Method for treating cancer using an anti-TIGIT antibody

Abstract

Provided is a method of treating cancer or increasing, enhancing, or stimulating an immune response by using an antibody that specifically binds to TIGIT (T cell immunoreceptor with Ig and ITIM domains, WUCAM, or Vstm3) and antigen-binding fragments thereof in combination with an anti-PD1 antibody.

Description

Technical Field

[0001] This application relates to an antibody that specifically binds to TIGIT (T cell immunoreceptor with Ig and ITIM domains) in combination with an anti-PD1 antibody 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 that 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 have been cloned and characterized in mice and humans. Full-length human TIGIT has a sequence of 244 amino acids (SEQ ID NO: 26), where 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), has a V-type Ig-like domain (corresponding to amino acids 39-127 of SEQ ID NO: 26), followed by a 21aa transmembrane sequence, and an 82aa 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). Within the ECD, human TIGIT shares only 59% and 87% aa sequence identity with mouse and cynomolgus monkey, 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), as well as 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] To date, two TIGIT ligands, CD155 (also known as poliovirus receptor or PVR) and CD112 (also known as poliovirus receptor-related 2, PVRL2, nectin-2), have been identified. These ligands are expressed mainly 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 within immune cells when it binds to its ligands CD155 and CD112. The binding affinity of TIGIT for CD155 (Kd: approximately 1 nM) is much higher than that for CD112, and whether the TIGIT:CD112 interaction is functionally relevant to the mediation of inhibitory signals remains unclear. The costimulatory receptor CD226 (DNAM-1) binds to the same ligands with a lower affinity (Kd: approximately 100 nM) but transmits a positive signal (Bottino C, et al., 2003 J Exp Med 198:557-67). Furthermore, the "TIGIT-like" receptor CD96 (TACTILE) also plays a similar inhibitory role in the same pathway (Chan CJ, et al., 2014 Nat.Immunol 15:431-8).

[0006] Upregulation of TIGIT expression in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) has been reported in many types of cancer, such as lung (Tassi, et al., Cancer Res. 2017 77:851-861), esophagus (Xie J, et al., Oncotarget 2016 7:63669-63678), breast (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 prognostic survival outcomes in patients (Kong Y et al., Clin Cancer Res. 2016 22:3057-66). Upregulation of TIGIT signaling plays an important role not only in immune tolerance to cancer but also in chronic viral infections. During HIV infection, the expression of TIGIT on T cells was significantly higher and was positively correlated with viral load and disease progression (Chew GM, et al., 2016 PLoS Pathog. 12:e1005349). Furthermore, blocking the TIGIT receptor alone or in combination with other blockers can functionally rescue "exhausted" T cells both in vitro and in vivo (Chauvin JM, et al., J Clin Invest. 2015 125:2046-2058, Chew GM, et al., 2016 PLoS Pathog. 12:e1005349, Johnston RJ, et al. Cancer Cell 2014 26:923-937). In the case of cancer and viral infection, activation of TIGIT signaling promotes immune cell dysfunction, leading to cancer growth or viral infection spread. Inhibition of TIGIT-mediated inhibitory signaling by therapeutic agents can restore the functional activity of immune cells such as T cells, NK cells, dendritic cells (DCs), and as a result, enhance immunity against cancer or chronic viral infection.

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

Summary of the Invention

[0008] The present disclosure relates to a method for treating cancer by administering an anti-TIGIT antibody.

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

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

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

[0012] In this method, the method comprises administering to a subject an effective amount of a pH-dependent antibody or an antigen-binding fragment thereof that 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] In this method, the anti-TIGIT antibody or an 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] In this method, the anti-TIGIT antibody enhances antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

[0015] In this method, the anti-TIGIT antibody has reduced fucosylation.

[0016] In this method, the anti-TIGIT antibody has changes in the Fc amino acids at S239D and I332E (EU number).

[0017] In this method, the anti-TIGIT antibody has changes in the Fc amino acids at S239D, I332E, and A330L (EU number).

[0018] In the method according to any one of the above, the anti-TIGIT antibody has an increased binding affinity for FcγRIIIA-V158 and FcγRIIIA-F158.

[0019] In the method according to any one of the above, the anti-TIGIT antibody has enhanced ADCC in regulatory T (Treg) cells.

[0020] In the method according to any one of the above, the anti-TIGIT antibody activates natural killer (NK) cells.

[0021] In the method according to any one of the above, the anti-TIGIT antibody has increased trogocytosis.

[0022] In the above method, this method further comprises administering an anti-PD1 antibody that specifically binds to human PD1 and comprises a heavy chain variable region containing 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 containing LCDR1 of SEQ ID NO: 18, LCDR2 of SEQ ID NO: 19, and LCDR3 of SEQ ID NO: 20.

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

[0024] In this method, the anti-PD1 antibody comprises an IgG4 constant domain containing SEQ ID NO: 23.

[0025] In this method, the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, gastric 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] In this method, the cancer is non-small cell lung cancer.

[0027] In this method, the head and neck cancer is hypopharyngeal cancer.

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

[0029] In this method, the cancer is uterine cancer.

[0030] In this method, the gastric cancer is gastric cancer or gastroesophageal junction cancer.

[0031] In this method, the cervical cancer is recurrent or metastatic cervical cancer.

[0032] In this method, the cancer is kidney cancer.

[0033] In this method, it further includes the administration of chemotherapy.

[0034] In this method, the chemotherapy is chemoradiotherapy.

[0035] In this method, the anti-PD1 antibody is administered at 200 mg every three weeks.

Brief Description of the Drawings

[0036]

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Modes for Carrying Out the Invention

[0037] Definitions Conservative amino acid substitutions of amino acids are generally known in the art. Generally, a conservative amino acid substitution means that an amino acid residue is replaced by another amino acid residue having a similar side chain.

[0038] Unless otherwise defined elsewhere in this specification, all other technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the technical field to which this invention pertains.

[0039] As used in this specification, including the appended claims, singular words such as "a", "an", and "the" include the corresponding plural referents unless the context clearly dictates otherwise.

[0040] The term "or" is used to mean "and / or" and is used synonymously with "and / or" unless the context clearly indicates otherwise.

[0041] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to mean including the recited amino acid sequences, DNA sequences, steps, or groups thereof but not excluding any other amino acid sequences, DNA sequences, steps. The term "comprising" as used herein may be replaced by the terms "containing", "including", or in some cases, "having".

[0042] The term "TIGIT" includes various mammalian isoforms such as human TIGIT, orthologs of human TIGIT, and analogs containing at least one epitope within TIGIT. The amino acid sequence of TIGIT, for example, human TIGIT, and the nucleotide sequence encoding it are known in the art (see Genbank AAI01289). The human TIGIT sequence (SEQ ID NO: 26) [Chemical formula]

[0043] As used herein, the terms "administer", "administering", "treat" and "treatment", when applied to animals, humans, subjects, cells, tissues, organs, or biological fluids, mean contacting an exogenous pharmaceutical, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ or biological fluid. Treatment of cells includes contacting the cells with a reagent, as well as contacting the reagent with a fluid, where the fluid contacts the cells. The terms "administer" or "treatment" further include, for example, in vitro and ex vivo treatment of cells with a reagent, diagnostic, binding compound, or another cell. As used herein, the term "subject" refers to any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human.

[0044] As used herein, the term "antibody" is used in the broadest sense and specifically includes antibodies (including full-length monoclonal antibodies) and antibody fragments, so long as they recognize an antigen (e.g., TIGIT). Antibodies are usually monospecific, but may be described as idiotypic, heterospecific, or multispecific. Antibody molecules bind to specific antigenic determinants or epitopes on an antigen by specific binding sites.

[0045] The term "monoclonal antibody" or "mAb" or "Mab" as used herein means a substantially homogeneous population of antibodies, i.e., the antibody molecules within the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in trace amounts. In contrast, conventional (polyclonal) antibody preparations usually contain a number of different antibodies having different amino acid sequences within the variable domains, particularly the complementarity determining regions (CDRs) that are often specific for different epitopes. The modifier "monoclonal" indicates the characteristic of an antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring the production of an antibody by any particular method. Monoclonal antibodies (mAbs) can be obtained by methods known to those skilled in the art. See, for example, 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: A LABORATORY 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 of their subclasses. Hybridomas producing mAbs can be cultured in vitro or in vivo. Production in vivo can result in high titers of mAbs, and cells from individual hybridomas are injected intraperitoneally into mice (e.g., initially primed Balb / c mice) to generate ascitic fluid containing high concentrations of the desired mAb. MAbs of isotype IgM or IgG can be purified from such ascitic fluid or culture supernatant using column chromatography methods well known to those skilled in the art.

[0046] Generally, the basic antibody structural unit contains a tetramer. Each tetramer contains a pair of two identical polypeptide chains, and each pair has one "light chain" (about 25 kDa) and one "heavy chain" (about 50 - 70 kDa). The amino-terminal portion of each chain contains a variable region of about 100 - 110 or more amino acids in length that is mainly involved in antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region that is mainly involved in effector functions. Usually, human light chains are classified into kappa light chains and lambda light chains. Furthermore, human heavy chains are usually classified as α, δ, ε, γ, or μ, and the isotypes of antibodies are defined as IgA, IgD, IgE, IgG, and IgM, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 10 amino acids.

[0047] The variable regions of each light chain / heavy chain (VL / VH) pair form the antibody binding site. Thus, generally, a complete antibody has two binding sites. Except for bispecific or bifunctional antibodies, the two binding sites are generally the same.

[0048] Generally, the variable domains of both the heavy and light chains contain three hypervariable regions, also called "complementary determining regions (CDRs)", which are located between relatively conserved framework regions (FRs). The CDRs are typically aligned by the framework regions and enable binding to a specific epitope. Generally, the variable domains of both the light and heavy chains sequentially include FR-1 (or FR1), CDR-1 (or CDR1), FR-2 (FR2), CDR-2 (CDR2), FR-3 (or FR,3), CDR-3 (CDR3) and FR-4 (or FR4) from the N-terminus to the C-terminus. The amino acid assignments to each domain generally follow the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md., 5th ed., NIH Publ. 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.

[0049] The term "hypervariable region" means the amino acid residues of an antibody that are involved in antigen binding. The hypervariable regions include the amino acid residues of the "CDRs" (i.e., VL-CDR1, VL-CDR2, and VL-CDR3 of the light chain variable domain, and VH-CDR1, VH-CDR2, and VH-CDR3 of 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. (definition of CDR regions of antibodies by sequence), and also see Chothia and Lesk (1987) J. Mol. Biol. 196:901-917 (definition of CDR regions of antibodies by structure). The term "framework" or "FR" residues means the variable domain residues other than the hypervariable region residues as defined herein as CDR residues.

[0050] 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 to which the full-length antibody binds, e.g., 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, bispecific antibodies, linear antibodies, single-chain antibody molecules (e.g., single-chain Fv (ScFv)), nanobodies, and multispecific antibodies formed from antibody fragments.

[0051] An antibody that binds specifically to a particular target protein is also described as binding specifically to the particular target protein. This means that the antibody shows preferential binding to its target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered "specific" for its intended target when its binding determines the presence of the target protein in a sample without producing unwanted results such as false positives. Antibodies or binding fragments thereof useful in the present invention will bind to the target protein with an affinity that is at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and most preferably at least 100-fold the affinity for non-target proteins. An antibody herein is said to bind specifically to a polypeptide containing a given amino acid sequence, e.g., the amino acid sequence of the mature human TIGIT molecule, but not to a protein lacking that sequence.

[0052] The expressions "pH-dependent binding", "pH-dependent target binding" and "pH-dependent antigen binding" are interchangeable in the present disclosure and indicate 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 signal to their antigen at a weakly acidic pH, such as pH 6.0, commonly found in the tumor microenvironment, compared to binding affinity and / or binding signal at physiological pH, such as pH 7.4. Methods for determining the binding affinity and / or strength of the binding signal of the antibodies of the present application are known in the art and include, but are not limited to, surface plasmon resonance (Biacore) or similar techniques. More specifically, the antibodies of the present application have a KD ratio at pH 7.4 / pH 6.0 of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more when measured by surface plasmon resonance (Biacore) or similar techniques. Alternatively, or additionally, the antibodies of the present application have an Rmax (RU) value at pH 6.0 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold higher than the Rmax at pH 7.4 measured by surface plasmon resonance (Biacore) or similar techniques. The binding affinity of the antibody can be measured at 25°C or 37°C. The tumor microenvironment has been found to exhibit a relatively acidic pH compared to the physiological state or normal tissues (Zhang et al., Focus on molecular Imaging 2010, Tannock and Rotin et al. Cancer Res 1989). Therefore, the antibodies of the present application having the above pH-dependent binding are advantageous as anti-TIGIT therapeutic agents that selectively target TIGIT-positive lymphocytes in the tumor microenvironment and have low toxicity associated with peripheral activation of lymphocytes.

[0053] As used herein, the term "human antibody" means an antibody that contains only human immunoglobulin protein sequences. A human antibody may contain mouse carbohydrate chains when produced in mice, mouse cells or hybridomas derived from mouse cells. Similarly, "mouse antibody" or "rat antibody" means an antibody that contains only mouse or rat immunoglobulin protein sequences, respectively.

[0054] The term "humanized antibody" also means an antibody form and a human antibody that contain the sequences of non-human (e.g., mouse) antibodies. Such antibodies contain a minimal sequence derived from non-human immunoglobulins. Generally, a humanized antibody contains substantially all of at least one, typically two, variable domains, and 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 the FR regions of human immunoglobulin sequences. A humanized antibody optionally also contains at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. When it is necessary to distinguish a humanized antibody from a parental rodent antibody, a prefix such as "hum", "hu", "Hu" or "h" is added to the name of the antibody clone. The humanized form of a rodent antibody generally contains the same CDR sequences as the parental rodent antibody, but may contain certain amino acid substitutions for reasons such as improved affinity, improved stability of the humanized antibody, or other reasons.

[0055] The antibodies of the present application have potential therapeutic applications in the treatment of cancer. As used herein, the term "cancer" or "tumor" typically means or describes a physiological state of a mammal characterized by disordered cell growth. Examples of cancer include, but are not limited to, lung cancer (including small cell lung cancer, 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 tumor, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or metastatic lesions of cancer.

[0056] Furthermore, the antibodies of the present application have potential therapeutic uses in the control of 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 a process leading to the depletion of the ability of immune cells to respond during cancer or chronic viral infections.

[0057] As used herein, the term "therapeutically effective amount" refers to an amount of an antibody sufficient to have an effect on the treatment of a disease, disorder, or at least one clinical symptom of a disease or disorder when administered to a subject for treating the disease, disorder, or symptom. A "therapeutically effective amount" can vary depending on the antibody, the disease, the disorder, and / or the symptoms of that disease or disorder, the severity of the disease or disorder, and / or the symptoms of the disease or disorder, the age of the subject being treated, and / or the weight of the subject being treated. The appropriate amount in any given case may be apparent to those of ordinary skill 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 the active agents included in the combination therapy for effective treatment of a disease, disorder, or condition.

[0058] As used herein, a "subject" is a mammal, such as a rodent or a primate, preferably a higher primate, such as a human (e.g., a patient having or at risk of having a disorder described herein).

[0059] Anti-LIGHT antibody The present invention provides antibodies that specifically bind to human TIGIT and antigen-binding fragments thereof. Furthermore, the present disclosure provides antibodies having desired pharmacokinetic properties and other desired attributes, and thus can be used to reduce the likelihood of cancer or treat cancer. The present disclosure further provides pharmaceutical compositions comprising the antibodies, and methods of making and using such pharmaceutical compositions for the prevention and treatment of cancer and related diseases.

[0060] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TIGIT. The antibody or antigen-binding fragment of the present disclosure includes, but is not limited to, an antibody or an antigen-binding fragment thereof generated as described in Table 1 below. [Table 1-1] [Table 1-2]

[0061] The present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, and the above antibody or antibody fragment (e.g., antigen-binding fragment) includes a VH domain having the amino acid sequence of SEQ ID NO: 7. The present invention further provides an antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, and the above antibody or antigen-binding fragment includes a VH CDR having the amino acid sequence of any one of the VH CDRs provided herein. In one aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, and the above antibody includes one, two, three, or more VH CDRs (or consists of) having the amino acid sequence of any one of the VH CDRs provided by the present invention.

[0062] The present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, and the above antibody or antigen-binding fragment includes a VL domain having the amino acid sequence of SEQ ID NO: 8. The present invention further provides an antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, and the above antibody or antigen-binding fragment includes a VL CDR having the amino acid sequence of any one of the VL CDRs listed herein. In particular, the present disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, and the above antibody or antigen-binding fragment includes one, two, three, or more VL CDRs (or consists of) having the amino acid sequence of any one of the VL CDRs of the present disclosure.

[0063] Other antibodies or antigen-binding fragments thereof of the present disclosure are mutated but contain amino acids having at least 60%, 70%, 80%, 90%, 95%, or 99% identity in the CDR regions shown in the sequences described herein. In some embodiments, it contains a mutant amino acid sequence in which 1, 2, 3, 4, or 5 or fewer amino acids are mutated in the CDR region when compared to the CDR regions disclosed in the provided sequences.

[0064] Other antibodies of the present disclosure include antibodies in which the amino acids or the nucleic acids encoding the amino acids are mutated but have at least 60%, 70%, 80%, 90%, 95%, or 99% identity to the sequences listed in Table 1. In some embodiments, it contains a mutant amino acid sequence in which 1, 2, 3, 4, or 5 or fewer amino acids are mutated in the variable region when compared to the variable regions shown in the sequences described herein while retaining substantially the same therapeutic activity.

[0065] Further modification of the framework of the Fc region In yet other embodiments, the Fc region is modified by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids can be substituted with different amino acid residues such that the antibody has an altered affinity for effector ligands but retains the antigen-binding ability of the parental antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described, for example, in U.S. Pat. Nos. 5,624,821 and 5,648,260 by Winter et al.

[0066] In another embodiment, one or more amino acid residues can be substituted with one or more different amino acid residues such that the antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described, for example, in U.S. Pat. No. 6,194,551 by Idusogie et al.

[0067] In yet another aspect, one or more amino acid residues are altered such that the ability of the antibody to fix complement is changed. This approach is described, for example, in PCT Publication WO 94 / 29351 by Bodmer et al. In certain aspects, one or more amino acids of the antibodies or antigen-binding fragments thereof are substituted with one or more allotype amino acid residues for IgG1 subclass and kappa isotype. Allotype amino acid residues further include, but are not limited to, the constant regions of the heavy chains of IgG1, IgG2, IgG3 subclasses, and the constant region of the light chain of the kappa isotype as described in Jefferis et al., MAbs. 1:332-338 (2009).

[0068] In another aspect, the Fc region is modified to enhance the ability of the antibody to mediate antibody-dependent cell cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids. This approach is described, for example, in PCT Publication WO00 / 42072 by Presta. Additionally, the binding sites for FcγRI, FcγRII, FcγRIII, 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).

[0069] In yet another aspect, the glycosylation of the antibody is modified. For example, non-glycosylated antibodies (i.e., antibodies lacking or having reduced glycosylation) can be created. By changing the glycosylation, for example, the affinity of the antibody for its "antigen" can be increased. Such carbohydrate modifications can be achieved, for example, by changing one or more glycosylation sites within the antibody sequence. For example, by making one or more amino acid substitutions, one or more variable region framework glycosylation sites are removed, thereby removing glycosylation at that site. Such non-glycosylation can increase the affinity of the antibody for its antigen. Such an approach is described, for example, in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0070] Additionally or alternatively, antibodies with altered glycosylation types can be generated (e.g., hypofucosylated antibodies with reduced amounts of fucosyl residues, or antibodies with increased bisecting GlcNAc structures). Such altered glycosylation patterns have been demonstrated to enhance the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in host cells with an altered glycosylation machinery. Cells with an altered glycosylation machinery have been described in the art and can be used as host cells for expressing recombinant antibodies, thereby producing antibodies with altered glycosylation. For example, EP1,176,195 by Hang et al. describes cell lines having a functionally disrupted FUT8 gene encoding fucosyltransferase, and as a result, antibodies expressed in such cell lines exhibit hypofucosylation. PCT Publication WO03 / 035835 by Presta describes a variant CHO cell line, Lec13 cells, which have a reduced ability to attach fucose to the Asn(297)-linked carbohydrate and also exhibit hypofucosylation of the antibodies expressed in those host cells (see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO99 / 54342 by Umana et al. describes cell lines engineered to express a glycoprotein-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that the antibodies expressed in the engineered cell lines show an increase in bisecting GlcNAc structures and, as a result, an increase in the ADCC activity of the antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

[0071] In another aspect, when reduction of ADCC is desired, the human antibody subclass IgG4 has been shown in many previous reports to have only moderate ADCC and little or no CDC effector function (Moore G L, et al. 2010 MAbs, 2:181 - 189). On the other hand, native IgG4 has been found to be less stable under stress conditions such as acidic buffer or elevated temperature (Angal 1993 Mol Immunol, 30:105 - 108, Dall’Acqua, et al., 1998 Biochemistry, 37:9266 - 9273, Aalberse et al., 2002 Immunol, 105:9 - 19). Reduction of ADCC can be achieved by operably linking an antibody to an IgG4 engineered with a combination of modifications that reduce or abrogate FcγR binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector functions. Considering the physicochemical properties of the antibody as a biological drug, one of the less desirable intrinsic properties of IgG4 is the formation of half - antibodies by the dynamic separation of its two heavy chains in solution, which has led 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). A mutation of serine to proline at position 228 (EU numbering system) appeared to be inhibitory to IgG4 heavy - chain separation (Angal 1993 Mol Immunol, 30:105 - 108, Aalberse et al., 2002 Immunol, 105:9 - 19).Some of the amino acid residues of the hinge and γFc region have been reported to affect antibody interaction 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). Furthermore, some IgG4 isotypes that rarely occur in the human population can also induce different physicochemical properties (Brusco et al., 1998 Eur J Immunogenet, 25:349-55, Aalberse et al., 2002 Immunol, 105:9-19). It is possible to modify the hinge and Fc region of human IgG4 and introduce a number of changes to produce TIGIT antibodies with low ADCC, CDC, and instability. These modified IgG4 Fc molecules can be found disclosed in SEQ ID NOs: 83-88, U.S. Patent No. 8,735,553.

[0072] Antibody production Anti-TIGIT antibodies and antigen-binding fragments thereof can be produced by any means 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 performed from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc.

[0073] The present disclosure further provides a polynucleotide encoding an antibody described herein, for example, a polynucleotide encoding a heavy or light chain variable region or segment comprising a complementarity determining region described herein. In some embodiments, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with the polynucleotide encoding the polypeptide of SEQ ID NO: 7. In some embodiments, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with the polynucleotide encoding the polypeptide of SEQ ID NO: 8.

[0074] The polynucleotide of the present invention can encode the variable region sequence of an anti-TIGIT antibody. It is also possible to encode both the variable region and the constant region of the antibody. A part of the polynucleotide sequence encodes a polypeptide comprising the variable regions of both the heavy and light chains of one of the exemplified anti-TIGIT antibodies. Some other polynucleotides encode two polypeptide segments that are substantially identical to the variable regions of one of the heavy and light chains of a mouse antibody, respectively.

[0075] Also provided herein are expression vectors and host cells for producing anti-TIGIT antibodies. The choice of expression vector will vary depending on the host cell in which the vector is to be expressed. Typically, an expression vector contains a promoter and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an anti-TIGIT antibody chain or an antigen-binding fragment thereof. In some embodiments, it is used to prevent expression of the inserted sequence, except when under the control of inducible conditions. Inducible promoters include, for example, the arabinose, lacZ, metallothionein promoter, or heat shock promoter. Cultures of the transformed organisms can be grown under non-inducing conditions without biasing the population due to a coding sequence for which the expression product is more favorably tolerated by the host cell. In addition to the promoter, other regulatory elements may be required or desired for efficient expression of the anti-TIGIT antibody or antigen-binding fragment. These elements typically include the ATG start codon and adjacent ribosome binding site or other sequences. Furthermore, the efficiency of expression can be enhanced by incorporating an enhancer appropriate for the cell line in use (see, for example, 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 CMV enhancer can be used to enhance expression in mammalian host cells.

[0076] Host cells for carrying and expressing an anti-TIGIT antibody chain may be prokaryotic cells or eukaryotic cells. E. coli is one prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other microbial hosts suitable for use include bacilli such as Bacillus subtilis, enterobacteria such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, typically, an expression vector containing an expression control sequence (e.g., origin of replication) compatible with the host cell can also be created. Furthermore, there are various well-known promoters such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the promoter system derived from phage lambda. The promoter typically optionally controls expression by 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 the anti-TIGIT polypeptide. Insect cells combined with a baculovirus vector can also be used.

[0077] In other embodiments, mammalian host cells are used to express and produce the anti-TIGIT antibodies of the invention. For example, they can be either a hybridoma cell line that expresses an endogenous immunoglobulin gene or a mammalian cell line with an exogenous expression vector. These include any normal lethal or normal or abnormal immortal animal or human cells. For example, numerous suitable host cell lines have been developed that can secrete intact immunoglobulins, including CHO cell lines, various COS cell lines, HEK293 cells, myeloma cell lines, transformed B cells, and hybridomas. The use of mammalian tissue cell cultures for expressing polypeptides is generally described, for example, in Winnacker, From Genes to Clones, VCH Publishers, NY, N.Y., 1987. Expression vectors for mammalian host cells can contain expression control sequences such as origins of replication, promoters, enhancers (see, for example, Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites such as ribosome binding sites, RNA splice 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 regulatable or inducible. Useful promoters include the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP Pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate early CMV promoter), the constitutive CMV promoter, and combinations of promoters-enhancers known in the art.

[0078] Methods of Detection and Diagnosis The antibodies or antigen-binding fragments of the present disclosure are useful for various applications, including but not limited to methods for detecting TIGIT. In one aspect, the antibody or antigen-binding fragment is useful for detecting the presence of TIGIT in a biological sample. As used herein, the term "detecting" includes quantitative detection or qualitative detection. In certain forms, the biological sample includes cells or tissues. In other aspects, such tissues include normal tissues and / or cancer tissues that express TIGIT at high levels compared to other tissues.

[0079] In one aspect, the present disclosure provides a method for detecting the presence of TIGIT in a biological sample. In certain embodiments, the method includes contacting the biological sample with an anti-TIGIT antibody under conditions that permit the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. The biological sample includes, but is not limited to, urine or blood samples.

[0080] Also included are methods for diagnosing disorders associated with TIGIT expression. In certain embodiments, the method includes contacting test cells with an anti-TIGIT antibody, determining the level of TIGIT expression (quantitative or qualitative) in the test cells by detecting the binding of the anti-TIGIT antibody to the TIGIT polypeptide, and comparing the expression level in the test cells to the TIGIT expression level in control cells (e.g., normal cells or non-TIGIT-expressing cells derived from the same tissue as the test cells), wherein an elevated level of TIGIT expression in the test cells compared to the control cells indicates the presence of a disorder associated with TIGIT expression.

[0081] Treatment methods The antibodies or antigen-binding fragments of the present disclosure are useful for various 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.

[0082] In one aspect, the present disclosure provides a method for treating cancer. In certain aspects, the method includes administering to a patient in need thereof an effective amount of an anti-TIGIT antibody or antigen-binding fragment. The cancer includes, 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.

[0083] The antibody or antigen-binding fragment of the invention can be administered by any suitable means, including, if desired in parenteral, intralung, intranasal, and local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. The dosing may be by any suitable route, such as by injection, for example, intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules (including, but not limited to, single or multiple administrations over various time points, bolus dosing, and pulse infusion) are contemplated herein.

[0084] The antibody or antigen-binding fragment of the invention is formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder to be treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site to which the agent is to be delivered, the method of administration, the dosing schedule, and other factors known to medical practitioners. The antibody, optionally but not necessarily, is formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will vary depending on the amount of antibody present in the formulation, the type of disease or treatment, and the other factors described above. These are generally used at the same dosage amounts, by the same route of administration as described herein, or at about 1 to 99% of the dosage amounts described herein, or at any dosage amount determined empirically / clinically to be appropriate, and by any route.

[0085] For the prevention or treatment of a disease, the appropriate dosage of the antibody or antigen-binding fragment of the present invention will vary depending on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapies, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient either once or over a series of treatments. Depending on the type and severity of the disease, an antibody in the range of about 1 μg / kg to 100 mg / kg can be an initial candidate dosage for administration to the patient, for example, by one or more individual administrations or by continuous infusion. A typical daily dosage can be in the range of about 1 μg / kg to 100 mg / kg or more, depending on the above factors. For repeated dosing over several days or longer, depending on the condition, the treatment will generally be continued until the desired suppression of the disease symptoms occurs. Such dosages can be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient receives about 2 to about 20 doses of the antibody, or for example, about 6 doses). A higher initial loading dose can be administered first, followed by one or more lower doses. However, other dosing regimens may also be useful. The progress of this treatment is easily monitored by conventional techniques and assays.

[0086] Combination therapy In one aspect, the TIGIT antibodies of the present disclosure can be used in combination with other therapeutic agents, such as anti-PD1 antibodies. Other therapeutic agents that can be used with the TIGIT antibodies of the present disclosure include chemotherapeutic agents (e.g., paclitaxel or paclitaxel formulations (e.g., Abraxane®), docetaxel, carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5-azacitidine, 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), multi-kinase inhibitors (e.g., MGCD265, RGB-286638), CD-20 targeting agents (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agents (e.g., alemtuzumab), prednisone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., oblimersen sodium), aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD-19 targeting 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., AMG102), EGEN-001, polo-like kinase 1 inhibitors (e.g., BI672), but are not limited thereto.

[0087] The TIGIT antibodies of the present disclosure can be used in combination with other therapeutic agents, such as anti-PD1 antibodies. Anti-PD1 antibodies include, but are not limited to, tislelizumab, pembrolizumab, and nivolumab. Tislelizumab is disclosed in U.S. Patent No. 8,735,553 and Table 2 below.

Table 2-1

Table 2-2

[0088] Pembrolizumab (formerly MK-3475), disclosed by Merck in U.S. Patent Nos. 8,354,509 and 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 is approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is being clinically studied for the treatment of head and neck squamous cell carcinoma (HNSCC) and refractory Hodgkin lymphoma (cHL). Nivolumab (disclosed by Bristol-Meyers Squibb) is a fully human IgG4-K monoclonal antibody. Nivolumab (clone 5C4) is disclosed in U.S. Patent No. 8,008,449 and WO2006 / 121168. Nivolumab is approved for the treatment of melanoma, lung cancer, kidney cancer, and Hodgkin lymphoma.

[0089] Pharmaceutical Compositions and Formulations Also provided are compositions comprising a pharmaceutical formulation comprising an anti-TIGIT antibody or antigen-binding fragment, or a polynucleotide encoding an anti-TIGIT antibody or antigen-binding fragment. In certain embodiments, the composition comprises one or more antibodies or antigen-binding fragments that bind to TIGIT, or one or more polynucleotides comprising a sequence encoding one or more antibodies or antigen-binding fragments that bind to TIGIT. These compositions may further comprise a suitable carrier, such as a pharmaceutically acceptable excipient, including buffers well known in the art.

[0090] The pharmaceutical formulations of the TIGIT antibodies or antigen-binding fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antigen-binding fragments having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, and they include buffering agents (such as phosphoric acid, citric acid, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl 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, and immunoglobulins), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, and lysine), monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, and dextrin, chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (such as Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited thereto.

[0091] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which includes a histidine-acetate buffer.

[0092] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include a semipermeable matrix of a solid hydrophobic polymer containing an antibody, which matrix is in the form of a shaped article, for example, a film or microcapsule.

[0093] Formulations used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through a sterile filtration membrane.

[0094] Pharmaceutical Compositions and Kits In some aspects, the disclosure provides a composition comprising an anti-TIGIT antibody described herein, formulated with at least one pharmaceutically acceptable excipient, for example, a pharmaceutically acceptable composition. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible. The excipient can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or topical administration (e.g., by injection or infusion).

[0095] The compositions herein can take a variety of forms. These include, for example, liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, liposomes, and liquid, semi-solid, and solid dosage forms such as suppositories. The appropriate form depends on the intended method of administration and therapeutic use. A typical appropriate composition is in the form of an injection solution and an infusion solution. One suitable method of administration is parenteral administration (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.

Examples

[0096] Example 1. Generation of Anti-TIGIT Monoclonal Antibodies Anti-TIGIT monoclonal antibodies (mAbs) were generated based on the conventional hybridoma fusion technology with some modifications (de St Groth and Sheidegger, 1980 J Immunol Methods 35:1, Mechetner, 2007 Methods Mol Biol 378:1). mAbs showing high binding activity in enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) were selected for further characterization.

[0097] Cloning and sequence analysis of TIGIT antibodies Based on the manufacturer's protocol, mouse hybridoma clones were harvested using the Ultrapure RNA kit (Catalog number 74104, QIAGEN, Germany) to prepare total cellular RNA. First-strand cDNA was synthesized using Invitrogen's cDNA synthesis kit (Catalog number 18080-051), and PCR amplification of the nucleotide sequences encoding the heavy-chain variable region (Vh) and kappa-chain variable region (Vk) of mouse mAbs was performed using a PCR kit (Catalog number CW0686, CWBio, Beijing, China). Oligo primers used for antibody cDNA cloning of Vh and Vk were synthesized by Invitrogen (Beijing, China) based on previously reported sequences (Brocks et al., 2001 Mol Med 7:461). Subsequently, the PCR products were subcloned into the pEASY-Blunt cloning vector (Catalog number C B101-02, TransGen, China) and sequenced by Genewiz (Beijing, China). The amino acid sequences of the Vh region and Vk region were deduced from the DNA sequence analysis results. Mu1217 was identified as a particular clone of interest.

[0098] Humanization of mouse anti-human TIGIT mAb mu1217 For the humanization of mu1217, the human germline IgG genes were searched for sequences sharing high homology with the cDNA sequence of the mu1217 variable region by comparison with the human immunoglobulin gene database in IMGT. Human IGVH and IGVκ genes that are present at high frequencies in the human antibody repertoire (Glanville et al., PNAS 106:20216-20221 2009) and have high homology with mu1217 were selected as templates for humanization.

[0099] Humanization was performed by CDR grafting (Methods in Molecular Biology, Vol 248:Antibody Engineering, Methods and Protocols, Humana Press). The humanized antibody (A1217) was designed as human IgG1 MF form using an in-house developed expression vector, and the sequence of this antibody is shown in Table 1.

[0100] Example 2. Binding Activity, Structure and Function of A1217 to TIGIT To better understand how the A1217 antibody shows high affinity for TIGIT, potently inhibits the TIGIT-PVR interaction, and can bind to TIGIT in a pH-dependent manner, especially while showing pharmacokinetic and molecular evaluation properties, the crystal structure of A1217 complexed with TIGIT was determined as described in detail below. Mutagenesis experiments at the TIGIT interface were also performed to identify functional epitope residues, particularly HIS76 of TIGIT for pH-dependent binding.

[0101] Expression, Purification, Crystallization of TIGIT and Fab Human TIGIT residues 23 - 128 with an N-terminal HIS tag were expressed as inclusion bodies in E. coli BL21(DE3)pLysS using the pET21a vector (Novagen). Site-directed mutagenesis of TIGIT was introduced by a QuickChange-based procedure (Xia et al., Nucleic Acids Res, 2015.43(2):p.e12) using Q5 DNA polymerase (New England Biolabs). Protein expression in the BL21(DE3) pLysS host strain was induced at 37 °C for 4 hours with 1 mM IPTG at an OD600 of 0.6 - 1.0. Cells were collected by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate (pH 7.0), 300 mM sodium chloride). Cells were lysed by sonication on ice. Inclusion bodies were collected by centrifugation (30 minutes at 20,000 rpm at 4 °C), dissolved in 8 M urea, 20 mM Tris (pH 8.0), 200 mM NaCl, 1 mM DTT, and stirred overnight. After removing the insoluble pellet by centrifugation (30 minutes at 20,000 rpm at 4 °C), the solubilized fraction was applied to a Ni-Penta™ affinity column (Marvelgent Biosciences Inc.) and washed with 10 column volumes of wash buffer (8 Mm urea, 20 mM Tris (pH 8.0), 200 mM NaCl, 5 mM imidazole). Next, the protein was eluted with elution buffer (8 M urea, 20 mM Tris (pH 8.0), 200 mM NaCl, 200 mM imidazole). The eluted protein was refolded by dialysis against a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 0.4 M L-arginine, 1 mM oxidized glutathione, 5 mM reduced glutathione, and further purified by gel filtration in buffer (20 mM Tris (pH 8.0), 100 mM NaCl) using a HiLoad 16 / 600 Superdex™ 75pg column (GE Healthcare Life Sciences). TIGIT mutants were purified in the same manner as the wild-type protein.

[0102] The DNA sequence of the Fab fragment of A1217 was synthesized by codon optimization in mammalian cells. The sequences of the heavy and light chains of the Fab were cloned into the pMAX vector, each with a C-terminal 6xHIS tag on the heavy chain. Plasmids containing 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 using TALON affinity resin (Clontech Laboratories) and subsequently further purified using a HiLoad 16 / 600 Superdex™ 75pg column (GE Healthcare Life Sciences). Similarly, plasmids of the full-length heavy and light chains of A1217 were transiently co-transfected into HEK293G cells for protein expression. The full-length antibody was purified using Mab Select SuRe™ affinity resin (GE Healthcare) and then further purified using a HiLoad 16 / 600 Superdex™ 200pg column (GE Healthcare Life Sciences).

[0103] The Fab of A1217 was concentrated to approximately 10 mg / ml with 20 mM Tris (pH 8.0) and 100 mM NaCl for initial crystallization screening. The A1217 Fab was incubated with a 1.5 molar excess of TIGIT on ice for 30 minutes and purified by a Superdex™ 75 Increase 10 / 300 GL column (GE Healthcare) in 20 mM Tris (pH 8.0) and 100 mM NaCl. The collected fractions were concentrated to approximately 10 mg / ml and used for the first crystallization screening. Crystals of A1217 Fab / TIGIT grew with 0.1 M citric acid (pH 4.6), 1 M lithium chloride, and 7% PEG6000. Crystals cryoprotected by stepwise addition of 5% glycerol to a final concentration of 20% were flash-frozen in liquid nitrogen. X-ray diffraction data were collected at the beamline BL45XU of Spring-8 (Japan Synchrotron Radiation Research Institute). For comparison, the tiragolumab Fab was generated in a similar manner.

[0104] Data collection and structure solution Diffraction data of the A1217 Fab / TIGIT and tirzepatide Fab / TIGIT complexes were collected using the automatic data collection system ZOO (Hirata et al., Acta Crystallogr D Struct Biol, 2019.75(Pt 2):p.138 - 150) at BL45XU of Spring-8 (Japan) and processed by KAMO (Yamashita et al., Acta Crystallographica Section D, Structural biology, 2018.74(Pt 5):p.441 - 449). For molecular replacement using PHASER (McCoy et al., J Appl Crystallogr, 2007.40(Pt 4):p.658 - 674), a rigid body search model from in-house solved Fab and TIGIT (PDB: 3UCR) was adopted. Structure refinement was performed by combining the REFMAC (Murshudov et al., Acta Crystallogr D Biol Crystallogr, 1997.53(Pt 3):p.240 - 55) and PHENIX (Adams et al., Acta Crystallogr D Biol Crystallogr, 2010.66(Pt 2):p. 213 - 21) programs with manual model building using COOT (Emsley et al., Acta Crystallogr D Biol Crystallogr, 2004.60(Pt 12 Pt 1):p.2126 - 32). Statistics of the crystal structure data are summarized in Table 3. All molecular graphics were created using PyMOL (Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.8.2015).

Table 3-1

Table 3-2

[0105] Example 3. Structure of A1217 Bound to Human TIGIT The A1217 Fab complexed with TIGIT crystallized in the P 2 21 21 space group, with four complexes in the asymmetric unit and diffracted to 2.45 Å. When the four individual complexes in the asymmetric unit were superimposed, only minor changes in the backbone conformation were observed between the individual copies. The structure of A1217 bound to human TIGIT (Figure 1A) shows that A1217 interacts sterically with PVR binding (Figure 1B). The buried surface area between A1217 and TIGIT is approximately 1556 Å 2 is. The epitope of TIGIT by A1217 consists of a number of discontinuous regions. 18 residues of A1217 Fab (paratope) and 15 residues of TIGIT (epitope) are involved in paratope-epitope formation (Figure 1C). The interactions at the A1217 / TIGIT interface are mainly non-polar, with a total of 11 hydrogen bonds and 3 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 bonds between TIGIT and A1217 are 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 LCDR33 side chain atoms of, and LEU65 TIGIT , LEU73 TIGIT , LYS53HCDR2 , GLY54 HCDR2 , GLY56 HCDR2 , ASN101 HCDR3 , TYR102 HCDR3 includes the backbone atoms of, while HIS76 TIGIT and ASP103 HCDR3 Two salt bridges are formed between and GLU60 TIGIT and LYS53 HCDR2 One salt bridge was found between and. Furthermore, only GLN64, ILE68, GLY74, TRP75, SER78, PRO79 among the residues of TIGIT are involved in van der Waals interactions, while the residues of A1217 involved in these interactions are SER57, TYR59, PHE104 of the heavy chain, and SER32, TYR49, TYR91 of the light chain.

[0106] The crystal structure of tirzolgomab was also generated in the same manner as above. In the epitope mapping of TIGIT by tirzolgomab Fab, many cleaved regions are shown. Thirteen residues of tirzolgomab Fab (paratope) and ten residues of TIGIT (epitope) are involved in paratope-epitope formation calculated at a cutoff distance of 3.7 Å. The paratope of tirzolgomab 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 tirzolgomab Fab are not directly involved in epitope-paratope interactions. The tirzolgomab epitope 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 bonds between TIGIT and tirzolgomab Fab are ASN58 TIGIT , GLU60 TIGIT , HIS76 TIGIT , SER80 TIGIT , LYS82 TIGIT , LYS58HCDR2 、TYR60 HCDR2 、ASP107 HCDR3 and THR100 LCDR3 's side-chain atoms, as well as PRO79 TIGIT 、ARG56 HCDR2 and THR100 LCDR3 are involved in the main-chain atoms of, while GLU60 TIGIT and LYS58 HCDR2 form two salt bridges between, and LYS82 TIGIT and ASP107 HCDR3 form two salt bridges between, and HIS76 TIGIT and ASP107 HCDR3 form one salt bridge between. Furthermore, the residues of TIGIT that are only involved in van der Waals interactions are GLN56, ILE77, SER78, and HIS111, and the residues of tiragolumab Fab that are involved 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, TYR60 of HCDR2 of tiragolumab Fab cover the exposed hydrophobic surface formed by THR55, GLN56, ASN58, GLU60, ASP63, GLN64, LEU65, ALA67, ILE68, ILE109, HIS111 of TIGIT. LEU109 of HCDR3 of tiragolumab Fab is inserted into the hydrophobic pocket formed by ALA67, ILE68, HIS76, ILE77, and SER78 of TIGIT. Comparative data on the interactions of A1217 (osperlimab), tiragolumab, and TIGIT are shown in FIGS. 2A - C and FIGS. 3A - B.

[0107] Based on the crystal structure of the A1217 / TIGIT complex, the residues of TIGIT that contact A1217 (i.e., the epitope residues of TIGIT to which A1217 binds) and the residues of A1217 that contact TIGIT (i.e., the paratopic residues of A1217 that contact TIGIT) were determined. Tables 4 and 5 below show the residues of TIGIT and the residues of the light or heavy chain of A1217 that they contact, which were evaluated using a contact distance strictness of 3.7 Å, which is the point at which van der Waals (non-polar) interaction forces are maximized.

Table 4

Table 5

[0108] Alanine scanning of the human TIGIT interface was also performed, and alanine mutations of TIGIT residues were created for LEU73 / HIS76, HIS76, ILE68, ASP63, LEU73, PRO79, LEU65, GLN56, ASN70, GLU60, LYS82, SER80, GLN64 (Table 6). The capture immobilization strategy in SPR was adopted to test the binding kinetics of A1217 to TIGIT variants. In this experiment, mutations of the TIGIT residues LEU73 / HIS76, HIS76, and ILE68 reduced the binding of A1217 by more than 10-fold (Table 6), and the mutation of HIS76 almost abolished the binding of A1217, suggesting that this residue is important for the interaction. The LEU73A / HIS76A double mutant completely inhibited A1217 binding, which indicates that LEU73 and HIS76 of TIGIT form several hydrogen bonds (H76 TIGIT -D103 HCDR3 、H76 TIGIT -T31 LCDR1 、L73 TIGIT -W50 LCDR2 ) and salt bridges (H76 TIGIT -D103 HCDR3) is consistent with the structural observation that the TRP50 of LCDR2 is sandwiched by strong hydrophobic interactions supplemented by (). The LEU73A mutation of TIGIT showed the same binding rate as the WT, but the dissociation rate was slightly faster, and the binding affinity for A1217 decreased by about 7-fold. Furthermore, the ILE68A mutation of TIGIT showed the same binding rate as the WT, but the dissociation rate was much faster than that of the WT, and the binding affinity with A1217 decreased by about 30-fold. From the structure of the complex, it was revealed that ILE68 of TIGIT is in the hydrophobic core of the IgV domain and interacts extensively with the HCDR3 of A1217. Furthermore, the ASP63A mutation indicated that the binding affinity with A1217 decreased by about 7-fold because ASP63 of TIGIT formed three hydrogen bonds with THR52, GLY54, and GLY56 of HCDR2 in A1217. Furthermore, the PRO79A mutation of TIGIT had a slower binding rate and a faster dissociation rate than the WT, so the binding affinity with A1217 decreased by about 3-fold. The complex structure showed that PRO79 of TIGIT formed strong hydrophobic interactions with PHE104 of HCDR3 and TYR91, SER92, and TYR94 of LCDR3 in A1217. Mutations of TIGIT at SER80 and GLN64 had no effect on the binding of A1217 (Table 6). This analysis was consistent with the crystal structure analysis, and it was found that the TIGIT residues that most affected the binding of A1217 interacted with A1217 within the structure.

[0109] Regarding the interaction with tiragolumab, the LYS82A mutant that formed extensive hydrophobic interactions with TYR31 / TYR38 / TYR98 of the light chain and a strong hydrogen bond / salt bridge with ASP107 of the heavy chain completely inhibited the binding to tiragolumab. The ILE68A mutant formed strong hydrophobic interactions with LEU108 and LEU109 of HCDR3 in tiragolumab. As a result, the binding affinity to tiragolumab decreased by about 40-fold (Table 6), and this site also affected the binding of A1217. The LEU65A mutation of TIGIT decreased the binding affinity to tiragolumab by about 25-fold, resulting in a slower binding rate and a faster dissociation rate compared to WT TIGIT. The HIS76A mutant slightly affected the binding of tiragolumab, but this mutation almost inhibited the binding of A1217. Similarly, the P79A / S80A single mutant that formed a hydrogen bond with THR100 had little effect on tiragolumab binding. The mutagenesis experiment showed that A1217 and tiragolumab have different epitopes when binding to TIGIT, suggesting that the antagonistic mechanisms of the two antibodies may be different.

Table 6-1

Table 6-2

[0110] A1217 has the property of binding to TIGIT in a pH-dependent manner Based on SPR data, it was found that when the pH decreased from 7.4 to 6.0, the binding affinity (KD) of A1217 to TIGIT increased by about 17-fold (Table 7). The amine coupling method was used to measure the binding affinity between A1217 and TIGIT under different pHs. In this complex structure, ASP103 HCDR3 was appropriately positioned at a distance of 2.7 Å from HIS76 TIGIT to form a strong electrostatic interaction (Figure 1D). In the tumor microenvironment, HIS76 TIGITThe imidazole ring is mainly protonated and carries a positive charge in an acidic environment, so the electrostatic interaction with ASP103 HCDR3 is strengthened. However, under physiological conditions, HIS76 TIGIT has fewer positive charges, weakening this interaction. In the A1217 / TIGIT complex structure, ASP103 HCDR3 is appropriately positioned at a distance of 2.7 Å from HIS76 TIGIT to form a strong electrostatic interaction. However, such a favorable structure surrounding HIS76 TIGIT was not observed in tirzolgomab / TIGIT. When the pH decreased from 7.4 to 6.0, the binding affinity (KD) of A1217 for TIGIT was found to increase by about 17-fold, while tirzolgomab did not show an obvious pH-dependent binding to TIGIT (Table 7). In conclusion, pH-sensitive antibodies may be superior to non-pH-sensitive antibodies in relaxing the balance between effectiveness against tumor cells and safety against normal cells.

Table 7

[0111] Example 4. Generation of Anti-TIGIT Antibodies with Reduced Fucosylation Removing core fucose from N-glycans attached to human IgG1 significantly enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) response (Shields, et al., (2002) J Biol Chem 277, 26733-26740; Shinkawa et al., (2003) J Biol Chem 278, 3466-3473). There are numerous approaches to reducing core fucosylation. In the study introduced herein, the approach of 2F-peracetylfucose, a fucosyltransferase (FUT) inhibitor, was utilized.

[0112] A1217 P5-2 3G11 is a stably transfected CHO-K1 cell line that expresses the A1217 antibody. A1217 P5-2 3G11 cells were cultured in shaker flasks and passaged every 3 - 4 days for maintenance. Regardless of the addition of 1.25% Glycosylation Adjust (Gal+)(Sigma-Aldrich, 14701C0), they were seeded in Hyclone ActiPro™ medium at 5x10 5 cells / mL. 2F-peracetylfucose at concentrations of 0, 50, 100, 150, 200 μM was added to the culture medium. Cell supernatants were collected on day 14 and filtered through a 0.2 μm filter for further analysis.

[0113] The A1217 antibody was purified by protein A chromatography capture step under platform conditions, and the glycan profile was analyzed by HILIC-UPLC oligosaccharides (Figure 4). As a result of glycan analysis, it was shown that the inhibitor decreased total fucosylation to 11% with 50 μM 2F-peracetylfucose and to less than 7% with 100, 150 or 200 μM 2F-peracetylfucose.

[0114] Example 5. Generation of anti-TIGIT effector variants Combinations of the Fc mutations S239D, I332E or S239D, I332E, A330L (EU number) were generated on a plasmid containing the DNA fragment encoding the heavy chain of A1217 according to the method provided by the Fast MultiSite Mutagenesis System™ (FM201-01, Transgenbiotech). Variant A1217DE contains the amino acid changes S239D, I332E (DE). Variant A1217DEL contains the amino acid changes S239D, I332E, A330L (DEL). Expi-CHO cells were transfected with a plasmid corresponding to the A1217 effector variant (A1217DE or A1217DEL) and cultured at 30 °C and 5% CO2 for 10 days. Subsequently, the supernatant was collected and the protein was purified using MabSelect SuRe™ (17543802, Cytiva).

[0115] Example 6. Binding Affinity of TIGIT Antibody to FcγR For the measurement of FcγRI binding, Protein A was bound to an activated CM5 biosensor chip (catalog number BR100530, GE Life Sciences), and 30 nM of A1217 or A1217 variant was flowed over the chip and captured by Protein A. A series of concentrations of FcγRI (0.0586 nM to 15 nM) were injected into the SPR running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4) at 30 μL / min. The binding response of A1217 or the mutant to FcγRI was calculated by subtracting RU from the reference flow cell without injecting A1217 or the mutant. For the determination of FcγRI binding affinity, kon and koff were calculated using a 1:1 Langmuir binding model, and KD was calculated as the ratio of koff / kon.

[0116] Anti-human kappa antibodies were conjugated to an activated CM5 biosensor chip (catalog number BR100530, GE Life Sciences), and A1217 or A1217 variants were flowed over the chip and captured on the anti-human kappa surface. Different concentrations of FcγR (FcγRIIA, FcγRIIB, FcγRIIIA) in a series were injected at 30 μL / min into the SPR running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4). The binding response of A1217 or the mutant to FcγR was calculated by subtracting RU from the reference flow cell without injecting A1217 or the mutant. For the determination of the binding affinities of FcγRIIA, FcγRIIB, and FcγRIIIA, all concentration data were fitted using the steady-state affinity model.

[0117] As shown in Table 8, compared to A1217, A1217AF had approximately a 10-fold increased binding affinity for FcγRIIIA-V158 and FcγRIIIA-F158. Compared to A1217, A1217DEL and A1217DE also increased the binding affinity for FcγRIIIA-V158 and FcγRIIIA-F158. Furthermore, A1217DEL and A1217DE increased the binding affinity for FcγRI and FcγRII.

Table 8

[0118] Example 7. Binding of a TIGIT antibody with reduced fucosylation to FcγRIII FcγRIIIA-V158 or FcγRIIIA-F158 overexpressing HEK293 cells were first incubated with A1217 or A1217AF and subsequently stained with secondary antibody, Alexa Fluor™ 647 anti-human IgG Fc (Biolegend, REF#409320). Cell samples were washed and fixed with 1% paraformaldehyde in DPBS. Immunofluorescence was detected using NovoCyte® flow cytometry (ACEA) and analyzed using Guava Soft™ 3.1.1 software. The results of this analysis are shown in FIGS. 5A - B. A1217AF increased binding to FcγRIIIA-V158 or FcγRIIIA-F158 overexpressing HEK293 cells compared to A1217, indicating enhanced Fc effector function of A1217AF compared to A1217 via both variants of FcγRIIIA.

[0119] Example 8. Binding of Anti-TIGIT Effector Variants to FcγRIII FcγRIIIA-V158 or FcγRIIIA-F158 overexpressing HEK293 cells were first incubated with A1217, A1217DE, or A1217DEL, and subsequently stained with a 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. Immunofluorescence was detected using Guava easyCyte™ 6HT (Merck-Millipore, USA) and analyzed using Guava Soft™ 3.1.1 software. The results are shown in FIGS. 6A-D, and A1217DE and A1217DEL increased binding to FcγRIIIA-V158 or FcγRIIIA-F158 overexpressing HEK293 cells compared to wild-type A1217, indicating that the Fc effector functions of A1217DE and A1217DEL were enhanced compared to A1217 via both variants of FcγRIIIA.

[0120] Example 9. Binding of anti-TIGIT effector variants to TIGIT TIGIT overexpressed in BW5147.3 cells was first incubated with A1217, A1217DE, or A1217DEL, and subsequently stained with a 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]. The cell samples were washed and fixed with 1% paraformaldehyde in DPBS. Immunofluorescence was detected using a Guava easyCyte™ 6HT (Merck-Millipore, USA) and analyzed using Guava Soft 3.1.1 software. This data is shown in Figure 7. In this result, A1217, A1217DE, and A1217DEL showed equivalent binding to TIGIT overexpressed in HEK293 cells, thus indicating that deletion does not change the TIGIT binding via FACS.

[0121] Example 10. Binding of anti-TIGIT antibodies with reduced fucosylation to complement C1q The C1q binding activity of A1217 or A1217AF was determined by sandwich ELISA. Briefly, serial dilutions of the designated A1217 or A1217AF variant were coated onto MaxiSorp immunoplates. C1q binding was tested by incubating human C1q in the antibody-coated wells. After washing, the bound C1q was detected with an anti-C1q monoclonal antibody and subsequently detected with an HRP-conjugated secondary antibody. The binding signal was measured by absorbance at 450 nm using a TMB (3,3′,5,5′-tetramethylbenzidine) substrate. The results are shown in Figure 8, and both A1217AF and A1217 showed equivalent binding to C1q.

[0122] Example 11. Anti-TIGIT antibodies and effector variants with reduced fucosylation enhanced ADCC 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 antibody bound to the target also binds to the FcγRIIIA receptor on the cell surface of effector cells such as NK cells, ADCC occurs due to cross-linking and activation of FcγRIIIA (Hogarth and Pietersz, (2012) Nat Rev Drug Discov 11, 311-331). The human FcγRIIIA gene shows polymorphism at the position encoding amino acid residue 158. The FcγRIIIA variant with valine at amino acid residue 158 (V158) has a high affinity for the IgG1 Fc portion, while the FcγRIIIA variant with phenylalanine (F158) has a low affinity for the IgG1 Fc portion. The killing of target cells by NK cells is usually used as a readout of ADCC activity in conventional ADCC assays. Due to the diversity of different donors and the polymorphism of FcγRIIIA, the responses of these cells can vary greatly. To use FcγRIIIA activation as an alternative readout for ADCC, FcγRIIIA activation is monitored in a more simplified and stable setting. Briefly, in the alternative ADCC assay, Jurkat cells were engineered to stably express the FcγRIIIA-V158 or FcγRIIIA-F158 variant, and a NFAT response element that promotes the expression of firefly luciferase (NFAT reporter luciferase). The resulting Jurkat / NFAT reporter luciferase / FcγRIIIA-V158 cells or Jurkat / NFAT reporter luciferase / FcγRIIIA-F158 cells were used as effector cells. BW5147.3 cells engineered to stably express TIGIT (BW5147.3 / TIGIT cells) were used as target cells. The biological activity of the antibody in the alternative ADCC assay is quantified through luciferase as a result of activation of the NFAT pathway. The luciferase activity in the effector cells is quantified by luminescence readings.

[0123] (A) Jurkat / NFAT reporter luciferase / FcγRIIIA-V158 cells (1x10 4 cells / well) were co-cultured overnight with BW5147.3 / TIGIT cells (1x10 4 cells / well). NFAT reporter activity was detected using the One-Step™ Luciferase Assay System from BPS Bioscience.

[0124] This experiment demonstrated that A1217AF, A1217DEL, and A1217DE had enhanced ADCC activity compared to A1217 wild-type (Figure 9A).

[0125] (B) Jurkat / NFAT reporter luciferase / FcγRIIIA-F158 cells (5x10 4 cells / well) were co-cultured overnight with BW5147.3 / TIGIT cells (1x10 4 cells / well). NFAT reporter activity was detected using the One-Glo™ Luciferase Assay System (Promega). A1217AF, A1217DEL, and A1217DE had enhanced ADCC activity compared to A1217 wild-type (Figure 9B).

[0126] Example 12. Anti-TIGIT decreased fucosylation and effector variants enhanced ADCC in Tregs

[0127] TIGIT + Tregs represent functionally distinct Treg subsets that are highly immunosuppressive (Joller et al., (2014) Immunity 40, 569-581). The expression of TIGIT is higher in intratumoral Tregs than in effector T cells, and also higher in Tregs derived from PBMCs of cancer patients than in Tregs derived from PBMCs of healthy donors (Preillon et al., (2021) Mol Cancer Ther 20, 121-131.). Since the Fc-functional competent TIGIT antibody A1217 binds to TIGIT +It is reasonable to consider that ADCC can be induced by Treg cells.

[0128] To measure the ADCC activity induced by A1217 or A1217AF against T cells, especially Treg, PBMCs from lung cancer patients were used as target cells. NK cells isolated from PBMCs of healthy donors were used as effector cells. A1217MF contains a silent Fc and reduces the amount of ADCC (SEQ ID NO: 12). Anti-TIGIT antibodies A1217, A1217AF or A1217MF were incubated overnight in a 96-well plate with target cells (5x10 4 cells / well) and NK effector cells (5x10 4 cells / well, purified using NK cell isolation kit, Miltenyi Biotec, catalog number 130-092-657). The cell samples were subjected to flow cytometry analysis. The results, as shown in FIGS. 10A-C, were that A1217AF enhanced the ADCC activity against Treg compared to A1217 wild type. As shown in FIG. 10A, the Treg frequency in CD3 + T cells was significantly decreased in a dose-dependent manner by treatment with A1217 or A1217 AF, but the ratio hardly changed in the A1217MF treatment group. Compared to A1217 wild type, A1217AF induced significantly higher ADCC against Treg. On the other hand, in the treatment with any of A1217 wild type, A1217MF, and A1217AF, the frequency of effector CD4 + T cells or CD8 + T cells was not changed (FIGS. 10B and 10C). The results clearly show that A1217AF can induce more potent ADCC against Treg cells in PBMCs derived from cancer patients compared to A1217 wild type. The enhanced Fc function can enhance the activity of TIGIT antibodies in the anti-tumor immune response through the reduction of Treg.

[0129] Example 13. Anti-TIGIT antibodies with reduced fucosylation activate NK cells 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 U S A 106, 17858-17863, Wang et al., (2015) Eur J Immunol 45, 2886-2897). The major FcγR expressed on NK cells is FcγRIIIA (Bruhns, (2012) Blood 119, 5640-5649). The co-ligation of TIGIT and FcγRIIIA in NK cells may converge downstream and lead to a stronger signal. To determine whether FcγR co-engagement ability is required for optimal NK cell activation by TIGIT antibodies 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) that expresses a high level of PVR in the presence of TIGIT antibodies of different Fc forms. Activation of NK cells was determined by measuring the NK cell degranulation marker CD107a by flow cytometry. The results showed that A1217AF had higher NK cell activation compared to A1217 wild type or A1217MF (Figures 11A-B).

[0130] 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 PBMC of healthy donors (5x10 4It was added overnight to the co-culture of (cells / wells). Before the co-culture assay, NK cells were pre-stimulated overnight with 25 U / mL of recombinant human IL-2 (Novoprotein, China, catalog number: C013). CD107a expression on NK cells was determined by FACS. In A1217AF, NK activation was significantly increased compared to wild-type A1217, suggesting that enhanced FcγR co-binding due to afucosylation of A1217 induces optimal NK cell activation in the presence of tumor cells.

[0131] Example 14. Trogsitosis Characteristics of a Reduced Fucosylated Anti-TIGIT Antibody Trogsitosis is the process by which cell surface molecules are transferred from donor cells to acceptor 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 trogsitosis via Fcγ receptors (FcγR) causes downregulation of cell surface receptors (Taylor and Lindorfer, (2015) Blood 125, 762 - 766). Downregulation of target receptors by trogsitosis can cause attenuation of signaling. To investigate whether a TIGIT antibody with Fc function can induce trogsitosis via FcγR to remove TIGIT from the cell surface and whether enhanced binding of Fc to FcγR due to DE / DEL mutations or reduced fucosylation can further promote trogsitosis, a trogsitosis assay was performed.

[0132] Jurkat / TIGIT / DNAM-1 cells (2x10 4 cells / well) were used as donor cells, and CFSE (Invitrogen, catalog number: C34554) - labeled HEK293 cells (4x10 4The (cells / well) were used as acceptor cells. Donor cells were pre-incubated with 10 μg / mL of CF633-labeled A1217 wild-type, A1217AF, or A1217MF for 30 minutes and washed. Then, the donor cells were incubated overnight with acceptor cells in a 96-well plate. The change in the mean fluorescence intensity (MFI) of TIGIT (CF633) on the donor cells was measured by FACS.

[0133] As shown in Figure 12, compared with A1217 wild-type, A1217AF significantly induced downregulation of TIGIT on Jurkat / TIGIT / DNAM-1 cells when the acceptor cells expressed FcγRIIIA-F158 and FcγRIIIA-V158, indicating that the Fc enhancement of A1217 by afucosylation can induce maximal downregulation of TIGIT via FcγRIIIA-binding-dependent trogocytosis. Interestingly, when the acceptor cells expressed FcγRI, A1217AF induced trogocytosis to the same extent as A1217 wild-type, which is consistent with the comparable binding affinity of A1217AF and A1217 for FcγRI.

[0134] Example 15. Trogocytosis characteristics of anti-TIGIT antibody mutants Jurkat / TIGIT / DNAM-1 (2x10 4 cells / well) cells were used as donor cells, and CFSE (Invitrogen, catalog number: C34554)-labeled HEK293 cells (4x10 4 cells / well) expressing different FcγRs were used as acceptor cells. Donor cells were pre-incubated with 10 μg / mL of CF633-labeled A1217 wild-type, A1217DE, A1217DEL, or A1217MF for 30 minutes and washed. Then, the donor cells were incubated overnight with acceptor cells in a 96-well plate. The change in the MFI of TIGIT (CF633) on the donor cells was measured by FACS.

[0135] The results of A1217DE and DEL effector variants were similar to those of A1217AF with reduced fucosylation. In cells expressing FcγRIIIA-F158 or FcγRIIIA-V158, trogocytosis of A1217DE and A1217DEL was greater compared to A1217 wild type. Fc-silent A1217MF was mostly or completely inactive as expected, and this data is shown graphically in Figure 13.

[0136] Example 16. A1217 antibody with reduced fucosylation in combination with an anti-PD1 antibody in a mouse tumor model In this experiment, 2x10 5 Renca cells were subcutaneously implanted into the right flank of humanized TIGIT mice on a BABL / c background. Renca cells are a mouse renal adenocarcinoma model. Next, mice were administered PBS as a control, 3 mg / kg of mPD-1 Ab, 10 mg / kg of A1217 wild type, or 10 mg / kg of A1217AF as monotherapy. In combination therapy, mice were administered 3 mg / kg of mPD-1 antibody and 10 mg / kg of A1217 wild type, or 3 mg / kg of mPD-1 Ab and 10 mg / kg of A1217AF. This treatment was administered intraperitoneally 5 days after tumor cell inoculation. Tumor volume was measured twice a week using the following formula, V = 0.5(a x b 2 ), where a and b are the major and minor diameters of the tumor, respectively. *p < 0.05.

[0137] This data is shown graphically in Figure 14. When comparing A1217 wild type and A1217AF, there was little difference when administered as monotherapy. However, when the A1217AF antibody was administered in combination with an anti-PD1 antibody, tumor shrinkage was improved compared to A1217 wild type administered in combination with the anti-PD1 antibody.

[0138] Example 17. Combination of A1217 antibody with reduced fucosylation and anti-PD1 antibody induces reduction of Tregs in a mouse tumor model To investigate whether the combination of the A1217AF antibody with reduced fucosylation and the anti-PD-1 antibody reduces Treg cells in the Renca model, 0.3 million RENCA tumor cells were subcutaneously implanted into the right flanks of female hTIGIT Balb / c mice (6 - 8 weeks old) on day 0, and when the average tumor volume reached 100 - 200 cm 3 ³, they were randomly divided into 8 groups according to body weight and tumor volume. The mice were treated with the anti-TIGIT antibody A1217 (10 mg / kg) with different Fc forms as a single agent, the mouse PD-1 inhibitory antibody Ch15mt (3 mg / kg) as a single agent, or a combination of the two antibodies at the indicated doses. Tumor samples were collected 48 hours after administration for isolation and FACS analysis of tumor-infiltrating lymphocytes (TIL). Treg cells were gated on CD45 + CD4 + FOXP3 + As shown in Figures 15A - C, there was no significant difference when comparing A1217 wild-type with A1217AF when administered as a single agent. However, when administered in combination with the anti-PD1 antibody, the A1217AF antibody significantly reduced intratumoral Treg compared to A1217 wild-type administered in combination with the anti-PD1 antibody, indicating that the afucosylation of A1217 can induce a more significant anti-tumor effect in combination with the PD-1 antibody by reducing Treg cells in the tumor microenvironment.

Claims

**Claim 1** A method for treating cancer, the method comprising administering to a subject an effective amount of a pH-dependent anti-TIGIT antibody or an antigen-binding fragment thereof. **Claim 2** The method according to claim 1, wherein the anti-TIGIT antibody binds to the TIGIT protein at amino acid histidine 76. **Claim 3** The method according to claim 2, wherein the anti-TIGIT antibody binds to the TIGIT protein at histidine 76 and leucine 73. **Claim 4** The method according to claim 3, wherein the method comprises administering to a subject an effective amount of a pH-dependent antibody or an antigen-binding fragment thereof that 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. **Claim 5** The method according to claim 4, wherein the anti-TIGIT antibody or an 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. **Claim 6** The method according to claim 1, wherein the anti-TIGIT antibody enhances antibody-dependent cell-mediated cytotoxicity (ADCC) activity. **Claim 7** The method according to claim 6, wherein the anti-TIGIT antibody has reduced fucosylation. **Claim 8** The method according to claim 6, wherein the anti-TIGIT antibody has changes in Fc amino acids at S239D and I332E (EU number). **Claim 9** The method according to claim 6, wherein the anti-TIGIT antibody has changes in Fc amino acids at S239D, I332E, and A330L (EU number). **Claim 10** The method according to any one of claims 7 to 9, wherein the anti-TIGIT antibody has increased binding affinity for FcγRIIIA-V158 and FcγRIIIA-F158. **Claim 11** The method according to any one of claims 7 to 9, wherein the anti-TIGIT antibody enhances ADCC in regulatory T (Treg) cells. **Claim 12** The method according to any one of claims 7 to 9, wherein the anti-TIGIT antibody activates natural killer (NK) cells. **Claim 13** The method according to any one of claims 7 to 9, wherein the anti-TIGIT antibody has increased trogocytosis. **Claim 14** The method further comprises administering an anti-PD1 antibody comprising a heavy chain variable region that specifically binds to human PD1 and comprises 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 that comprises LCDR1 of SEQ ID NO: 18, LCDR2 of SEQ ID NO: 19, and LCDR3 of SEQ ID NO: 20, the method according to claim 1.

15. The anti-PD1 antibody or an 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, the method according to claim 14.

16. The anti-PD1 antibody comprises an IgG4 constant domain comprising SEQ ID NO: 23, the method according to claim 14 or 15.

17. The cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, gastric 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, the method according to claim 1.

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

19. The method according to claim 17, wherein the head and neck cancer is hypopharyngeal cancer.

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

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

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

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

24. The method according to claim 17, wherein the cancer is kidney cancer.

25. The method according to claim 14, further comprising administering chemotherapy.

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

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

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