Methods of cancer treatment using anti-TIGIT antibodies

Abstract

Methods of treating cancer or increasing, enhancing or stimulating an immune response with antibodies and antigen-binding fragments thereof that specifically bind to TIGIT (T cell immune receptor with Ig and ITIM domains, WUCAM, or Vstm3) in combination with an anti-PD1 antibody are provided.

Description

Technical Field

[0001] The present application relates to antibodies that specifically bind to TIGIT (T cell immunoreceptor with Ig and ITIM domains) in combination with anti-PD1 antibodies for the treatment of cancer. Background Art

[0002] TIGIT (T cell immunoglobulin and ITIM domain) is a type I transmembrane protein and a member of the CD28 protein family. It plays an important role in inhibiting T cell and NK cell-mediated functional activities in anti-tumor immunity (Boles KS, et al., 2009 Eur. J Immunol, 39: 695-703; Stanietsky N, et al., 2009 PNAS 106: 17858-63; Yu X, et al. 2009 Nat. Immunol, 10: 48-57).

[0003] The gene and cDNA encoding TIGIT were cloned and characterized in mice and humans. Full-length human TIGIT has a sequence of 244 amino acids in length (SEQ ID NO: 26), of which the first 21 amino acids consist of a signal peptide. The amino acid sequence of mature human TIGIT contains 223 amino acid (aa) residues (NCBI accession number: NM_173799). The extracellular domain (ECD) of mature human TIGIT consists of 120 amino acid residues (corresponding to amino acids 22-141 of SEQ ID NO: 26), which has a V-type Ig-like domain (corresponding to amino acids 39-127 of SEQ ID NO: 26), followed by a 21-aa transmembrane sequence and an 82-aa cytoplasmic domain with an immunoreceptor tyrosine-based inhibitory motif (ITIM) (Yu X, et al. 2009 Nat. Immunol, 10: 48-57; Stengel KF, et al. 2012 PNAS 109: 5399-04). In the ECD, human TIGIT shares only 59% and 87% amino acid sequence identity with mice and cynomolgus monkeys, respectively.

[0004] TIGIT is expressed on T cells (including activated T cells, memory T cells, regulatory T (Treg) cells and follicular T helper (Tfh) cells) and NK cells (Boles KS, et al., 2009 Eur J Immunol, 39:695-703; Joller N, et al., 2014 Immunity 40:569-81; Levin SD, et al., 2011 Eur J Immunol, 41:902-15; Stanietsky N, et al., 2009 PNAS 106:17858-63; Yu X, et al., 2009 Nat. Immunol, 10:48-57).

[0005] So far, two TIGIT ligands have been identified: CD155 (also known as poliovirus receptor or PVR) and CD112 (also known as poliovirus receptor-related 2, PVRL2, nectin-2). These ligands are mainly expressed on APCs (such as dendritic cells and macrophages) and tumor cells (Casado JG, et al., 2009 Cancer Immunol Immunother 58: 1517-26; Levin SD, et al., 2011 Eur. J Immunol, 41: 902-15; Mendelsohn CL et al., 1989 56: 855-65; Stanietsky N, et al., 2009 PNAS 106: 17858-63; Yu X, et al. 2009 Nat. Immunol, 10: 48-57). As an immune "checkpoint" molecule, TIGIT initiates inhibitory signaling in immune cells when it engages with its ligands CD155 and CD112. The binding affinity of TIGIT to CD155 (Kd: about 1nM) is much higher than that to CD112, and whether TIGIT:CD112 interaction has functional relevance in mediating inhibitory signals remains to be determined. Co-stimulatory receptor CD226 (DNAM-1) binds to the same ligand with lower affinity (Kd: about 100nM), but delivers positive signals (Bottino C, et al., 2003J Exp Med 198: 557-67). In addition, CD96 (TACTILE), a "TIGIT-like" receptor, also plays a similar inhibitory role in the same pathway (Chan CJ, et al., 2014Nat.Immunol15: 431-8).

[0006] Upregulation of TIGIT expression in tumor infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) has been reported to be present in many types of cancers, such as lung cancer (Tassi, et al., Cancer Res. 2017 77: 851-861), esophageal cancer (Xie J, et al., Oncotarget 20167: 63669-63678), breast cancer (Gil Del Alcazar CR, et al. 2017 Cancer Discov.), acute myeloid leukemia (AML) (Kong Y et al., Clin Cancer Res. 2016 22: 3057-66) and melanoma (Chauvin JM, et al., J Clin Invest. 2015 125: 2046-2058). Increased expression of TIGIT in AML is associated with poor prognosis of patient survival outcomes (Kong Y et al., Clin Cancer Res. 2016 22: 3057-66). The upregulation of TIGIT signaling plays an important role not only in immune tolerance to cancer, but also in immune tolerance to chronic viral infection. During HIV infection, the expression of TIGIT on T cells is significantly higher and positively correlated with viral load and disease progression (Chew GM, et al., 2016PLoS Pathog.12:e1005349). In addition, blocking of TIGIT receptors alone or in combination with other blocking can rescue functional "exhausted" T cells in vitro and in vivo (Chauvin JM, et al., J Clin Invest.2015 125:2046-2058; Chew GM, et al., 2016PLoS Pathog.12:e1005349; Johnston RJ, et al. Cancer Cell 2014 26:923-937). In the case of cancer and viral infection, the activation of TIGIT signaling promotes immune cell dysfunction, leading to cancer hyperplasia or prolonged viral infection. Inhibition of TIGIT-mediated inhibitory signaling by therapeutic agents can restore the functional activity of immune cells, including T cells, NK cells, and dendritic cells (DCs), and thereby enhance immunity against cancer or chronic viral infections.

[0007] Therefore, anti-TIGIT antibodies with enhanced effector functions may induce effective immune responses in the treatment of cancer or chronic viral infections. Summary of the invention

[0008] The present disclosure relates to methods of cancer treatment administering anti-TIGIT antibodies.

[0009] A method of cancer treatment, comprising administering to a subject an effective amount of a pH-dependent anti-TIGIT antibody or an antigen-binding fragment thereof.

[0010] The method, wherein the anti-TIGIT antibody binds to the TIGIT protein at amino acid histidine 76.

[0011] The method, wherein the anti-TIGIT antibody binds to the TIGIT protein at histidine 76 and leucine 73.

[0012] The method, wherein the method comprises administering to a subject an effective amount of a pH-dependent antibody or an antigen-binding fragment thereof, wherein the pH-dependent antibody or antigen-binding fragment thereof specifically binds to human TIGIT and comprises: a heavy chain variable region comprising HCDR (heavy chain complementarity determining region) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising LCDR (light chain complementarity determining region) 1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6.

[0013] The method, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising SEQ ID NO:7, and a light chain variable region (VL) comprising SEQ ID NO:8.

[0014] The method, wherein the anti-TIGIT antibody has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

[0015] The method, wherein the anti-TIGIT antibody has reduced fucosylation.

[0016] The method, wherein the anti-TIGIT antibody has Fc amino acid changes at S239D and I332E (EU numbering).

[0017] The method, wherein the anti-TIGIT antibody has Fc amino acid changes at S239D, I332E, and A330L (EU numbering).

[0018] The method of any of the above, wherein the anti-TIGIT antibody has increased binding affinity for FcγRIIIA-V158 and FcγRIIIA-F158.

[0019] The method described in any of the above items, wherein the anti-TIGIT antibody has enhanced ADCC in T-regulatory (Treg) cells.

[0020] The method described in any of the above items, wherein the anti-TIGIT antibody activates natural killer (NK) cells.

[0021] The method described in any of the above items, wherein the anti-TIGIT antibody has increased trogocytosis.

[0022] The above method, wherein the method further comprises administering an anti-PD1 antibody, which specifically binds to human PD1 and comprises: a heavy chain variable region comprising HCDR1 of SEQ ID NO: 15, HCDR2 of SEQ ID NO: 16, and HCDR3 of SEQ ID NO: 17; and a light chain variable region comprising LCDR1 of SEQ ID NO: 18, LCDR2 of SEQ ID NO: 19, and LCDR3 of SEQ ID NO: 20.

[0023] The above method, wherein the anti-PD1 antibody or antigen-binding fragment thereof specifically binds to human PD1 and comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 22.

[0024] The method, wherein the anti-PD1 antibody comprises: an IgG4 constant domain comprising SEQ ID NO:23.

[0025] The method, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, stomach cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma or sarcoma.

[0026] The method, wherein the cancer is non-small cell lung cancer.

[0027] The method, wherein the head and neck cancer is nasopharyngeal carcinoma.

[0028] The method, wherein the esophageal cancer is esophageal squamous cell carcinoma (ESCC).

[0029] The method, wherein the cancer is uterine cancer.

[0030] The method, wherein the gastric cancer is gastric or gastroesophageal junction cancer.

[0031] The method, wherein the cervical cancer is recurrent or metastatic cervical cancer.

[0032] The method, wherein the cancer is renal cancer.

[0033] The method further comprises administering chemotherapy.

[0034] The method, wherein the chemotherapy is chemoradiotherapy.

[0035] The method, wherein the anti-PD1 antibody is administered at 200 mg every three weeks. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1A -D shows the crystal structure of A1217 (Ociperlimab) Fab bound to human TIGIT. Figure 1A is the crystal structure of A1217 Fab bound to human TIGIT, where the A1217 heavy chain (HC) and light chain (LC) regions are colored in black and gray, while TIGIT is represented by a white surface. Figure 1B The crystal structure of A1217 Fab bound to human TIGIT is shown superimposed with the known PVR / TIGIT complex structure (PDB 3UDW), showing steric clashes and competitive binding of A1217 and PVR to TIGIT in the grey ribbon. Figure 1C The atomic interactions on the binding surface of the A1217 / TIGIT complex identified certain key residues of A1217 (paratope residues as indicated by lines) and certain key residues of TIGIT (epitope residues as indicated by bars with underlined text). For clarity, the non-CDR regions of A1217 were removed and TIGIT was represented by a white transparent surface. The framed residues were identified as functionally important epitope residues by alanine scanning mutagenesis. Figure 1D Display A1217 in HIS76 TIGIT The surrounding residues of HIS76 TIGIT With ASP103 HCDR3 The interaction between HIS76 and TIGIT is critical for pH-dependent binding to TIGIT. TIGIT and ASP103 HCDR3 Highlight with underlined text.

[0037] Figure 2A -C shows the blocking comparison between A1217 (Ospelizumab) Fab and Tiragolumab Fab. Figure 2A Superposition of Osperizumab Fab / TIGIT and Tislelizumab Fab / TIGIT complexes is shown. Osperizumab Fab and Tislelizumab Fab are colored in black and white, respectively. TIGIT is shown in white surface representation. Figure 2BThe epitopes of Osperizumab Fab or Tislelizumab Fab are shown. The overlapping epitopes between the two antibodies are marked with underlined black text. The unique epitope of Osperizumab Fab is colored black with white text, while the unique epitope of Tislelizumab Fab is colored gray with black text. Figure 2C Observed from the side Figure 2B (Rotated 90°).

[0038] Figure 3A -B depicts the atomic interactions on the binding surface of the osperlimab Fab / TIGIT and tislelizumab Fab / TIGIT complexes. Figure 3A The binding interface between Osperizumab Fab and TIGIT is depicted. The HCDR1, HCDR2, and HCDR3 of the heavy chain are marked with bold italic black text, black text, and underlined black text, respectively. The LCDR1, LCDR2, and LCDR3 of the light chain are marked with bold italic gray text, gray text, and underlined gray text, respectively. Figure 3B Depict the binding interface between Tislelizumab_Fab and TIGIT. The HCDR2 and HCDR3 of the heavy chain are marked with black text and underlined black text, respectively. The LCDR1 and LCDR3 of the light chain are marked with gray text and underlined gray text, respectively. TIGIT is marked with bold white text in both (a) and (b).

[0039] Figure 4 is a table summarizing the production of afucosylated A1217 antibody.

[0040] Figure 5A -B. Figure 5A The binding of A1217AF antibody to FcγRIIIA-F158 was shown, while Figure 5B Binding of the A1217AF antibody to FcγRIIIA-V158 is depicted.

[0041] Fig. 6A -D. Fig. 6A -B is a comparison of A1217WT with A1217AF and mutated effector variant antibodies (including Fc silent antibodies) and how they bind to FcγRIIIA-V158 and FcγRIIIA-F158. Figure 6C -D depicts the binding of A1217 antibody to FcγRIIIA-V158 and FcγRIIIA-F158 expressed on HEK293 cells.

[0042] Figure 7 A1217, A1217DE, and A1217DEL were shown to have comparable binding to TIGIT-overexpressing HEK293 cells.

[0043] Figure 8 It was indicated that A1217AF and A1217 showed comparable binding to C1q.

[0044] Fig.9A -B shows that A1217AF, A1217DEL and A1217DE have enhanced ADCC activity when compared to A1217 wild type.

[0045] Fig. 10A -C shows that A1217AF has enhanced ADCC activity against Tregs.

[0046] Fig.11A -B indicates the activation of NK cells by A1217AF.

[0047] Fig.12 These results suggest that TIGIT is downregulated by A1217AF.

[0048] Fig.13 The cytotoxicity properties of A1217 were shown.

[0049] Fig.14 This is a mouse model of A1217 combined with anti-PD1 antibody.

[0050] Fig.15A -C is a mouse model of A1217 combined with anti-PD1 antibody and Treg depletion. DETAILED DESCRIPTION

[0051] definition

[0052] Conservative amino acid substitutions of amino acids are well known in the art. Generally, conservative amino acid substitutions mean that one amino acid residue is replaced by another amino acid residue with a similar side chain.

[0053] Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0054] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their plural counterparts unless the context clearly dictates otherwise.

[0055] Unless the context clearly dictates otherwise, the term "or" is used to mean, and is used interchangeably with, the term "and / or."

[0056] In this specification and the claims that follow, unless the context requires otherwise, the words "comprise" and variants such as "include" and "containing" should be understood to mean including the stated amino acid sequence, DNA sequence, steps or groups thereof, but not excluding any other amino acid sequence, DNA sequence, steps. When used herein, the term "comprising" can be replaced by the terms "containing", "including" or sometimes "having".

[0057] The term "TIGIT" includes various mammalian isoforms, e.g., human TIGIT, orthologs of human TIGIT, and analogs comprising at least one epitope within TIGIT. The amino acid sequence of TIGIT (e.g., human TIGIT) and the nucleotide sequence encoding it are known in the art (see Genbank AAI01289). Human TIGIT sequence (SEQ ID NO: 26)

[0058]

[0059] As used herein, the terms "administration," "administering," "treating," and "treatment" when applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. Treatment of cells encompasses contact of an agent with a cell, as well as contact of an agent with a fluid, wherein the fluid is in contact with the cell. The terms "administering" or "treatment" also include in vitro and ex vivo treatment of a cell, for example, by an agent, a diagnostic agent, a binding compound, or by another cell. The term "subject" herein refers to any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human.

[0060] The term "antibody" is used in the broadest sense herein and specifically encompasses antibodies (including full-length monoclonal antibodies) and antibody fragments, as long as they recognize an antigen, e.g., TIGIT. Antibodies are typically monospecific, but may also be described as individual specific, heterospecific, or multispecific. Antibody molecules bind to specific antigenic determinants or epitopes on an antigen through specific binding sites.

[0061] The term "monoclonal antibody" or "mAb" or "Mab" herein means a substantially homogeneous antibody population, i.e., the antibody molecules contained in the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in small amounts. In contrast, conventional (polyclonal) antibody preparations typically include a variety of different antibodies having different amino acid sequences in their variable domains, particularly their complementary determining regions (CDRs), which are typically specific for different epitopes. The modifier "monoclonal" indicates the characteristic that an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring the production of antibodies by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, e.g., Kohler G et al., Nature 1975 256:495-497; U.S. Pat. No. 4,376,110; Ausubel FM et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992; Harlow E et al., ANTIBODIES: ALABORATORY MANUAL, Cold spring Harbor Laboratory 1988; and Colligan JE et al., CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The mAbs disclosed herein can be of any immunoglobulin class, including IgG, IgM, IgD, IgE, IgA, and any subclass thereof. The hybridomas producing mAbs can be cultured in vitro or in vivo. High titers of mAbs can be obtained by in vivo production, wherein cells from individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb / c mice, to produce ascites containing high concentrations of the desired mAb. MAbs of the isotype IgM or IgG can be purified from this ascites fluid or from the culture supernatant using column chromatography methods well known to those skilled in the art.

[0062] Typically, the basic antibody structural unit includes a tetramer. Each tetramer includes two pairs of identical polypeptide chains, each pair having a "light chain" (about 25kDa) and a "heavy chain" (about 50-70kDa). The amino terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The carboxyl terminal portion of the heavy chain can define a constant region that is primarily responsible for effector function. Typically, human light chains are classified as κ and λ light chains. In addition, human heavy chains are typically classified as α, δ, ε, γ or μ, and the isotypes of antibodies are defined as IgA, IgD, IgE, IgG and IgM, respectively. In the light chain and the heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, wherein the heavy chain also includes a "D" region of about 10 or more amino acids.

[0063] The variable regions of each light chain / heavy chain (VL / VH) pair form an antibody binding site. Thus, typically an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are typically the same.

[0064] Typically, the variable regions of both the heavy and light chains contain three hypervariable regions, also referred to as "complementarity determining regions (CDRs)", which are located between relatively conserved framework regions (FRs). The CDRs are typically arranged by the framework regions to enable binding to specific epitopes. Typically, from N-terminus to C-terminus, the light chain variable domain and the heavy chain variable domain sequentially contain FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR3), CDR-3 (CDR3), and FR-4 (or FR4). Amino acids are generally assigned to each domain according to the following definitions of protein sequences of immunological interest: Kabat, et al., National Institutes of Health, Bethesda, Md.; 5th Edition; NIH Publication No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252: 6609-6616; Chothia, et al., (1987) J Mol. Biol. 196: 901-917 or Chothia, et al., (1989) Nature 342: 878-883.

[0065] The term "hypervariable region" means the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "CDR" (i.e., VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable domain and VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md (defining the CDR regions of antibodies by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of antibodies by structure). The term "framework" or "FR" residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0066] Unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen that binds to a full-length antibody, such as a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as single-chain Fv (ScFv); nanobodies and multispecific antibodies formed from antibody fragments.

[0067] Antibodies that specifically bind to a specified target protein are also described as specifically binding to a specific target protein. This means that compared to other proteins, the antibody shows preferential binding to the target, but this specificity does not require absolute binding specificity. If the binding of the antibody is a determining factor for the presence of the target protein in the sample, for example, no undesirable results, such as false positives, are produced, then the antibody is considered to be "specific" for its intended target. The antibodies or binding fragments thereof that can be used in the present invention will bind to the target protein with at least twice, preferably at least 10 times, more preferably at least 20 times, and most preferably at least 100 times the affinity for the non-target protein. If the antibody herein binds to a polypeptide comprising a given amino acid sequence (e.g., the amino acid sequence of a mature human TIGIT molecule) but does not bind to a protein lacking the sequence, then it is considered that the antibody specifically binds to a polypeptide comprising the sequence.

[0068] The expressions "pH-dependent binding", "pH-dependent target binding" and "pH-dependent antigen binding" are interchangeable in the present disclosure, indicating that the antibodies of the present application bind to their target / antigen, i.e., human TIGIT, in a pH-dependent manner. Specifically, the antibodies of the present application exhibit higher binding affinity and / or binding signals to their antigens at a mildly acidic pH (e.g., pH 6.0) than at a physiological pH (e.g., pH 7.4), which is typically found in a tumor microenvironment. Methods for determining the binding affinity and / or binding signal strength of the antibodies of the present application are well known in the art, including but not limited to surface plasmon resonance (Biacore) or similar techniques. More specifically, the antibodies of the present application have a K of greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more at pH 7.4 / pH 6.0 as measured by surface plasmon resonance (Biacore) or similar techniques. DRatio. Alternatively or additionally, as measured by surface plasmon resonance (Biacore) or similar techniques, the antibody of the present application has an Rmax (RU) value at pH 6.0 that is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times that of Rmax at pH 7.4. The binding affinity of the antibody can be measured at 25°C or 37°C. It has been found that the tumor microenvironment shows a relatively more acidic pH than physiological conditions or normal tissues (Zhang et al., Focus on molecular Imaging 2010; Tannock and Rotin et al. Cancer Res 1989). Therefore, the antibody of the present application having the above-mentioned pH-dependent binding is advantageous as an anti-TIGIT therapeutic agent for selectively targeting TIGIT-positive lymphocytes in the tumor microenvironment and having lower toxicity associated with peripheral activation of lymphocytes.

[0069] The term "human antibody" as used herein means an antibody comprising only human immunoglobulin protein sequences. If produced in a mouse, mouse cell, or hybridoma derived from a mouse cell, a human antibody may contain murine carbohydrate chains. Similarly, a "mouse antibody" or a "rat antibody" means an antibody comprising only mouse or rat immunoglobulin protein sequences, respectively.

[0070] The term "humanized antibody" means an antibody form that contains sequences from non-human (e.g., mouse) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. In general, a humanized antibody will contain substantially all of at least one, usually two, variable domains, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. The humanized antibody will optionally also contain at least a portion of an immunoglobulin constant region (Fc), usually at least a portion of an immunoglobulin constant region (Fc) of a human immunoglobulin. When it is necessary to distinguish a humanized antibody from a parent rodent antibody, the prefix " hum ”, “ hu , "Hu", or "h" is added to the antibody clone name. A humanized form of a rodent antibody will usually contain the same CDR sequences as the parent rodent antibody, but may include certain amino acid substitutions to increase affinity, increase the stability of the humanized antibody, or for other reasons.

[0071] The antibodies of the present application have potential therapeutic uses in treating cancer. The term "cancer" or "tumor" herein means or describes a physiological condition characterized by unregulated cell growth in mammals. Examples of cancer include, but are not limited to, lung cancer (including small cell lung cancer or non-small cell lung cancer), adrenal cancer, liver cancer, gastric cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, leukemia, bladder cancer, bone cancer, brain cancer, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumors, or metastatic lesions of cancer.

[0072] In addition, the antibodies of the present application have potential therapeutic uses in controlling viral infections and other human diseases that are mechanistically involved in immune tolerance or "exhaustion". In the context of the present application, the term "exhaustion" refers to the process that leads to the exhaustion of the ability of immune cells to respond during cancer or chronic viral infection.

[0073] As used herein, the term "therapeutically effective amount" refers to an amount of an antibody that is sufficient to affect such treatment of a disease, disorder or symptom when administered to a subject to treat a disease or condition or at least one clinical symptom of a disease or condition. A "therapeutically effective amount" may vary with the antibody, the disease, disorder, and / or symptoms of the disease or condition, the severity of the disease, disorder and / or symptoms of the disease or condition, the age of the subject to be treated, and / or the weight of the subject to be treated. In any given case, the appropriate amount may be apparent to one skilled in the art or may be determined by routine experimentation. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of active agents contained in the combination for the effective treatment of a disease, disorder or condition.

[0074] As used herein, a "subject" is a mammal, such as a rodent or a primate, preferably a higher primate, such as a human (eg, a patient having or at risk of having a disorder described herein).

[0075] Anti-TIGIT antibodies

[0076] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to human TIGIT. In addition, the present disclosure provides antibodies that have desirable pharmacokinetic characteristics and other desirable properties, and therefore can be used to reduce the likelihood of cancer or treat cancer. The present disclosure also provides pharmaceutical compositions comprising antibodies and methods for preparing and using such pharmaceutical compositions for the prevention and treatment of cancer and related conditions.

[0077] The present disclosure provides antibodies or antigen-binding fragments thereof that specifically bind to TIGIT. The antibodies or antigen-binding fragments of the present disclosure include, but are not limited to, antibodies or antigen-binding fragments thereof produced as described in Table 1 below.

[0078] Table 1. A1217 (osperlimab) sequence

[0079]

[0080]

[0081] The present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody or antibody fragment (e.g., antigen-binding fragment) comprises a VH domain having an amino acid sequence of SEQ ID NO: 7. The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody or antigen-binding fragment comprises a VH CDR having an amino acid sequence of any one of the VH CDRs provided herein. In one aspect, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody comprises (or alternatively, consists of) one, two, three or more VH CDRs having an amino acid sequence of any one of the VH CDRs provided in the present disclosure.

[0082] The present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody or antigen-binding fragment comprises a VL domain having an amino acid sequence of SEQ ID NO: 8. The present disclosure also provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody or antigen-binding fragment comprises a VL CDR having an amino acid sequence of any one of the VL CDRs listed herein. Specifically, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody or antigen-binding fragment comprises (or alternatively, consists of) one, two, three or more VL CDRs having an amino acid sequence of any one of the VL CDRs of the present disclosure.

[0083] Other antibodies or antigen-binding fragments thereof disclosed herein comprise mutated amino acids, but have at least 60%, 70%, 80%, 90%, 95% or 99% identity in the CDR regions to the CDR regions depicted in the sequences described herein. In some aspects, it includes mutant amino acid sequences, wherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regions when compared to the CDR regions disclosed in the provided sequences.

[0084] Other antibodies of the present disclosure include antibodies in which the amino acids or nucleic acids encoding the amino acids have been mutated; but have at least 60%, 70%, 80%, 90%, 95% or 99% identity percentage to the sequences described in Table 1. In some aspects, it comprises a mutant amino acid sequence in which no more than 1, 2, 3, 4 or 5 amino acids have been mutated in the variable region when compared to the variable region depicted in the sequences described herein, while retaining substantially the same therapeutic activity.

[0085] Further changes to the Fc region framework

[0086] In other aspects, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids can be replaced with different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand to which its affinity is changed can be, for example, an Fc receptor or the C1 component of complement. This method is described in, for example, U.S. Patent Nos. 5,624,821 and 5,648,260 by Winter et al.

[0087] In another aspect, one or more amino acid residues can be replaced with one or more different amino acid residues such that the antibody has altered CIq binding and / or reduced or abolished complement dependent cytotoxicity (CDC).This approach is described, for example, in U.S. Patent No. 6,194,551 to Idusogie et al.

[0088] On the other hand, one or more amino acid residues are changed to change the ability of the antibody to fix complement. The method is described in, for example, PCT publication WO 94 / 29351 by Bodmer et al. In a specific aspect, for IgG1 subclass and κ isotype, one or more amino acids of the antibody or its antigen-binding fragment of the present disclosure are replaced by one or more allotypic amino acid residues. Allotypic amino acid residues also include, but are not limited to, the constant region of the heavy chain of IgG1, IgG2 and IgG3 subclasses and the constant region of the light chain of the κ isotype as described in Jefferis et al., MAbs.1:332-338 (2009).

[0089] In another aspect, the Fc region is modified by modifying one or more amino acids to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors. This method is described, for example, in PCT Publication WO00 / 42072 by Presta. In addition, the binding sites for FcγRI, FcγRII, FcγRIII and FcRn on human IgG1 have been mapped, and variants with improved binding have been described (see Shields et al., J. Biol. Chem. 276: 6591-6604, 2001).

[0090] On the other hand, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (i.e., the antibody lacks or has reduced glycosylation) can be prepared. Glycosylation can be changed, for example, to increase the affinity of the antibody to the "antigen". Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made, resulting in the elimination of one or more variable region framework glycosylation sites, thereby eliminating the glycosylation of the site. This aglycosylation can increase the affinity of the antibody to the antigen. This method is described in, for example, U.S. Patent Nos. 5,714,350 and 6,350,861 of Co et al.

[0091] Additionally or alternatively, antibodies with altered glycosylation types can be prepared, such as low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies are expressed to produce antibodies with altered glycosylation. For example, EP 1,176,195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene that encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibit low fucosylation. PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes a cell line engineered to express a glycoprotein-modifying glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell line exhibit increased bisecting GlcNac structures, which results in increased ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0092] On the other hand, if it is desired to reduce ADCC, many previous reports show that human antibody subclass IgG4 has only moderate ADCC and almost no CDC effector function (Moore GL et al. 2010 MAbs, 2: 181-189). On the other hand, it has been found that native IgG4 is less stable under stress conditions, such as in acidic buffer or at elevated temperatures (Angal 1993 Mol Immunol, 30: 105-108; Dall'Acqua, et al., 1998 Biochemistry, 37: 9266-9273; Aalberse et al., 2002 Immunol, 105: 9-19). Reduced ADCC can be achieved by operably linking the antibody to IgG4, which is engineered with a combination of changes to have reduced or ineffective FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Considering the physicochemical properties of antibodies as biopharmaceuticals, one of the less desirable inherent properties of IgG4 is that its two heavy chains dynamically separate in solution to form half antibodies, which leads to the generation of bispecific antibodies in vivo via a process called "Fab arm exchange" (Van der Neut Kolfschoten et al., 2007 Science, 317: 1554-157). The mutation of serine at position 228 to proline (EU numbering system) appears to be inhibitory to the separation of IgG4 heavy chains (Angal 1993 Mol Immunol, 30: 105-108; Aalberse et al., 2002 Immunol, 105: 9-19). It is reported that some amino acid residues in the hinge region and the γFc region have an effect on the interaction of antibodies with Fcγ receptors (Chappel et al., 1991 Proc. Natl. Acad. Sci. USA, 88: 9036-9040; Mukherjee et al., 1995 FASEB J, 9: 115-119; Armour et al., 1999 Eur J Immunol, 29: 2613-2624; Clynes et al., 2000 Nature Medicine, 6: 443-446; Arnold 2007 Annu Rev immunol, 25: 21-50). In addition, some IgG4 isotypes that rarely appear in the human population can also cause different physicochemical properties (Brusco et al., 1998 Eur J Immunogenet, 25: 349-55; Aalberse et al., 2002 Immunol, 105: 9-19). To generate TIGIT antibodies with low ADCC, CDC, and instability, the hinge and Fc regions of human IgG4 can be modified and many changes can be introduced.These modified IgG4 Fc molecules can be found disclosed in SEQ ID NOs: 83-88 of US Pat. No. 8,735,553.

[0093] Antibody production

[0094] Anti-TIGIT antibodies and antigen-binding fragments thereof can be produced by any method known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, while full-length monoclonal antibodies can be obtained, for example, by hybridoma or recombinant production. Recombinant expression can be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0095] The present disclosure also provides polynucleotides encoding antibodies described herein, such as polynucleotides encoding heavy or light chain variable regions or fragments comprising complementary determining regions as described herein. In some aspects, the polynucleotides encoding the heavy chain variable region have at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with the polynucleotides encoding the polypeptide of SEQ ID NO: 7. In some aspects, the polynucleotides encoding the light chain variable region have at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with the polynucleotides encoding the polypeptide of SEQ ID NO: 8.

[0096] The polynucleotides disclosed herein can encode variable region sequences of anti-TIGIT antibodies. They can also encode both the variable region and the constant region of an antibody. Some polynucleotide sequences encode polypeptides comprising the variable regions of both the heavy chain and the light chain of one of the exemplified anti-TIGIT antibodies. Some other polynucleotides encode two polypeptide fragments that are substantially identical to the variable regions of the heavy chain and the light chain of one of the murine antibodies, respectively.

[0097] The present disclosure also provides expression vectors and host cells for producing anti-TIGIT antibodies. The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, the expression vector contains a promoter and other regulatory sequences (e.g., enhancers), which are operably linked to a polynucleotide encoding an anti-TIGIT antibody chain or an antigen-binding fragment thereof. In some aspects, except for the case under induction conditions, an inducible promoter is used to prevent the expression of the inserted sequence. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. The culture of the transformed organism can be amplified under non-inducing conditions without deviating from the population of coding sequences whose expression products are better tolerated by host cells. In addition to the promoter, other regulatory elements may also be required or desired for the effective expression of anti-TIGIT antibodies or their antigen-binding fragments. These elements typically include an ATG start codon and an adjacent ribosome binding site or other sequence. In addition, expression efficiency can be enhanced by including an enhancer appropriate for the cell system being used (see, e.g., Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

[0098] The host cell for carrying and expressing the anti-TIGIT antibody chain can be prokaryotic or eukaryotic. Escherichia coli (E. coli) is a prokaryotic host that can be used to clone and express the polynucleotides of the present disclosure. Other suitable microbial hosts include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia and various species of Pseudomonas. In these prokaryotic hosts, expression vectors can also be prepared, which usually contain expression control sequences (e.g., replication origins) compatible with the host cells. In addition, there will be any number of well-known promoters, such as lactose promoter systems, tryptophan (trp) promoter systems, β-lactamase promoter systems, or promoter systems from bacteriophage λ. The promoter is usually optionally expressed with an operator sequence, and has a ribosome binding site sequence, etc., for initiating and completing transcription and translation. Other microorganisms, such as yeast, can also be used to express anti-TIGIT polypeptides. A combination of insect cells and baculovirus vectors can also be used.

[0099] In other aspects, mammalian host cells are used to express and produce the anti-TIGIT antibodies of the present disclosure. For example, they can be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These cells include any normal dead or normal or abnormal immortal animal or human cells. For example, many suitable host cell lines capable of secreting complete immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is generally discussed in, for example, Winnacker, From Genes to Clones, VCH Publishers, NY, NY, 1987. Expression vectors for mammalian host cells may include expression control sequences, such as replication origins, promoters and enhancers (see, for example, Queen et al., Immunol. Rev. 89: 49-68, 1986), and necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific and / or adjustable or regulatable. Useful promoters include but are not limited to metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRPpolIII promoters, constitutive MPSV promoters, tetracycline-inducible CMV promoters (such as people's immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

[0100] Testing and diagnostic methods

[0101] The antibodies or antigen-binding fragments disclosed herein can be used in a variety of applications, including but not limited to methods for detecting TIGIT. In one aspect, the antibodies or antigen-binding fragments can be used to detect the presence of TIGIT in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In some aspects, the biological sample includes cells or tissues. In other aspects, such tissues include normal and / or cancerous tissues that express TIGIT at higher levels relative to other tissues.

[0102] In one aspect, the present disclosure provides a method for detecting the presence of TIGIT in a biological sample. In certain aspects, the method includes contacting the biological sample with an anti-TIGIT antibody under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample may include, but is not limited to, a urine or blood sample.

[0103] Also included is a method for diagnosing a disease associated with TIGIT expression. In some aspects, the method includes contacting a test cell with an anti-TIGIT antibody; determining the expression level of TIGIT in the test cell (quantitatively or qualitatively) by detecting the binding of the anti-TIGIT antibody to the TIGIT polypeptide; and comparing the expression level in the test cell with the TIGIT expression level in a control cell (e.g., a normal cell or non-TIGIT expressing cell having the same tissue origin as the test cell), wherein a higher TIGIT expression level in the test cell compared to the control cell indicates the presence of a disease associated with TIGIT expression.

[0104] Treatment

[0105] The antibodies or antigen-binding fragments of the present disclosure can be used in a variety of applications, including but not limited to methods for treating TIGIT-related disorders or diseases. In one aspect, the TIGIT-related disorder or disease is cancer.

[0106] In one aspect, the present disclosure provides a method for treating cancer. In certain aspects, the method comprises administering an effective amount of an anti-TIGIT antibody or antigen-binding fragment to a patient in need thereof. Cancer may include, but is not limited to, breast cancer, head and neck cancer, gastric cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

[0107] Antibodies or antigen-binding fragments of the present invention can be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if necessary for local treatment, can be administered intralesionally. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-lived or long-term. Various dosage regimens including but not limited to single or multiple administrations, push injection administration and pulse infusions at various time points are contemplated herein.

[0108] The antibodies or antigen-binding fragments of the present invention will be formulated, dosed and applied in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the agent, the method of administration, the administration schedule, and other factors known to practitioners. The antibody need not but is optionally formulated with one or more agents currently used to prevent or treat the condition in question. The effective amount of such other agents depends on the amount of the antibody present in the formulation, the type of condition or treatment, and other factors discussed above. These are usually used with the same dosage and with the administration route as described herein, or about 1% to 99% of the dosage described herein, or with any dosage and by any route determined to be suitable through experience / clinical practice.

[0109] For the prevention or treatment of a disease, the appropriate dosage of the antibody or antigen-binding fragment of the present invention will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is used for prevention or treatment purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitable for administration to the patient once or over a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 100 mg / kg can be an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dose range can be about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over a few days or longer (depending on the conditions), treatment will generally be maintained until the desired suppression of disease symptoms occurs. Such doses can be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives about two to about twenty, or, for example, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimens are also useful. The progress of this treatment is easily monitored by conventional techniques and assays.

[0110] Combination therapy

[0111] In one aspect, the TIGIT antibodies of the present disclosure may be used in combination with other therapeutic agents, such as anti-PD1 antibodies. Other therapeutic agents that may be used with the TIGIT antibodies of the present disclosure include, but are not limited to, chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents; (e.g., ), docetaxel; carboplatin; topotecan; cisplatin; irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD-20 targeted agents (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeted agents (e.g., alemtuzumab), prednisolone, alfa-dabepol statins, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD-19 targeted agents (e.g., MEDI-551, MOR208), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), HGF / SF inhibitors (e.g., AMG 102), EGEN-001, Polo-like kinase 1 inhibitors (e.g., BI 672).

[0112] The TIGIT antibodies disclosed herein can be used in combination with other therapeutic agents, such as anti-PD1 antibodies. Anti-PD1 antibodies may include, but are not limited to, tislelizumab, pembrolizumab, or nivolumab. Tislelizumab is disclosed in US 8,735,553 and in Table 2 below.

[0113] Table 2. - Tislelizumab sequence

[0114]

[0115]

[0116] Pembrolizumab (formerly known as MK-3475), as disclosed by Merck in US 8,354,509 and US 8,900,587, is a humanized IgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of the PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is being studied in clinical trials for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin's lymphoma (cHL). Nivolumab (as disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Pat. No. US 8,008,449 and WO 2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, renal cancer, and Hodgkin's lymphoma.

[0117] Pharmaceutical compositions and preparations

[0118] Also provided are compositions comprising anti-TIGIT antibodies or antigen-binding fragments, or polynucleotides, including pharmaceutical preparations, wherein the polynucleotides comprise sequences encoding anti-TIGIT antibodies or antigen-binding fragments. In certain embodiments, the composition comprises one or more antibodies or antigen-binding fragments that bind to TIGIT, or one or more polynucleotides comprising sequences encoding one or more antibodies or antigen-binding fragments that bind to TIGIT. These compositions may also comprise suitable carriers, such as pharmaceutically acceptable excipients, including buffers well known in the art.

[0119] Pharmaceutical formulations of TIGIT antibodies or antigen-binding fragments as described herein are prepared by mixing such antibodies or antigen-binding fragments having the desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0120] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0121] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg films, or microcapsules.

[0122] Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0123] Pharmaceutical compositions and kits

[0124] In some aspects, the present disclosure provides a composition, such as a pharmaceutically acceptable composition, comprising an anti-TIGIT antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible, etc. The excipient may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

[0125] The compositions herein can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. Suitable forms depend on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusible solutions. A suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0126] Example

[0127] Example 1: Generation of anti-TIGIT monoclonal antibodies

[0128] Anti-TIGIT monoclonal antibodies (mAbs) were generated based on conventional hybridoma fusion technology (de St Groth and Sheidegger, 1980 J Immunol Methods 35:1; Mechetner, 2007 Methods Mol Biol 378:1) with minor modifications. mAbs with high binding activity in enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) assays were selected for further characterization.

[0129] Cloning and sequence analysis of TIGIT antibodies

[0130] According to the manufacturer's protocol, Ultrapure RNA kit (catalog number: 74104, QIAGEN, Germany) was used to harvest mouse hybridoma clones to prepare total cell RNA. The first chain cDNA was synthesized using a cDNA synthesis kit (catalog number: 18080-051) from Invitrogen, and PCR kit (catalog number: CW0686, CWBio, Beijing, China) was used to encode the nucleotide sequence of the heavy chain variable region (Vh) and kappa chain variable region (Vk) of mouse mAb. Oligo primers for antibody cDNA cloning of Vh and Vk were synthesized by Invitrogen (Beijing, China) based on previously reported sequences (Brocks et al., 2001Mol Med 7: 461). The PCR product was then subcloned into a pEASY-Blunt cloning vector (catalog number: CB101-02, TransGen, China) and sequenced by Genewiz (Beijing, China). The amino acid sequences of the Vh and Vk regions were derived from the DNA sequencing results. Mu1217 was identified as the specific clone of interest.

[0131] Humanization of mouse anti-human TIGIT mAb mu1217

[0132] For the humanization of mu1217, we searched for sequences in human germline IgG genes that were highly homologous to the cDNA sequences of mu1217 variable regions by comparing them with the human immunoglobulin gene database in IMGT. We selected human IGVH and IGVH sequences that were highly homologous to mu1217 and were present at high frequencies in the human antibody library (Glanville et al., PNAS 106:20216-20221 2009). Genes serve as templates for humanization.

[0133] Humanization was performed by CDR-grafting (Methods in Molecular Biology, Vol. 248: Antibody Engineering, Methods and Protocols, Humana Press), and the humanized antibody (A1217) was engineered into a human IgG1MF format using an expression vector developed in-house, and the sequence of the antibody is shown in Table 1.

[0134] Example 2: Binding activity, structure and function of A1217 on TIGIT

[0135] To better understand how the A1217 antibody is able to have high affinity for TIGIT, robust blockade of the TIGIT-PVR interaction, and particularly pH-dependent binding to TIGIT, while exhibiting pharmacokinetic and molecular evaluation properties, the crystal structure of A1217 in complex with TIGIT was determined as detailed below. Mutagenesis experiments of the TIGIT interface were also performed to identify functional epitope residues, particularly HIS76 of TIGIT for pH-dependent binding.

[0136] TIGIT and Fab expression, purification and crystallization

[0137] Human TIGIT residues 23-128 with a C-terminal HIS tag were expressed as inclusion bodies in Escherichia coli BL21 (DE3) pLysS using the pET21a vector (Novagen). Site-directed mutations of TIGIT were introduced using a QuickChange-based program (Xia et al., Nucleic Acids Res, 2015.43(2): Page e12) using Q5 DNA polymerase (New England Biolabs). Protein expression in the BL21 (DE3) pLysS host strain was induced at OD600 of 0.6-1.0 with 1mM IPTG for 4h at 37°C. Cells were collected by centrifugation and resuspended in lysis buffer (50mM sodium phosphate pH 7.0, 300mM sodium chloride). The cells were lysed under ultrasonic treatment on ice. The inclusion bodies were recovered by centrifugation (20,000 rpm for 30 min at 4°C) and dissolved in 8 M urea, 20 mM Tris pH 8.0, 200 mM NaCl, 1 mM DTT, followed by stirring overnight. After removing the undissolved precipitate by centrifugation (20,000 rpm for 30 min at 4°C), the dissolved fraction was applied to Ni-Penta TM Affinity column (Marvelgent Biosciences Inc.) and washed with 10 column volumes of wash buffer (8M urea, 20mM Tris pH 8.0, 200mM NaCl, 5mM imidazole). The protein was then eluted with elution buffer (8M urea, 20mM Tris pH 8.0, 200mM NaCl, 200mM imidazole). The eluted protein was refolded by dialysis against a buffer containing 20mM Tris pH 8.0, 200mM NaCl, 0.4M L-arginine, 1mM oxidized glutathione, 5mM reduced glutathione and eluted using HiLoad 16 / 600 Superdex TMFurther purification was performed by gel filtration on a 75 pg column (GE Healthcare Life Sciences) in buffer (20 mM Tris pH 8.0, 100 mM NaCl). TIGIT mutants were purified similarly to the wild-type protein.

[0138] The DNA sequence of the Fab fragment of A1217 was synthesized in mammalian cells by codon optimization. The heavy and light chain sequences of the Fab were cloned into pMAX vectors with a C-terminal 6xHIS tag of the heavy chain. The plasmids carrying the heavy and light chains of the Fab were transiently co-transfected into HEK293G cells for protein expression. The supernatant containing the secreted Fab was purified by TALON affinity resin (Clontech Laboratories) and then using HiLoad 16 / 600 Superdex TM 75pg column (GE Healthcare Life Sciences) for further purification. Similarly, the plasmids of the full-length heavy and light chains of A1217 were transiently co-transfected into HEK293G cells for protein expression. TM The full-length antibody was purified using affinity resin (GE Healthcare) and then purified using HiLoad 16 / 600 Superdex TM The column was further polished using a 200 pg column (GE Healthcare Life Sciences).

[0139] A1217 Fab was concentrated to approximately 10 mg / ml in 20 mM Tris pH 8.0, 100 mM NaCl for initial crystallization screening. A1217 Fab was incubated with 1.5 molar excess of TIGIT on ice for 30 min and purified by Superdex. TM 75Increase 10 / 300GL column (GE Healthcare) was purified in 20mM Tris pH 8.0, 100mM NaCl. The combined fractions were concentrated to about 10mg / ml and used for initial crystal screening. Crystals of A1217Fab / TIGIT were grown in 0.1M citric acid pH 4.6, 1M lithium chloride, 7% PEG6000. Crystals cryoprotected to a final concentration of 20% with stepwise 5% glycerol were quickly frozen in liquid nitrogen. X-ray diffraction data were collected at beamline BL45XU at Spring-8 (Japan SynchrotronRadiation Research Institute). For comparison, tislelizumab Fab was produced by a similar method.

[0140] Data collection and structure elucidation

[0141] Diffraction data of A1217 Fab / TIGIT and tislelizumab Fab / TIGIT complexes were collected in BL45XU of Spring-8 (Japan) using the automatic data collection system ZOO (Hirata et al., Acta Crystallogr D StructBiol, 2019.75 (Pt 2): pp. 138-150), and processed by KAMO plus (Yamashita et al., Acta Crystallographica Section D, Structural biology, 2018.74 (Pt 5): pp. 441-449). Molecular replacement using PHASER (McCoy et al., J Appl Crystallogr, 2007.40 (Pt 4): pp. 658-674) used rigid body search models of Fab and TIGIT (PDB: 3UCR) resolved internally. The structure was refined using the programs REFMAC (Murshudov et al., Acta Crystallogr D Biol Crystallogr, 1997.53 (Pt 3): pp. 240-55) and PHENIX (Adams et al., Acta Crystallogr D Biol Crystallogr, 2010.66 (Pt 2): pp. 213-21) and manual model building in COOT (Emsley et al., Acta Crystallogr D Biol Crystallogr, 2004.60 (Pt 12Pt 1): pp. 2126-32). Crystallographic data statistics are summarized in Table 3. All molecular graphics were made with PyMOL (Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.8.2015).

[0142] Table 3. Data collection and detailed statistics

[0143]

[0144]

[0145] a The values ​​in parentheses are those for the highest resolution shell.

[0146] b Calculated from approximately 5% of the reflections left out during refinement

[0147] c rmsd, root mean square deviation

[0148] Example 3: Structure of A1217 bound to human TIGIT

[0149] A1217 Fab in complex with TIGIT crystallized in the P 2 21 21 space group with four complexes in the asymmetric unit and diffracted to Superposition of the four individual complexes in the asymmetric unit showed only minor variations in backbone positioning between the individual copies. Structure of A1217 bound to human TIGIT ( Figure 1A ) shows the spatial interface of A1217 and PVR ( Figure 1B The buried surface area between A1217 and TIGIT is approximately The TIGIT epitope of A1217 is composed of many discontinuous regions. Eighteen residues of A1217 Fab (paratope) and fifteen residues of TIGIT (epitope) participate in the paratope-epitope formation ( Figure 1C ). The interactions at the A1217 / TIGIT interface are primarily nonpolar in nature, with a total of eleven hydrogen bonds and three salt bridges. The paratope of A1217 consists of TYR33 of HCDR1; THR52, LYS53, GLY54, GLY56, SER57, TYR59 of HCDR2; ASN101, TYR102, ASP103, PHE104 of HCDR3; THR31, SER32 of LCDR1; TYR49, TRP50 of LCDR2; TYR91, SER92, TYR94 of LCDR3 ( Figure 1C The A1217 epitope of TIGIT contains GLN56, GLU60, ASP63, GLN64, LEU65, ILE68, ASN70, LEU73, GLY74, TRP75, HIS76, SER78, PRO79, SER80, LYS82 ( Figure 1C ). The hydrogen bond between TIGIT and A1217 involves GLN56 TIGIT 、ASP63 TIGIT 、ASN70 TIGIT 、HIS76 TIGIT SER80 TIGIT LYS82 TIGIT TYR33 HCDR1 、THR52 HCDR2 、ASP103 HCR3 、THR31 LCDR1 、TRP50 LCDR2 SER92 LCDR3 TYR94 LCDR3Side chain atoms and LEU65 TIGIT 、LEU73 TIGIT LYS53 HCDR2 、GLY54 HCDR2 、GLY56 HCDR2 、ASN101 HCDR3 TYR102 HCDR3 The main chain atoms in HIS76 TIGIT With ASP103 HCDR3 Two salt bridges are formed between GLU60 TIGIT With LYS53 HCDR2 A salt bridge was found between them. In addition, the residues of TIGIT that only participate in van der Waals interactions are GLN64, ILE68, GLY74, TRP75, SER78, and PRO79, while the residues of A1217 that participate in these interactions are SER57, TYR59, and PHE104 of the heavy chain and SER32, TYR49, and TYR91 of the light chain.

[0150] The crystal structure of tislelizumab was also generated by a similar method as described above. Epitope mapping of tislelizumab Fab to TIGIT showed many disconnected regions. Thirteen residues (paratopes) of tislelizumab Fab and ten residues (epitopes) of TIGIT participate in paratope-epitope formation, using The cutoff distance was calculated. The paratope of tislelizumab Fab consists of ARG56, PHE57, LYS58 and TYR60 of HCDR2; TYR106, ASP107, LEU108 and LEU109 of HCDR3; TYR31 and TYR38 of LCDR1; and TYR98, SER99 and THR100 of LCDR3. Therefore, HCDR1 and LCDR2 of tislelizumab Fab are not directly involved in epitope-paratope interactions. The epitope of tislelizumab consists of GLN56, ASN58, GLU60, HIS76, ILE77, SER78, PRO79, SER80, LYS82 and HIS111. Eight hydrogen bonds, four salt bridges and van der Waals forces contribute to the formation of the binding interface. The hydrogen bond between TIGIT and tislelizumab Fab involves ASN58 TIGIT 、GLU60 TIGIT 、HIS76 TIGIT SER80 TIGIT LYS82 TIGIT LYS58 HCDR2 TYR60 HCDR2 、ASP107 HCDR3 and THR100 LCDR3 Side chain atoms and PRO79 TIGIT 、ARG56HCDR2 and THR100 LCDR3 The main chain atoms of GLU60 TIGIT With LYS58 HCDR2 Two salt bridges are formed between LYS82 TIGIT With ASP107 HCDR3 Two salt bridges are formed between them, and HIS76 TIGIT With ASP107 HCDR3 A salt bridge is formed between the two residues. In addition, the residues of TIGIT that only participate in van der Waals interactions are GLN56, ILE77, SER78, and HIS111, while the residues of tislelizumab Fab that participate in this interaction are PHE57, TYR106, LEU108, and LEU109 of the heavy chain and TYR31, TYR38, TYR98, and SER99 of the light chain. The amino acids ARG56, PHE57, LYS58, and TYR60 of HCDR2 in tislelizumab Fab cover the exposed hydrophobic surface formed by THR55, GLN56, ASN58, GLU60, ASP63, GLN64, LEU65, ALA67, ILE68, ILE109, and HIS111 of TIGIT. LEU109 of HCDR3 in tislelizumab Fab inserts into the hydrophobic pocket formed by ALA67, ILE68, HIS76, ILE77 and SER78 of TIGIT. Comparative data between the interaction of A1217 (ospelizumab) and tislelizumab with TIGIT are shown in Figure 2A -C and Figure 3A -B.

[0151] Based on the crystal structure of the A1217 / TIGIT complex, the residues of TIGIT that contact A1217 (i.e., the epitope residues of TIGIT that bind to A1217) and the residues of A1217 that contact TIGIT (i.e., the paratope residues of A1217 that contact TIGIT) were determined. Tables 4 and 5 below show the residues of TIGIT and the light chain or heavy chain residues of A1217 that they contact, as shown using The van der Waals (non-polar) interaction forces, as assessed strictly by the contact distance, are highest at this point.

[0152] Table 4. Epitope residues of TIGIT and their corresponding paratope residues on the light chain of A1217

[0153] TIGIT Residue number A1217 light chain Residue number ILE 68 TRP 50 ASN 70 TRP 50 LEU 73 TYR 49 TRP 50 GLY 74 TRP 50 TRP 75 TRP 50 HIS 76 THR 31 SER 32 TRP 50 PRO 79 TYR 91 SER 92 SER 80 TYR 94 LYS 82 SER 92

[0154] Table 5. Epitope residues of TIGIT and their corresponding paratope residues on the heavy chain of A1217

[0155] TIGIT Residue number A1217 heavy chain Residue number GLN 56 ASN 101 TYR 102 GLU 60 LYS 53 ASP 63 TYR 33 THR 52 GLY 54 GLY 56 GLN 64 TYR 33 SER 57 LEU 65 TYR 33 ILE 68 ASP 103 ASN 70 TYR 102 HIS 76 ASP 103 PHE 104 SER 78 PHE 104 SER 80 TYR 59

[0156] Alanine scanning of the human TIGIT interface was also performed, wherein alanine mutations of TIGIT residues were performed on LEU73 / HIS76, HIS76, ILE68, ASP63, LEU73, PRO79, LEU65, GLN56, ASN70, GLU60, LYS82, SER80, and GLN64 (Table 6). The binding kinetics of A1217 to TIGIT variants were tested using a capture immobilization strategy in SPR. In this experiment, mutations of TIGIT residues LEU73 / HIS76, HIS76, and ILE68 reduced A1217 binding to less than one-tenth (Table 6), and HIS76 mutations almost eliminated A1217 binding, indicating that this residue is critical for the interaction. The double mutant LEU73A / HIS76A completely abolished A1217 binding, which is consistent with the strong hydrophobic interaction between LEU73 and HIS76 of TIGIT that sandwiches TRP50 of LCDR2 and supplements it with several hydrogen bonds (H76 TIGIT -D103 HCDR3 、H76 TIGIT -T31 LCDR1 、L73 TIGIT -W50 LCDR2 ) and salt bridge (H76 TIGIT -D103 HCDR3). The LEU73A mutation in TIGIT has a similar association rate to WT, but a slightly faster dissociation rate, resulting in a binding affinity with A1217 reduced to about one-seventh. In addition, the ILE68A mutation in TIGIT has a similar association rate compared to WT, but a much faster dissociation rate, resulting in a binding affinity with A1217 reduced to about one-thirtieth. The composite structure reveals that ILE68 of TIGIT is located in the hydrophobic core of the IgV domain and interacts extensively with the HCDR3 of A1217. In addition, the ASP63A mutation shows a binding affinity with A1217 reduced to about one-seventh, because ASP63 of TIGIT forms three hydrogen bonds with THR52, GLY54 and GLY56 of HCDR2 in A1217. In addition, the PRO79A mutation in TIGIT has a slower association rate and a faster dissociation rate compared to WT, resulting in a binding affinity with A1217 as low as about one-third. The composite structure shows that PRO79 of TIGIT forms strong hydrophobic interactions with PHE104 of HCDR3 and TYR91, SER92 and TYR94 of LCDR3 in A1217. Mutations of TIGIT at SER80 and GLN64 do not affect A1217 binding (Table 6). This analysis is consistent with the crystal structure analysis, in which the TIGIT residues that most affect A1217 binding were found to interact with A1217 in the structure.

[0157] For the interaction with tislelizumab, the LYS82A mutant, which has extensive hydrophobic interactions with TYR31 / TYR 38 / TYR 98 of the light chain and forms a strong hydrogen bond / salt bridge with ASP107 of the heavy chain, completely abolished tislelizumab binding. The ILE68A mutant forms strong hydrophobic interactions with LEU108 and LEU109 of HCDR3 in tislelizumab, resulting in a binding affinity with tislelizumab as low as about 40 times (Table 6), and this site also affects A1217 binding. The LEU65A mutation in TIGIT results in a binding affinity with tislelizumab as low as about 25 times, resulting in a slower association rate and faster dissociation rate compared to WT TIGIT. The HIS76A mutant has a slight effect on tislelizumab binding, while this mutation almost abolishes A1217 binding. Similarly, the P79A / S80A single mutant, which forms hydrogen bonds with THR100, had only a slight effect on tislelizumab binding. Mutagenesis experiments showed that A1217 and tislelizumab have different epitopes when binding to TIGIT, which means that the antagonistic mechanisms of the two antibodies may be different.

[0158] Table 6. Binding kinetics of TIGIT to A1217 and tislelizumab

[0159]

[0160]

[0161] a HB / SB represent hydrogen bond and salt bridge, respectively.

[0162] A1217 has pH-dependent binding properties for TIGIT

[0163] Based on our SPR data, we observed that the binding affinity (KD) of A1217 for TIGIT increased by approximately 17-fold when the pH was decreased from 7.4 to 6.0 (Table 7). The binding affinity between A1217 and TIGIT at different pH was measured using an amine coupling method. HCDR3 Towards HIS76 TIGIT by This results in a strong electrostatic interaction ( Figure 1D ). In the tumor microenvironment, HIS76 TIGIT The imidazole ring can be mostly protonated and carry a positive charge in this acidic environment, which enhances the affinity with ASP103. HCDR3 However, under physiological conditions, HIS76 TIGIT will carry less positive charge and weaken this interaction. In the A1217 / TIGIT complex, ASP103 HCDR3 Relative to HIS76 TIGIT by The distance between HIS76 and tislelizumab / TIGIT was well positioned and formed a strong electrostatic interaction, while no TIGIT This favorable structure. We found that when the pH dropped from 7.4 to 6.0, the binding affinity (KD) of A1217 for TIGIT increased by approximately 17 times, while tislelizumab did not show obvious pH-dependent binding to TIGIT (Table 7). In summary, pH-sensitive antibodies can better alleviate the balance between efficacy against tumor cells and safety against normal cells compared to non-pH-sensitive antibodies.

[0164] Table 7. Binding kinetics of TIGIT and A1217 compared with tislelizumab at different pH

[0165]

[0166] Example 4: Production of anti-TIGIT antibodies with reduced fucosylation

[0167] Removal of core fucose from N-glycans attached to human IgG1 significantly enhances antibody-dependent cellular cytotoxicity (ADCC) responses (Shields, et al., (2002) J Biol Chem 277, 26733-26740); Shinkawa et al., (2003) J Biol Chem 278, 3466-3473). There are many methods to reduce core fucosylation. The work presented herein utilizes the method of 2F-peracetylfucose, an inhibitor of fucosyltransferase (FUT).

[0168] A1217 P5-2 3G11 is a stably transfected CHO-K1 cell line expressing the A1217 antibody. A1217 P5-2 3G11 cells were cultured in shake flasks, passaged every 3-4 days, and maintained at 5x10 5 Cells / mL were seeded in Hyclone ActiPro with or without 1.25% Glycosylation Adjust (Gal+) (Sigma-Aldrich, 14701C0). TM 2F-peracetylfucose was added to the culture at concentrations of 0, 50, 100, 150, and 200 μM. Cell supernatants were harvested on day 14 and filtered through a 0.2 μm filter for further analysis.

[0169] The A1217 antibody was purified using a protein A chromatography capture step under platform conditions and the glycan profile was analyzed by HILIC-UPLC oligosaccharide analysis ( Figure 4 ). Glycan analysis showed that the inhibitor reduced total fucosylation to 11% at 50 μM 2F-peracetylfucose and to less than 7% at 100, 150 or 200 μM 2F-peracetylfucose.

[0170] Example 5: Generation of anti-TIGIT effector variants

[0171] According to the Fast MultiSite Mutagenesis System TM(FM201-01, Transgen biotech) provided method, S239D, I332E or S239D, I332E, A330L (EU numbering) Fc mutation combination was generated on a plasmid containing a DNA fragment encoding the heavy chain of A1217. Variant A1217DE contains amino acid changes S239D, I332E (DE). Variant A1217DEL contains amino acid changes S239D, I332E, A330L (DEL). Expi-CHO cells were transfected with the corresponding plasmids of A1217 effector variants (A1217DE or A1217DEL) and cultured for 10 days at 30°C and 5% CO2. The supernatant was then harvested and the supernatant was purified using MabSelect SuRe TM (17543802, Cytiva) to purify the protein.

[0172] Example 6: Binding affinity of TIGIT antibodies to FcγR

[0173] To determine FcγRI binding, protein A was coupled to an activated CM5 biosensor chip (Catalog No. BR100530, GE Life Sciences), and 30 nM of A1217 or A1217 variants were flowed through the chip and captured by protein A. A series of concentrations of FcγRI (from 0.0586 nM to 15 nM) were injected at 30 μL / min into SPR running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween20, pH 7.4). The binding response of A1217 or mutants to FcγRI was calculated by subtracting RU from a reference flow cell into which A1217 or mutants were not injected. To determine FcγRI binding affinity, kon and koff were calculated using a one-to-one Langmuir binding model; KD was calculated as the ratio of koff / kon.

[0174] Anti-human κ antibodies were coupled to activated CM5 biosensor chips (Catalog No. BR100530, GE LifeSciences), and A1217 or A1217 variants were flowed through the chip and captured by the anti-human κ surface. A series of different FcγRs (FcγRIIA, FcγRIIB, FcγRIIIA) at 30 μL / min were injected into SPR running buffer (10mM HEPES, 150mMNaCl, 3mM EDTA, 0.05% Tween20, pH7.4). The binding response of A1217 or mutants to FcγR was calculated by subtracting RU from the reference flow cell without injection of A1217 or mutants. In order to determine the binding affinity of FcγRIIA, FcγRIIB and FcγRIIIA, the data from all concentrations were fitted using a steady-state affinity model.

[0175] As shown in Table 8, A1217AF showed an approximately 10-fold increase in binding affinity to FcγRIIIA-V158 and FcγRIIIA-F158 compared to A1217. A1217DEL and A1217DE also increased binding affinity to FcγRIIIA-V158 and FcγRIIIA-F158 compared to A1217. In addition, A1217DEL and A1217DE increased binding affinity to FcγRI and FcγRII.

[0176] Table 8 Binding affinity of TIGIT antibody to FcγR measured by SPR

[0177]

[0178] Example 7: Reduced binding of fucosylated TIGIT antibodies to FcγRIII

[0179] HEK293 cells overexpressing FcγRIIIA-V158 or FcγRIIIA-F158 were first incubated with A1217 or A1217AF and then with secondary antibodies Alexa Fluor TM 647 anti-human IgG Fc, Biolegend, REF#409320). The cell samples were washed and fixed with 1% paraformaldehyde in DPBS. Immunofluorescence was detected by flow cytometry (ACEA) and Guava Soft TM 3.1.1 software was used for analysis. The results of this analysis are shown in Figure 5A-B. A1217AF showed increased binding to HEK293 cells overexpressing FcγRIIIA-V158 or FcγRIIIA-F158 compared to A1217, indicating that A1217AF has enhanced Fc effector function through both variants of FcγRIIIA compared to A1217.

[0180] Example 8: Binding of anti-TIGIT effector variants to FcγRIII

[0181] HEK293 cells overexpressing FcγRIIIA-V158 or FcγRIIIA-F158 were first incubated with A1217, A1217DE, or A1217DEL, and then incubated with secondary antibody Alexa Fluor. 488F(ab')2 fragment goat anti-human IgG (F(ab')2 fragment specific, Jackson ImmunoResearch, catalog number 109-546-097). Cell samples were washed and fixed with 1% paraformaldehyde in DPBS. TM Immunofluorescence was detected by 6HT (Merck-Millipore, USA) and analyzed using Guava Soft TM 3.1.1 software for analysis. The results are shown in Fig. 6A -D, A1217DE and A1217DEL showed increased binding to HEK293 cells overexpressing FcγRIIIA-V158 or FcγRIIIA-F158 compared with wild-type A1217, indicating that A1217DE and A1217DEL have enhanced Fc effector functions through the two variants of FcγRIIIA compared with A1217.

[0182] Example 9: Binding of anti-TIGIT effector variants to TIGIT

[0183] BW5147.3 cells overexpressing TIGIT were first incubated with A1217, A1217DE, or A1217DEL, and then incubated with secondary antibody Alexa Fluor. 488 F(ab')2 fragment goat anti-human IgG [F(ab')2 fragment specific, Jackson ImmunoResearch, catalog number 109-546-097]. Cell samples were washed and fixed with 1% paraformaldehyde in DPBS. TM Immunofluorescence was detected by 6HT (Merck-Millipore, USA) and analyzed using Guava Soft 3.1.1 software. The data are shown in Figure 7In this result, A1217, A1217DE, and A1217DEL showed comparable binding to HEK293 cells overexpressing TIGIT, thus indicating that the deletion did not produce a change in TIGIT binding via FACS.

[0184] Example 10: Reduced binding of fucosylated anti-TIGIT antibodies to complement C1q

[0185] The C1q binding activity of A1217 or A1217AF was determined by sandwich ELISA. Briefly, serial dilutions of the indicated A1217 or A1217AF variants were plated on MaxiSorp immunoplates. C1q binding was tested by incubating human C1q with the antibody-coated wells. After washing, bound C1q was detected with an anti-C1q monoclonal antibody followed by HRP-conjugated secondary antibody. The binding signal was measured by absorbance at 450 nm using TMB (3,3′,5,5′-tetramethylbenzidine) substrate. The results are shown in Figure 8 In which both A1217AF and A1217 showed comparable binding to C1q.

[0186] Example 11: Anti-TIGIT Antibodies and Effector Variants with Reduced Fucosylation Have Enhanced ADCC

[0187] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism for killing target cells. Antibodies bind to target antigens on the surface of target cells. When the Fc portion of the target-binding antibody also binds to the FcγRIIIA receptor on the cell surface of effector cells such as NK cells, the cross-linking and activation of FcγRIIIA lead to ADCC, Hogarth and Pietersz, (2012) Nat Rev Drug Discov 11, 311-331. The human FcγRIIIA gene exhibits dimorphism at the position encoding amino acid residue 158. FcγRIIIA variants with valine (V158) at amino acid residue 158 have high affinity for IgG1 Fc part, and FcγRIIIA variants with phenylalanine (F158) have lower affinity for IgG1 Fc part. NK cells are generally used as a readout of ADCC activity in classical ADCC assays to kill target cells. Due to the variability of different donors and the polymorphism of FcγRIIIA, these cells can be highly variable in response. In order to use FcγRIIIA activation as an alternative reading of ADCC, we monitor FcγRIIIA activation with a more simplified and stable setting. In short, in the alternative ADCC assay, Jurkat cells are engineered to stably express FcγRIIIA-V158 or FcγRIIIA-F158 variants, and the NFAT response element (NFAT-reporter gene-luciferase) that drives firefly luciferase expression. The resulting Jurkat / NFAT-reporter gene-luciferase / FcγRIIIA-V158 or Jurkat / NFAT-reporter gene-luciferase / FcγRIIIA-F158 cells are used as effector cells. The BW5147.3 cells (BW5147.3 / TIGIT cells) engineered to stably express TIGIT are used as target cells. The antibody bioactivity in the alternative ADCC assay is quantified by the luciferase as a result of NFAT pathway activation. The luciferase activity in the effector cells is quantitatively measured with luminescent readings.

[0188] (A) Jurkat / NFAT-reporter-luciferase / FcγRIIIA-V158 cells (1 x 10 4 cells / well) and BW5147.3 / TIGIT cells (1x10 4 cells / well) overnight. TM Luciferase assay system (BPS Bioscience) was used to detect NFAT reporter gene activity.

[0189] This experiment showed that A1217AF, A1217DEL and A1217DE had enhanced ADCC activity when compared to A1217 wild type ( Fig.9A ).

[0190] (B) Jurkat / NFAT-reporter gene-luciferase / FcγRIIIA-F158 cells (5x10 4 cells / well) and BW5147.3 / TIGIT cells (1x 10 4 cells / well) overnight. TM NFAT reporter gene activity was detected using the luciferase assay system (Promega). A1217AF, A1217DEL, and A1217DE had enhanced ADCC activity when compared to the A1217 wild type ( Fig. 9B ).

[0191] Example 12: Anti-TIGIT reduced fucosylation and effector variants have enhanced ADCC in Tregs

[0192] TIGIT + Tregs represent a highly immunosuppressive functionally distinct Treg subset (Joller et al., (2014) Immunity 40, 569-581). Since TIGIT expression on tumor Tregs is higher than that on effector T cells, and expression on PBMC-derived Tregs in cancer patients is higher than that on PBMC-derived Tregs in healthy donors (Preillon et al., (2021) Mol Cancer Ther 20, 121-131.), it is reasonable to speculate that the Fc-functional TIGIT antibody A1217 can be used in TIGIT. + Induces ADCC in Treg cells.

[0193] To measure the ADCC activity induced by A1217 or A1217AF against T cells, especially Tregs, PBMCs from lung cancer patients were used as target cells. NK cells isolated from PBMCs from healthy donors were used as effector cells. A1217MF contains a silent Fc, which reduces the amount of ADCC (SEQ ID NO: 12). Anti-TIGIT antibodies A1217, A1217AF or A1217MF were co-administered with target cells (5x10 4 cells / well) and NK effector cells (5x10 4 10 cells / well, purified using NK cell isolation kit, Miltenyi Biotec, catalog number 130-092-657) and incubated overnight in a 96-well plate. Cell samples were analyzed by flow cytometry.

[0194] The results showed that A1217AF enhanced ADCC activity against Tregs when compared with A1217 wild-type. Fig. 10A -C. Fig. 10A As shown, after A1217 or A1217AF treatment, CD3 + The frequency of Tregs in T cells was significantly reduced in a dose-dependent manner, while the percentage remained largely unchanged in the A1217MF-treated group. A1217AF induced significantly higher ADCC against Tregs compared to A1217 wild-type. On the other hand, neither A1217 wild-type, A1217MF, or A1217AF treatment altered the frequency of effector CD4+T cells or CD8+T cells ( Fig. 10B and 10C ). The results clearly showed that A1217AF was able to induce stronger ADCC against Treg cells in PBMCs derived from cancer patients compared with A1217 wild-type. Enhanced Fc function can enhance the activity of TIGIT antibodies in anti-tumor immune responses through Treg reduction.

[0195] Example 13: Anti-TIGIT antibodies with reduced fucosylation activate NK cells

[0196] TIGIT is constitutively expressed on natural killer (NK) cells, and the interaction between TIGIT and its ligands PVR and PVR-L2 inhibits NK cell-mediated cytotoxicity (Stanietsky et al., (2009) Proc Natl Acad Sci US A106, 17858-17863; Wang et al., (2015) Eur J Immunol 45, 2886-2897). The dominant FcγR expressed on NK cells is FcγRIIIA (Bruhns, (2012) Blood 119, 5640-5649). The additive and / or synergistic effects of co-engagement of TIGIT and FcγRIIIA on NK cells can be concentrated downstream, resulting in stronger signals. To confirm whether optimal NK activation by TIGIT antibodies requires the ability to co-engage FcγRs, and whether Fc-enhanced A1217 can further promote NK cell activation, purified primary NK cells were co-cultured with the human breast cancer cell line SK-BR-3 (ATCC HTB-30) expressing high levels of PVR in the presence of TIGIT antibodies with different Fc forms. NK cell activation was determined by measuring the NK cell degranulation marker CD107a via flow cytometry. The results showed that A1217AF had greater activation of NK cells (PVR) when compared with A1217 wild type or A1217MF. Fig.11A -B).

[0197] In the co-culture assay, anti-TIGIT antibodies A1217 wild-type, A1217AF, or A1217MF were added to SK-BR-3 (5x10 4 cells / well) and primary NK cells isolated from PBMCs from healthy donors (5x10 4 Cells / well) were cultured overnight. NK cells were pre-stimulated with 25U / mL recombinant human IL-2 (Novoprotein, China, catalog number C013) before co-culture assay. CD107a expression on NK cells was determined by FACS. When compared with A1217 wild type, A1217AF significantly increased NK activation, indicating that FcγR co-engagement enhanced by A1217 afucosylation induces optimal NK cell activation in the presence of tumor cells.

[0198] Example 14: Cytotoxicity properties of anti-TIGIT antibodies with reduced fucosylation

[0199] Cytotoxicity is the process by which cell surface molecules are transferred from donor cells to recipient cells (Beum et al., (2008) J Immunol 181, 8120-8132; Joly and Hudrisier, (2003) Immunity 40, 569-581; Machlenkin et al., (2008) Cancer Res 68, 2006-2013; Rossi et al., (2013) Blood 122, 3020-3029). Antibody-mediated cytotoxicity via Fcγ receptors (FcγRs) causes downregulation of receptors on the cell surface (Taylor and Lindorfer, (2015) Blood 125, 762-766). Downregulation of target receptors by cytotoxicity can cause attenuated signal transduction. To investigate whether TIGIT antibodies with Fc function can induce FcγR-mediated endocytosis to remove TIGIT from the cell surface and whether enhanced Fc binding to FcγR by DE / DEL mutation or reduced fucosylation can further promote endocytosis, endocytosis assays were performed.

[0200] Jurkat / TIGIT / DNAM-1 cells (2x10 4 cells / well) were used as donor cells, and HEK293 cells (4×10 4Cells / well) were used as recipient cells. Donor cells were pre-incubated with 10 μg / mL CF633-labeled A1217 wild type, A1217AF or A1217MF for 30 minutes and washed. Donor cells were then incubated with recipient cells in 96-well plates overnight. The changes in the mean fluorescence intensity (MFI) of TIGIT (CF633) on donor cells were measured by FACS.

[0201] like Fig.12 As shown, A1217AF significantly induced TIGIT downregulation on Jurkat / TIGIT / DNAM-1 cells when recipient cells expressed FcγRIIIA-F158 and FcγRIIIA-V158 when compared to A1217 wild-type, indicating that A1217 can induce maximal TIGIT downregulation through FcγRIIIA binding-dependent cytotoxicity via afucosylated Fc-enhancement. Interestingly, A1217AF induced cytotoxicity levels comparable to A1217 wild-type when recipient cells expressed FcγRI, consistent with the comparable binding affinity of A1217AF and A1217 to FcγRI.

[0202] Example 15: Cytotoxicity properties of anti-TIGIT antibody mutants

[0203] Jurkat / TIGIT / DNAM-1(2x10 4 cells / well) were used as donor cells, and HEK293 cells (4×10 4 Cells / well) were used as recipient cells. Donor cells were pre-incubated with 10 μg / mL CF633-labeled A1217 wild type, A1217DE, A1217DEL or A1217MF for 30 minutes and washed. Donor cells were then incubated overnight with recipient cells in 96-well plates. Changes in TIGIT (CF633) MFI on donor cells were measured by FACS.

[0204] Results for the A1217DE and DEL effector variants were similar to those for A1217AF with reduced fucosylation. On cells expressing FcγRIIIA-F158 or FcγRIIIA-V158, A1217DE and A1217DEL had greater cytotoxicity when compared to A1217 wild-type. Fc-silenced A1217MF had little or no activity as expected, and this data is presented in graphical form. Fig.13 middle.

[0205] Example 16: A1217 antibody with reduced fucosylation combined with anti-PD1 antibody in a mouse tumor model

[0206] For this experiment, 2x10 5 Renca cells were implanted subcutaneously into the right flank of humanized TIGIT mice. Renca cells are a model of murine renal adenocarcinoma. The mice were then administered PBS as a control, 3mg / kg mPD-1Ab, 10mg / kg A1217 wild type, or 10mg / kg A1217AF as a single agent therapy. For combination therapy, 3mg / kg mPD-1 antibody was administered to mice in combination with 10mg / kg A1217 wild type or 3mg / kg mPD-1Ab and 10mg / kg A1217AF. The treatment was administered intraperitoneally on day 5 after tumor cell inoculation. Tumor volume was determined twice a week using the following formula: V = 0.5 (a x b 2 ) where a and b are the long and short diameters of the tumor, respectively. *p<0.05.

[0207] The data are presented in graphical form in Fig.14 In comparing A1217 wild-type and A1217AF, there was little difference when administered as a single agent. However, when administered in combination with anti-PD1 antibody, the A1217AF antibody had improved tumor reduction compared to A1217 wild-type administered in combination with anti-PD1 antibody.

[0208] Example 17: A1217 antibody with reduced fucosylation combined with anti-PD1 antibody induces Treg reduction in a mouse tumor model

[0209] To investigate whether A1217AF antibody with reduced fucosylation combined with anti-PD-1 antibody reduces Treg cells in the Renca model, 300,000 RENCA tumor cells were implanted subcutaneously in the right flank of female hTIGIT Balb / c mice (6-8 weeks old) on day 0 and when the mean tumor volume reached 100-200 cm 3 Mice were randomly divided into 8 groups according to body weight and tumor volume. Mice were treated with anti-TIGIT antibody A1217 (10 mg / kg) with different Fc forms as a single agent, murine PD-1 blocking antibody Ch15mt (3 mg / kg) as a single agent, or a combination of both antibodies at the indicated doses. Tumor samples were collected 48 hours after dosing for tumor infiltrating lymphocyte (TIL) isolation and FACS analysis. + CD4 + FOXP3 + Set a gate for Treg cells. Fig.15AAs shown in Figure 1-C, when comparing A1217 wild type and A1217AF, there was no significant difference when administered as a single agent. However, when administered in combination with anti-PD1 antibody, A1217AF antibody significantly reduced intratumoral Tregs compared to A1217 wild type and anti-PD1 antibody combination, indicating that afucosylation of A1217 can induce a more significant anti-tumor effect by reducing Treg cells in the tumor microenvironment in combination with PD-1 antibody.

Claims

1. A method for cancer treatment, comprising administering to a subject an effective amount of a pH-dependent anti-TIGIT antibody or an antigen-binding fragment thereof.

2. The method of claim 1, wherein the anti-TIGIT antibody binds to the TIGIT protein at amino acid histidine 76.

3. The method of claim 2, wherein the anti-TIGIT antibody binds to the TIGIT protein at histidine 76 and leucine 73.

4. The method of claim 3, wherein the method comprises administering to the subject an effective amount of a pH-dependent antibody or an antigen-binding fragment thereof, wherein the pH-dependent antibody or antigen-binding fragment thereof specifically binds to human TIGIT and comprises: a heavy chain variable region comprising HCDR (heavy chain complementarity determining region) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising LCDR (light chain complementarity determining region) 1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO:

6.

5. The method of claim 4, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 7, and a light chain variable region (VL) comprising SEQ ID NO:

8.

6. The method of claim 1, wherein the anti-TIGIT antibody has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

7. The method of claim 6, wherein the anti-TIGIT antibody has reduced fucosylation.

8. The method of claim 6, wherein the anti-TIGIT antibody has Fc amino acid changes at S239D and I332E (EU numbering).

9. The method of claim 6, wherein the anti-TIGIT antibody has Fc amino acid changes at S239D, I332E, and A330L (EU numbering).

10. The method of any one of claims 7 to 9, wherein the anti-TIGIT antibody has increased binding affinity for FcγRIIIA-V158 and FcγRIIIA-F158.

11. The method of any one of claims 7 to 9, wherein the anti-TIGIT antibody has enhanced ADCC in T-regulatory (Treg) cells.

12. The method of any one of claims 7 to 9, wherein the anti-TIGIT antibody activates natural killer (NK) cells.

13. The method of any one of claims 7 to 9, wherein the anti-TIGIT antibody has increased cytotoxicity.

14. The method of claim 1, wherein the method further comprises administering an anti-PD1 antibody that specifically binds to human PD1 and comprises: a heavy chain variable region comprising HCDR1 of SEQ ID NO: 15, HCDR2 of SEQ ID NO: 16, and HCDR3 of SEQ ID NO: 17; and a light chain variable region comprising LCDR1 of SEQ ID NO: 18, LCDR2 of SEQ ID NO: 19, and LCDR3 of SEQ ID NO:

20.

15. The method of claim 14, wherein the anti-PD1 antibody or antigen-binding fragment thereof specifically binds to human PD1 and comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

22.

16. The method of claim 14 or 15, wherein the anti-PD1 antibody comprises: an IgG4 constant domain comprising SEQ ID NO:

23.

17. The method of claim 1, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, stomach cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, or sarcoma.

18. The method of claim 17, wherein the cancer is non-small cell lung cancer.

19. The method of claim 17, wherein the head and neck cancer is nasopharyngeal carcinoma.

20. The method of claim 17, wherein the esophageal cancer is esophageal squamous cell carcinoma (ESCC).

21. The method of claim 17, wherein the cancer is uterine cancer.

22. The method of claim 17, wherein the gastric cancer is gastric or gastroesophageal junction cancer.

23. The method of claim 17, wherein the cervical cancer is recurrent or metastatic cervical cancer.

24. The method of claim 17, wherein the cancer is renal cancer.

25. The method of claim 14, further comprising administering chemotherapy.

26. The method of claim 20, wherein the chemotherapy is chemoradiotherapy.

27. The method of claim 1, wherein the anti-PD1 antibody is administered at 200 mg every three weeks.

Images