29527 generated on the mouse IgG2a isotype (modified so that residue 27 of VH FR3 was mutated from L to V, and residue 6 of VH FR4 was mutated from M to T), which was given at 20 μg / mouse. Anti-4-1BB (clone 3H3, BioXCell, BE0239) was given at 5ug / mouse, a-OX-40 (clone OX-86, BioXCell, BE0031) was given at 20ug / mouse, a-GITR (clone DTA-1, BioXCell, BE0063) was given at 10ug / mouse; and a-ICOS (clone 7E.17G9, BioXCell, BE0059) was given at 200ug / mouse. Tumor growth was monitored, and tumor volume was measured three times a week with an electronic caliper from day 7 to day 35. When the tumor volume exceeded 2000mm 3 Mice were sacrificed at 4 hr. Tumor growth curves were statistically analyzed by linear mixed models based on log-transformed tumor volume. Differences between treatment groups were assessed by testing the interaction of time*treatment group. This resulted in a good model fit for the vast majority of the data, except for very small tumor volumes (less than 10 mm 3). Therefore, these small tumor volumes were considered missing values. In order to test the synergistic effect of combining anti-TIGIT antibodies with corresponding immune checkpoint antibodies (IC-i.e., anti-41BB, anti-OX40, anti-GITR, and anti-ICOS), the treatment groups were recoded with a combination of two variables: anti-TIGIT (yes / no) and IC (yes / no). In addition to the additive effect of each treatment (anti-TIGIT*time and IC*time), the synergistic effect was evaluated by testing the interaction term of anti-TIGIT*IC*time.

[0568] Fig.33 A shows the median tumor growth curve for each group and the individual growth curves of mice treated with anti-TIGIT in monotherapy or in combination with anti-4-1BB. Tumor growth in mice treated with anti-TIGIT+anti-4-1BB was significantly inhibited compared with anti-TIGIT or anti-4-1BB monotherapy (p=0.0005 and p<0.0001, respectively). The combination of anti-TIGIT and anti-4-1BB antibodies resulted in 6 / 10 mice showing complete responses (where tumors <30 mm) compared with 1 / 10 or 0 / 10 complete responses in groups treated with a-TIGIT or a-4-1BB as single agents, respectively. 3 These data demonstrate the significant anti-tumor efficacy of anti-TIGIT therapy in combination with anti-4-1BB for the treatment of pre-established tumors.

[0569] Fig.33 B shows the median tumor growth curve for each group and the individual growth curves of mice treated with anti-TIGIT as a monotherapy or in combination with anti-OX-40. Tumor growth in mice treated with anti-TIGIT + anti-OX-40 was significantly inhibited compared with anti-TIGIT or anti-OX-40 monotherapy (p = 0.0002 and p < 0.0001, respectively). The combination of anti-TIGIT + anti-OX-40 achieved a synergistic anti-tumor efficacy that exceeded the additive effect of the two monotherapy treatments (p = 0.02). The combination of anti-TIGIT and anti-OX-40 antibodies resulted in 7 / 10 mice showing a complete response, compared to 1 / 10 or 0 / 10 complete responses in the groups treated with a-TIGIT or a-OX-40 as a single agent, respectively. These data demonstrate the significant synergistic anti-tumor efficacy of anti-TIGIT therapy in combination with anti-OX-40 for the treatment of pre-established tumors.

[0570] Fig.33C shows the median tumor growth curve for each group and the individual growth curves of mice treated with anti-TIGIT as a monotherapy or in combination with anti-GITR. Tumor growth in mice treated with anti-TIGIT+anti-GITR was significantly inhibited compared to anti-TIGIT or anti-GITR monotherapy (p<0.0001). The combination of anti-TIGIT+anti-GITR achieved a synergistic anti-tumor efficacy that exceeded the additive effect of the two monotherapy treatments (p=0.01). The combination of anti-TIGIT and anti-GITR antibodies resulted in 6 / 10 mice showing a complete response, compared to 1 / 10 or 0 / 10 in the groups treated with anti-TIGIT or anti-GITR as a single agent, respectively. These data demonstrate the significant and synergistic anti-tumor efficacy of anti-TIGIT therapy in combination with anti-GITR for the treatment of pre-established tumors.

[0571] Fig.33 D shows the median tumor growth curve for each group and the individual growth curves for mice treated with anti-TIGIT as a monotherapy or in combination with anti-ICOS. Tumor growth in mice treated with anti-TIGIT + anti-ICOS was significantly inhibited compared to anti-TIGIT or anti-ICOS monotherapy (p = 0.003 and p = 0.0001, respectively). The combination of anti-TIGIT and anti-ICOS antibodies resulted in 1 / 10 mice showing a complete response (with tumors < 30 mm) compared to 1 / 10 or 0 / 10 in the groups treated with anti-TIGIT or anti-ICOS antibodies as single agents, respectively. 3 These data demonstrate a significant synergistic anti-tumor efficacy of anti-TIGIT therapy in combination with anti-ICOS for the treatment of pre-established tumors.

[0572] Example 29: Activity of anti-TIGIT antagonist antibodies on γδ T cells

[0573] γδ (gamma-delta or g / d) T cells are an unconventional T cell population with described anti-tumor activity (Zhao et al. 2018. J Transl Med. 16:122) and anti-viral activity (e.g. CMV infection), and are also involved in autoimmune diseases (Malik S et al., 2016. Front Immunol. 7:14).

[0574] Flow cytometry analysis was performed to evaluate TIGIT expression on γδ T cells from freshly isolated PBMCs from healthy individuals with seronegative or seropositive status for cytomegalovirus (CMV) (CMV status was assessed by EFS Nouvelle Aquitaine, Bordeaux, France). Cells were stained using filtered FACS buffer (PBS + 2mM EDTA + 0.1% BSA) according to the manufacturer's instructions. Acquisition was performed on a FACS Fortessa (BD Biosciences) and analysis was performed using BD FACSDIVA software (BD Biosciences). Cells were gated based on forward and side scatter and viability. The following γδ T cells were selected: CD3 + TCRγδ + Vδ2 - (Vδ2 - γδ T cells), gated using the following antibodies: anti-TCRγδAPC, clone REA591 from Miltenyi #130-109-280; anti-TCR Vδ2-PE-Vio 770, clone REA771 from Miltenyi, #130-111-012; BV421 mouse anti-human CD3, clone UCHT1 from BD Biosciences, #560365; Zombie Aqua Fixable Viability Kit #423101 from Biolegend.

[0575] Similar to conventional αβ T cells, unconventional Vδ2-γδ T cells expressed TIGIT (anti-TIGIT, clone MBSA43 from eBioscience, #12-98500-42) in both CMV-negative and -positive populations ( Fig.34 A). To characterize the functional consequences of blocking the TIGIT receptor on this cell population, magnetically isolated Vδ1+γδT cells (anti-TCR Vd1-FITC, clone REA173 #130-100-532 and anti-FITC microbeads #130-048-701, both from Miltenyi) or total PBMCs from CMV-positive donors were activated with anti-Vδ1 (10 ug / ml) (clone R9.1, #IM1761 from Beckman Coulter) and IL-15 (20 ng / ml, #200-15-50 UG from Peprotech) in the presence or absence of the TIGIT-ligand CD155 (#9174-CD-050 from R&D Systems), and IL-2 (100 U / ml, #200-02-1MG Peprotech) was additionally added to the isolated Vδ1+γδT cells. Fig.34 B shows a dose-dependent reduction in IFNγ secretion (ELISA kit, #3420-1h-20 from Mabtech) mediated by the addition of TIGIT-ligand CD155 (0, 0.1, 1 and 10 ug / ml), with maximum inhibition achieved at 1 ug / ml of CD155. Addition of anti-TIGIT antibody clone 31282 (10 ug / ml) completely restored IFNγ production to the same or higher levels than in the absence of CD155 ligand, while the human IgG1 isotype control had a very limited effect. Fig.34 C shows a similar inhibitory effect mediated by CD155 (10 μg / ml) after full restoration of anti-Vδ1 activation and IFNγ secretion of total PBMCs when α-TIGIT clone 31282 was added to the mixture. These data demonstrate that, similar to αβ T cells, CD155 ligation to TIGIT impairs the activity of γδ T cells and that anti-TIGIT antibodies completely prevent this inhibitory effect.

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to human TIGIT, wherein the antibody or antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the combination is selected from the group consisting of: (i) a HCDR1 comprising SEQ ID NO: 16, a HCDR2 comprising SEQ ID NO: 17, a HCDR3 comprising SEQ ID NO: 18, a LCDR1 comprising SEQ ID NO: 61, a LCDR2 comprising SEQ ID NO: 62, and a LCDR3 comprising SEQ ID NO: 63; (ii) HCDR1 comprising SEQ ID NO:4, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO:6, LCDR1 comprising SEQ ID NO:49, LCDR2 comprising SEQ ID NO:50, and LCDR3 comprising SEQ ID NO:51; (iii) HCDR1 comprising SEQ ID NO:7, HCDR2 comprising SEQ ID NO:8, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:52, LCDR2 comprising SEQ ID NO:53, and LCDR3 comprising SEQ ID NO:54; (iv) HCDR1 comprising SEQ ID NO: 10, HCDR2 comprising SEQ ID NO: 11, HCDR3 comprising SEQ ID NO: 12, LCDR1 comprising SEQ ID NO: 55, LCDR2 comprising SEQ ID NO: 56, and LCDR3 comprising SEQ ID NO: 57; (v) HCDR1 comprising SEQ ID NO: 13, HCDR2 comprising SEQ ID NO: 14, HCDR3 comprising SEQ ID NO: 15, LCDR1 comprising SEQ ID NO: 58, LCDR2 comprising SEQ ID NO: 59, and LCDR3 comprising SEQ ID NO: 60; (vi) HCDR1 comprising SEQ ID NO: 1, HCDR2 comprising SEQ ID NO: 2, HCDR3 comprising SEQ ID NO: 3, LCDR1 comprising SEQ ID NO: 46, LCDR2 comprising SEQ ID NO: 47, and LCDR3 comprising SEQ ID NO: 48; (vii) HCDR1 comprising SEQ ID NO: 19, HCDR2 comprising SEQ ID NO: 20, HCDR3 comprising SEQ ID NO: 21, LCDR1 comprising SEQ ID NO: 64, LCDR2 comprising SEQ ID NO: 65, and LCDR3 comprising SEQ ID NO: 66; (viii) HCDR1 comprising SEQ ID NO:22, HCDR2 comprising SEQ ID NO:23, HCDR3 comprising SEQ ID NO:24, LCDR1 comprising SEQ ID NO:67, LCDR2 comprising SEQ ID NO:68, and LCDR3 comprising SEQ ID NO:69; (ix) a HCDR1 comprising SEQ ID NO:25, a HCDR2 comprising SEQ ID NO:26, a HCDR3 comprising SEQ ID NO:27, a LCDR1 comprising SEQ ID NO:70, a LCDR2 comprising SEQ ID NO:71, and a LCDR3 comprising SEQ ID NO:72; (x) a HCDR1 comprising SEQ ID NO:28, a HCDR2 comprising SEQ ID NO:29, a HCDR3 comprising SEQ ID NO:30, a LCDR1 comprising SEQ ID NO:73, a LCDR2 comprising SEQ ID NO:74, and a LCDR3 comprising SEQ ID NO:75; (xi) a HCDR1 comprising SEQ ID NO:31, a HCDR2 comprising SEQ ID NO:32, a HCDR3 comprising SEQ ID NO:33, a LCDR1 comprising SEQ ID NO:76, a LCDR2 comprising SEQ ID NO:77, and a LCDR3 comprising SEQ ID NO:78; (xii) a HCDR1 comprising SEQ ID NO:34, a HCDR2 comprising SEQ ID NO:35, a HCDR3 comprising SEQ ID NO:36, a LCDR1 comprising SEQ ID NO:79, a LCDR2 comprising SEQ ID NO:80, and a LCDR3 comprising SEQ ID NO:81; (xiii) HCDR1 comprising SEQ ID NO:37, HCDR2 comprising SEQ ID NO:38, HCDR3 comprising SEQ ID NO:39, LCDR1 comprising SEQ ID NO:82, LCDR2 comprising SEQ ID NO:83, and LCDR3 comprising SEQ ID NO:84; (xiv) a HCDR1 comprising SEQ ID NO:40, a HCDR2 comprising SEQ ID NO:41, a HCDR3 comprising SEQ ID NO:42, a LCDR1 comprising SEQ ID NO:85, a LCDR2 comprising SEQ ID NO:86, and a LCDR3 comprising SEQ ID NO:87; (xv) a HCDR1 comprising SEQ ID NO:43, a HCDR2 comprising SEQ ID NO:44, a HCDR3 comprising SEQ ID NO:45, a LCDR1 comprising SEQ ID NO:88, a LCDR2 comprising SEQ ID NO:89, and a LCDR3 comprising SEQ ID NO:90; (xvi) a HCDR1 comprising SEQ ID NO: 271, a HCDR2 comprising SEQ ID NO: 272, a HCDR3 comprising SEQ ID NO: 273, a LCDR1 comprising SEQ ID NO: 283, a LCDR2 comprising SEQ ID NO: 284, and a LCDR3 comprising SEQ ID NO: 285; (xvii) a HCDR1 comprising SEQ ID NO: 274, a HCDR2 comprising SEQ ID NO: 275, a HCDR3 comprising SEQ ID NO: 276, a LCDR1 comprising SEQ ID NO: 286, a LCDR2 comprising SEQ ID NO: 287, and a LCDR3 comprising SEQ ID NO: 288; (xviii) HCDR1 comprising SEQ ID NO:277, HCDR2 comprising SEQ ID NO:278, HCDR3 comprising SEQ ID NO:279, LCDR1 comprising SEQ ID NO:289, LCDR2 comprising SEQ ID NO:290, and LCDR3 comprising SEQ ID NO:

291.

2. The antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable domain having an amino acid sequence selected from the group consisting of SEQ ID No: 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 327, 329 and 331, and amino acid sequences that exhibit at least 90%, 95%, 97%, 98% or 99% sequence identity thereto; and optionally comprises a light chain variable domain having an amino acid sequence selected from the group consisting of: SEQ ID No: 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 328, 330 and 332, and amino acid sequences that exhibit at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

3. The isolated antibody or antigen-binding fragment thereof according to claim 2, comprising a combination of a heavy chain variable domain and a light chain variable domain, wherein the combination is selected from the group consisting of: (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 211, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 212, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 213, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 214, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 215, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 216, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 217, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 218, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (v) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 219, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 220, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (vi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 221, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 222, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (vii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 223, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 224, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (viii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 225, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 226, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (ix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 227, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 228, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (x) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 229, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 230, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (xi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 231, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 232, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (xii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 233, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 234, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (xiii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 235, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 236, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (xiv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 237, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 238, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (xv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 239, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 240, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (xvi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 327, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 328, or an amino acid sequence that is at least 90% identical to said amino acid sequence; (xvii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 329, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 330, or an amino acid sequence that is at least 90% identical to said amino acid sequence; and (xviii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 331, or an amino acid sequence that is at least 90% identical to said amino acid sequence, and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 332, or an amino acid sequence that is at least 90% identical to said amino acid sequence.

4. The antibody or antigen-binding fragment according to claim 1, wherein: HCDR1 comprises SEQ ID NO: 16 (YTFTSYYMH), HCDR2 comprises SEQ ID NO: 17 (VIGPSGASTSYAQKFQG), HCDR3 comprises SEQ ID NO: 18 (ARDHSDYWSGIMEV), LCDR1 comprises SEQ ID NO:61 (RASQSVRSSYLA), LCDR2 comprises SEQ ID NO:62 (GASSRAT), and LCDR3 comprises SEQ ID NO:63 (QQYFSPPWT).

5. The antibody or antigen-binding fragment of claim 4, wherein the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 221, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 222, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

6. An isolated antibody or antigen-binding fragment thereof, which binds to human TIGIT and does not compete with CD155 for binding to TIGIT, wherein the antibody or antigen-binding fragment comprises: a heavy chain variable domain comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein HCDR1 comprises SEQ ID NO: 280, HCDR2 comprises SEQ ID NO: 281, and HCDR3 comprises SEQ ID NO: 282; And the antibody or antigen-binding fragment further comprises: a light chain variable domain, wherein the light chain variable domain comprises light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), wherein LCDR1 comprises SEQ ID NO:292, LCDR2 comprises SEQ ID NO:293, and LCDR3 comprises SEQ ID NO:

294.

7. The antibody or antigen-binding fragment of claim 6, wherein the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 333, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 334, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

8. An isolated antibody or antigen-binding fragment thereof, which binds to human TIGIT and does not compete with CD155 for binding to TIGIT, wherein the antibody or antigen-binding fragment comprises: a heavy chain variable domain comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3), wherein HCDR1 comprises SEQ ID NO: 353, HCDR2 comprises SEQ ID NO: 354, and HCDR3 comprises SEQ ID NO: 355; And the antibody or antigen-binding fragment further comprises: a light chain variable domain, wherein the light chain variable domain comprises light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3), wherein LCDR1 comprises SEQ ID NO:356, LCDR2 comprises SEQ ID NO:357, and LCDR3 comprises SEQ ID NO:

358.

9. The antibody or antigen-binding fragment of claim 8, wherein the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 367, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto, and the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 368, or an amino acid sequence that exhibits at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.

10. An isolated antibody or antigen-binding fragment thereof, which cross-competes with the antibody of any one of claims 1-9 for binding to human TIGIT.

11. An isolated antibody or antigen-binding fragment thereof, which binds to human TIGIT at the same epitope as the antibody according to any one of claims 1-9.

12. An isolated antibody or antigen-binding fragment thereof, which binds to human TIGIT at an epitope comprising TIGIT residues Q56 and I109, optionally comprising residues Q56, N58 and I109, optionally comprising residues Q56, N58, E60, I68L73, H76 and I109.

13. An isolated antibody or antigen-binding fragment thereof, which binds to human TIGIT at an epitope consisting of TIGIT residues Q56, N58, E60, I68, L73, H76 and I109.

14. An isolated antibody or antigen-binding fragment thereof, which binds to human TIGIT at the same epitope as an antibody comprising: HCDR1 comprising SEQ ID NO:16, HCDR2 comprising SEQ ID NO:17, HCDR3 comprising SEQ ID NO:18, LCDR1 comprising SEQ ID NO:61, LCDR2 comprising SEQ ID NO:62, and LCDR3 comprising SEQ ID NO:

63.

15. The antibody or antigen-binding fragment according to any preceding claim, which is a human IgG antibody, preferably a human IgG1 antibody.

16. An antibody or antigen-binding fragment according to any preceding claim, which exhibits one or more effector functions selected from the following: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP) against cells expressing human TIGIT on the cell surface.

17. The antibody or antigen-binding fragment of any preceding claim, which selectively depletes Treg cells expressing TIGIT.

18. The antibody or antigen-binding fragment according to any preceding claim, which exhibits equivalent affinity for Treg cells expressing TIGIT and for CD8+ T cells expressing TIGIT.

19. The antibody or antigen-binding fragment of any preceding claim, which reduces TIGIT expression on CD8 T cells and / or on Treg cells.

20. An isolated polynucleotide or combination of isolated polynucleotides encoding the antibody or antigen-binding fragment according to any preceding claim.

21. An isolated polynucleotide encoding the VH domain and / or VL domain of an anti-TIGIT antibody, wherein the polynucleotide comprises one or more sequences selected from the group consisting of SEQ ID Nos: 241-270, 335-342 and 369-370.

22. An isolated polynucleotide or a combination of isolated polynucleotides, which encodes the VH domain and VL domain of an anti-TIGIT antibody, wherein the isolated polynucleotide or the combination of isolated polynucleotides comprises SEQ ID NO:251 and SEQ ID NO:

252.

23. An expression vector comprising a polynucleotide or combination of polynucleotides according to claim 20, claim 21 or claim 22 operably linked to a regulatory sequence that allows the antibody or antigen-binding fragment to be expressed in a host cell or a cell-free expression system.

24. A host cell or a cell-free expression system, comprising the expression vector according to claim 23.

25. A method for producing a recombinant antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell or cell-free expression system of claim 24 under conditions permissive for expression of the antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

26. The antibody or antigen-binding fragment of any one of claims 1-19 for use in therapy.

27. The antibody or antigen-binding fragment of any one of claims 1-19 for use in a method for treating cancer.

28. The antibody or antigen binding fragment of any one of claims 1-19 for use in a method for treating CMV infection.

29. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 19 and at least one pharmaceutically acceptable carrier or excipient.

30. The pharmaceutical composition of claim 29 for use in therapy.

31. The pharmaceutical composition of claim 29, for use in a method for treating cancer.

32. The pharmaceutical composition of claim 29 for use in a method of treating CMV infection.

33. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1-19 or the pharmaceutical composition of claim 29, thereby treating the cancer.

34. The method of treating cancer according to claim 33, wherein the method further comprises administering one or more additional therapeutic agents.

35. The method for treating cancer according to claim 34, wherein the one or more therapeutic agents are selected from: chemotherapeutic agents, anti-PD1 antibodies, anti-PD-L1 antibodies, anti-41BB antibodies, anti-OX40 antibodies, anti-GITR antibodies and anti-ICOS antibodies.

36. A method for promoting T cell activity, the method comprising contacting a population of T cells with the antibody or antigen-binding fragment of any one of claims 1-19.

37. The method for promoting T cell activity according to claim 36, wherein the method promotes αβ T cell activity.

38. The method for promoting T cell activity according to claim 36, wherein the method promotes γδ T cell activity.

39. The method for promoting T cell activity according to any one of claims 36-38, wherein the method is performed in vitro.

40. The method for promoting T cell activity according to any one of claims 36-38, wherein the method is performed in vivo in a human subject.

41. The method of promoting T cell activity according to claim 40, wherein the human subject has cancer.

42. The method of promoting T cell activity according to claim 40, wherein the human subject has a viral infection, optionally wherein the viral infection is a CMV infection.

43. The method for promoting T cell activity according to any one of claims 36-42, wherein the method further comprises contacting the T cell population with one or more of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody, an anti-GITR antibody, and an anti-ICOS antibody.

44. A method of treating a viral infection in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment of any one of claims 1-19 or the pharmaceutical composition of claim 29, thereby treating the viral infection.

45. The method of treating a viral infection according to claim 44, wherein the method further comprises administering one or more additional therapeutic agents.

46. ​​The method of treating a viral infection according to claim 45, wherein the one or more therapeutic agents are selected from the group consisting of: a chemotherapeutic agent, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody, an anti-GITR antibody, and an anti-ICOS antibody.

47. The method of treating a viral infection according to any one of claims 44-46, wherein the viral infection is a CMV infection.

48. A combination comprising an anti-TIGIT antibody or an antigen-binding fragment thereof and one or more of the following: a chemotherapeutic agent, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-41BB antibody, an anti-OX40 antibody, an anti-GITR antibody, and an anti-ICOS antibody.

49. The combination of claim 48, wherein the anti-TIGIT antibody or antigen-binding fragment is the antibody or antigen-binding fragment of any one of claims 1-19.

50. The combination of claim 48 or 49, wherein the anti-TIGIT antibody or antigen-binding fragment comprises a combination of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: HCDR1 comprises SEQ ID NO: 16 (YTFTSYYMH), HCDR2 comprises SEQ ID NO: 17 (VIGPSGASTSYAQKFQG), HCDR3 comprises SEQ ID NO: 18 (ARDHSDYWSGIMEV), LCDR1 comprises SEQ ID NO:61 (RASQSVRSSYLA), LCDR2 comprises SEQ ID NO:62 (GASSRAT), and LCDR3 comprises SEQ ID NO:63 (QQYFSPPWT).

51. The combination according to any one of claims 48-50, for use in a method of treating cancer or a viral infection, optionally wherein the viral infection is a CMV infection.

52. A combination according to any one of claims 48-50 for use in a method according to any one of claims 35, 36 and 38-44.

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