Anti-TIGIT antibodies and uses thereof

Abstract

The present invention provides anti-TIGIT antibodies and fragments thereof. These antibodies and fragments thereof specifically bind to TIGIT protein. Also provided are methods for using these antibodies or fragments thereof to treat and diagnose diseases such as cancer and viral infection.

Description

Background Art

[0001] The T cell immunoreceptor with Ig and ITIM domains (TIGIT) protein is a member of the PVR (poliovirus receptor) family of immunoglobulins. It is expressed on several classes of T cells, including follicular B helper T cells (TFH). TIGIT has been shown to bind PVR with high affinity, and this binding is thought to facilitate the interaction between TFH and dendritic cells to regulate T cell-dependent B cell responses.

[0002] TIGIT is a member of the immunoglobulin superfamily that possesses an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail and is expressed on subsets of activated T cells and natural killer (NK) cells. TIGIT is known to interact with CD155 (also known as PVR and necl-5), CD112 (also known as PVRL2 and nectin-2), and possibly CD113 (also known as PVRL3 and nectin-3). Binding of TIGIT to its high-affinity ligand CD155 expressed on antigen-presenting cells has been reported to inhibit T cell and NK cell function. TIGIT has also been reported to indirectly inhibit T cells by regulating cytokine production by dendritic cells.

[0003] Tumors constitute a highly suppressive microenvironment in which infiltrating T cells are exhausted and NK cells are silenced by checkpoint molecules such as PD-1 and TIGIT to evade immune responses. It has been reported that high levels of TIGIT expression on CD8+T cells are associated with poor clinical outcomes in AML subjects. It has been reported that the functional defects of exhausted TIGIT+CD8+T cells from AML subjects were reversed by siRNA-mediated knockdown of TIGIT expression. It has also been reported that effector CD8+T cells exhibit higher levels of TIGIT during HIV infection in the blood and SIV infection in lymphoid tissues.

[0004] TIGIT is also involved in viral infection. During human immunodeficiency virus (HIV) infection, CD8+T cells expressing TIGIT have been shown to amplify in HIV-infected individuals of different groups and are associated with clinical markers of HIV disease progression. Even in those HIV-infected individuals with undetectable viral load, elevated TIGIT levels persist, and most HIV-specific CD8+T cells simultaneously express both TIGIT and another negative checkpoint receptor, programmed death protein 1 (PD-1), and retain several features of exhausted T cells. Blocking these pathways with targeted monoclonal antibodies can restore HIV-specific CD8+T cell responses. This pathway can be potentially targeted during the "shock and kill" HIV treatment method to enhance the killing of HIV-infected cells. Summary of the invention

[0005] The present disclosure provides antibodies and antigen-binding fragments specific for human TIGIT protein, which can be used to treat various cancers and other diseases such as viral infections.

[0006] One embodiment of the present disclosure provides an antibody or a fragment thereof, wherein the antibody or fragment thereof is specific to a T cell immunoreceptor (TIGIT) protein having Ig and ITIM domains, and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO:1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO:3, and the light chain variable region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO:4, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:5, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:6.

[0007] In some embodiments, VH comprises an amino acid sequence selected from SEQ ID NOs: 9-14, and VL comprises an amino acid sequence selected from SEQ ID NOs: 15-18. In some embodiments, VH comprises an amino acid sequence of SEQ ID NO: 11, and VL comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, VH comprises an amino acid sequence of SEQ ID NO: 10, and VL comprises an amino acid sequence of SEQ ID NO: 16.

[0008] In some embodiments, the antibody or fragment thereof has antibody-dependent cellular cytotoxicity (ADCC) capability.

[0009] Methods are also provided. In one embodiment, a method for treating cancer in a patient in need is provided, the method comprising administering an antibody or fragment thereof disclosed herein to the patient. In some embodiments, the cancer is selected from bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer and thyroid cancer. In some embodiments, the method of treatment or use also includes a second agent, such as an immune checkpoint inhibitor (e.g., an antibody or antigen binding fragment specific to PD-1, PD-L1, CTLA-4, LAG-3, CD28, CD122, 4-1BB or BTLA).

[0010] In another embodiment, a method of treating or inhibiting an infection in a patient in need thereof is provided, the method comprising administering to the patient an antibody or fragment thereof of the present disclosure. In some embodiments, the infection is a viral, bacterial, fungal or parasitic infection. In some embodiments, the infection is an HIV infection.

[0011] Further, one embodiment provides a method of treating cancer in a patient in need thereof, comprising: (a) treating T cells in vitro with an antibody or fragment thereof disclosed herein; and (b) administering the treated T cells to the patient. In some embodiments, the method further comprises, prior to step (a), isolating the T cells from the individual. In some embodiments, the T cells are tumor infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof.

[0012] In one embodiment, a method for detecting expression of TIGIT in a sample is also provided, the method comprising contacting the sample with an antibody or fragment thereof of the present disclosure under conditions such that the antibody or fragment thereof binds to TIGIT, and detecting binding indicative of expression of TIGIT in the sample.

[0013] In one embodiment, there is further provided a method for identifying a patient suitable for treatment with an anti-TIGIT therapy, comprising isolating cells from a cancer patient and detecting the presence of a TIGIT protein using an antibody or fragment thereof disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Elisa EC for antibody 76D12B10 binding to human TIGIT protein is shown. 50 .

[0015] Figure 2 It was shown that the 76D12B10 antibody can bind to cynomolgus monkey TIGIT with high affinity, but cannot bind to rat or mouse TIGIT.

[0016] Figure 3 The binding kinetics of 76D12B10 to recombinant TIGIT are shown.

[0017] Figure 4 It was shown that all humanized antibodies tested had comparable binding efficacy to human TIGIT compared to the chimeric antibody.

[0018] Figure 5 It was shown that Hu02 antibody and Hu06 antibody had comparable binding efficacy to human TIGIT as the chimeric antibody.

[0019] Figure 6 The binding kinetics of Hu02 antibody and Hu06 antibody to recombinant TIGIT are shown.

[0020] Figure 7 It was shown that Hu02 antibody and Hu06 antibody could block the interaction between TIGIT and CD155 in a dose-dependent manner.

[0021] Figure 8 It was shown that Hu02 antibody and Hu06 antibody dose-dependently inhibited the binding of CD155 to its receptor TIGIT by cell-based functional assay.

[0022] Fig. 9 It was shown that Hu02 antibody has higher efficacy than Tiragolumab in the MC38 syngeneic mouse model.

[0023] Fig.10 It was shown that LP010-02 had comparable binding efficacy to that of tisleliumab on human TIGIT protein.

[0024] Fig.11 It was shown that LP010-02 and tirilimumab antibodies can effectively inhibit the binding of human TIGIT protein to human CD155.

[0025] Fig.12 It was shown that LP010-02 had better binding efficacy to cells overexpressing human TIGIT protein compared to tirilimumab.

[0026] Fig.13 It was shown that the LP010-02 antibody blocked the interaction of TIGIT and CD155 with higher activity than tisleliumab and 22G2.

[0027] Fig.14 It was shown that in vivo in the CT26 syngeneic mouse model, the combination of LP010-02 and HX008 had improved efficacy compared to each antibody alone.

[0028] Fig.15 It was shown that in vitro, LP010-02 had higher ADCC activity than tisleliumab.

[0029] Fig.16 It was shown that in vitro, LP010-02 had higher CDC activity than tisleliumab. DETAILED DESCRIPTION

[0030] definition

[0031] It should be noted that the term "a" or "an" entity refers to one or more entities in that entity; for example, "antibody" should be understood to represent one or more antibodies. Therefore, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0032] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Such examples include, but are not limited to, a heavy or light chain complementary determining region (CDR) or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein.

[0033] As used herein, the term "antibody fragment" or "antigen binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0034] As used herein, the term "antibody" includes polypeptides of various major classes that can be distinguished biochemically. It will be appreciated by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ or ε), with some subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, which is IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgG5, etc. have been fully characterized and are known to confer functional specialization. In view of the present disclosure, modified forms of each of these classes and isotypes are easily discernible to those skilled in the art and are therefore within the scope of the present disclosure. All immunoglobulin classes are obviously within the scope of the present disclosure, and the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight of approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight of approximately 53,000-70,000 Daltons. The four chains are usually connected by disulfide bonds in a "Y" configuration, with the light chains surrounding the heavy chains, starting at the mouth of the "Y" and continuing through the variable region.

[0035] The antibodies, antigen-binding polypeptides, variants or derivatives thereof disclosed herein include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv)), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising VK or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

[0036] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be combined with a kappa or lambda light chain. Typically, the light and heavy chains are covalently bound to each other, and when the immunoglobulin is produced by a hybridoma, a B cell, or a genetically engineered host cell, the "tail" portions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.

[0037] Both light and heavy chains are divided into structural and functional homologous regions. The terms "constant" and "variable" are used functionally. In this regard, it should be understood that the variable domains of the light chain (VK) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CK) and heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of the constant region domains increases as they are farther and farther away from the antigen binding site or the amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CK domains actually contain the carboxyl termini of the heavy and light chains, respectively.

[0038] As shown above, the variable region allows the antibody to selectively recognize and specifically bind to the epitope on the antigen. That is, the VK domain and the VH domain or the subset of the complementary determining region (CDR) of the antibody are combined to form a variable region that defines a three-dimensional antigen binding site. The four-level antibody structure forms an antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each chain in the VH chain and the VK chain. In some cases, for example, certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, complete immunoglobulin molecules can be composed only of heavy chains, without light chains. See, for example, Hamers-Casterman et al., Nature, Vol. 363: pp. 446-448 (1993).

[0039] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, discontinuous amino acid sequences that are specifically positioned to form an antigen binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen binding domain (referred to as "framework" regions) show less intermolecular variability. The framework region primarily adopts a -folded conformation, and the CDRs form loops that connect the -folded conformation and in some cases form a portion of the -folded conformation. Therefore, the framework region serves to form a scaffold that positions the CDRs in the correct orientation by interchain non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface that is complementary to an epitope on an immunoreactive antigen. The complementary surface promotes the non-covalent binding of an antibody to its cognate epitope. For any given heavy or light chain variable region, one of ordinary skill in the art can readily identify the amino acids comprising the CDRs and framework regions, respectively, as they have been precisely defined (see "Sequences of Proteins of Immunological Interest", Kabat, E et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., Vol. 196: pp. 901-917 (1987)).

[0040] In the case where there are two or more definitions of terms used and / or accepted in the art, unless there is a clear contrary statement, the definition of the term as used herein is intended to include all such meanings. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the discontinuous antigen binding sites found in the variable regions of both heavy and light chain polypeptides. This particular region has been described in Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J.MoI.Biol. Vol. 196: pp. 901-917 (1987), which are incorporated herein by reference in their entirety. The definition of CDR according to Kabat and Chothia includes overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDR of an antibody or its variants is intended to fall within the scope of the term as defined and used herein. The appropriate amino acid residues comprising the CDR defined by each of the above-mentioned references are listed in the table below as a comparison. The exact number of residues comprising a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

[0041] Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

[0042] Kabat et al. also defined a numbering system applicable to the variable domain sequence of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0043] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 starts at the fifteenth residue after the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 starts at about the thirty-third amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 starts at about residue 24 (i.e., after the cysteine ​​residue); includes about 10-17 residues; and ends at the next tryptophan residue. CDR-L2 starts at about the sixteenth residue after the end of CDR-L1, and includes about 7 residues. CDR-L3 begins at approximately the thirty-third residue after the end of CDR-L2 (ie, after the cysteine ​​residue); includes approximately 7-11 residues, and ends at the sequence F or WGXG, where X is any amino acid.

[0044] The antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In some embodiments, the variable region may be of condricthoid origin (e.g., from sharks).

[0045] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. The polypeptide comprising the heavy chain constant region includes at least one of the following: a CH1 domain, a hinge (e.g., an upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, the antigen-binding polypeptide used in the present disclosure may include a polypeptide chain containing a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the present disclosure includes a polypeptide chain containing a CH3 domain. In addition, the antibody used in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As noted above, those skilled in the art will appreciate that the heavy chain constant regions may be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0046] The heavy chain constant region of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may include a hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the heavy chain portion may include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0047] As used herein, the term "light chain constant region" includes an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

[0048] A "light chain-heavy chain pair" refers to a collection of light chains and heavy chains that can form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0049] As indicated above, the subunit structures and three-dimensional configurations of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0050] As used herein, the term "CH2 domain" includes the portion of the heavy chain molecule extending from about residue 244 to residue 360 ​​of, for example, an antibody using a conventional numbering scheme (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The uniqueness of the CH2 domain is that it is not tightly paired with another domain. Instead, two N-linked branched sugar chains are inserted between the two CH2 domains of a complete natural IgG molecule. It has also been well documented that the CH3 domain extends from the CH2 domain of the IgG molecule to the C-terminus and comprises approximately 108 residues.

[0051] As used herein, the term "hinge region" includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three different domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol, Vol. 161: p. 4083 (1998)).

[0052] As used herein, the term "disulfide bond" includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 region and the CK region are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

[0053] As used herein, the term "chimeric antibody" will be taken to refer to any antibody in which the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be complete, partial or modified, according to the present disclosure) is obtained from a second species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate), and the constant region is human.

[0054] As used herein, "percent humanization" is calculated by determining the number of framework amino acid differences (ie, non-CDR differences) between the humanized domain and the germline domain, subtracting that number from the total number of amino acids, then dividing by the total number of amino acids and multiplying by 100.

[0055] "Specific binding" or "having specificity" generally refers to an antibody binding to an epitope through its antigen binding domain, and that binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" an epitope when it binds to an epitope through its antigen binding domain more easily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which an antibody binds to an epitope. For example, antibody "A" may be considered to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be considered to have a higher binding specificity for epitope "C" than for a related epitope "D."

[0056] As used herein, the term "treatment" refers to both therapeutic treatment and preventive or defensive measures, wherein the goal is to prevent or slow down (mitigate) undesirable physiological changes or diseases, such as cancer progression. Beneficial or desired clinical results include, but are not limited to, the alleviation of detectable or undetectable symptoms, the weakening of the extent of the disease, the stable (i.e., non-deterioration) state of the disease, the delay or slowing of disease progression, the improvement or alleviation of the disease state, and (partial or complete) relief. "Treatment" may also refer to a prolonged survival period compared to the expected survival period when not receiving treatment. Individuals in need of treatment include individuals who have already suffered from a disorder or disease and individuals who are susceptible to a disorder or disease or individuals who want to prevent a disorder or disease.

[0057] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, domesticated animals, farm animals, and zoo, sport or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0058] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects (including human subjects), who would benefit from administration of an antibody or composition of the present disclosure for, e.g., detection, diagnostic procedures and / or therapy.

[0059] Anti-TIGIT antibodies

[0060] Similar to PD-1 and PD-L1, the T cell immune receptor with Ig and ITIM domains (TIGIT) is frequently overexpressed on immune cells in cancer patients. TIGIT is considered an immune checkpoint, and its blockade can lead to increased cell proliferation, cytokine production, and degranulation of TA-specific CD8+ T cells and TIL CD8+ T cells. Targeting TIGIT is also thought to enhance the killing of HIV-infected cells.

[0061] Antibodies targeting TIGIT are being developed and show some clinical promise. For example, Bristol-Myers Squibb has disclosed the anti-TIGIT antibody 22G2 (see, e.g., U.S. Patent Application Publication No.: US20160176963), which was used as a control in the experimental examples. Another reference antibody used for comparison is tirilimumab (RG6058), a fully human monoclonal antibody developed by Roche for the treatment of non-small cell lung cancer. However, no molecules targeting TIGIT have yet been approved.

[0062] The present disclosure provides anti-TIGIT antibodies with high affinity and inhibitory activity for human TIGIT protein. Importantly, as demonstrated in Examples 7-8, the newly disclosed anti-TIGIT antibodies are superior to the control antibody tirilimumab. Like tirilimumab, the antibodies of the present invention have effective binding affinity. However, unexpectedly, the antibodies of the present invention exhibit significantly higher activity than 22G2 and tirilimumab in inhibiting the interaction between TIGIT and CD155 (see, Example 8, Fig.13 In addition, the antibodies of the present invention exhibit higher antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) than tisleliumab (see Examples 10-11, Figure 15-16 ). Therefore, the antibody of the present invention has more excellent properties than the antibodies that have been developed.

[0063] Therefore, in some embodiments, the present disclosure provides an anti-TIGIT antibody or fragment thereof having binding specificity to a human TIGIT protein. In some embodiments, the antibody or fragment thereof comprises (a) a VH CDR1 comprising SEQ ID NO: 1; (b) a VH CDR2 comprising SEQ ID NO: 2; (c) a VH CDR3 comprising SEQ ID NO: 3; (d) a VL CDR1 comprising SEQ ID NO: 4; (e) a VL CDR2 comprising SEQ ID NO: 5; and (f) a VL CDR3 comprising SEQ ID NO: 6. In some embodiments, the anti-TIGIT antibody or fragment thereof comprises: (a) a VH CDR1 of SEQ ID NO: 1, or a variant of SEQ ID NO: 1 having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1; (b) a VH CDR2 of SEQ ID NO: 2, or a variant of SEQ ID NO: 2 having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 2; (c) a VH CDR3 of SEQ ID NO: 3, or a variant of SEQ ID NO: 3 having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 3; (d) a VL CDR1 of SEQ ID NO: 4, or a variant of SEQ ID NO: 4 having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 4. NO:4 a variant of SEQ ID NO:4 having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity; (e) VL CDR2 of SEQ ID NO:5; and (f) VLCDR3 of SEQ ID NO:6, or a variant of SEQ ID NO:6 having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:6.

[0064] In some embodiments, the anti-TIGIT antibody or fragment thereof comprises: (a) a VH CDR1 of SEQ ID NO:1, or a variant of SEQ ID NO:1 having a single substitution, deletion, or insertion relative to SEQ ID NO:1; (b) a VH CDR2 of SEQ ID NO:2, or a variant of SEQ ID NO:2 having a single substitution, deletion, or insertion relative to SEQ ID NO:2; (c) a VH CDR3 of SEQ ID NO:3, or a variant of SEQ ID NO:3 having a single substitution, deletion, or insertion relative to SEQ ID NO:3; (d) a VL CDR1 of SEQ ID NO:4, or a variant of SEQ ID NO:4 having a single substitution, deletion, or insertion relative to SEQ ID NO:4; (e) a VL CDR2 of SEQ ID NO:5, or a variant of SEQ ID NO:5 having a single substitution, deletion, or insertion relative to SEQ ID NO:5; and (f) a VL CDR3 of SEQ ID NO:6, or a variant of SEQ ID NO:6 having a single substitution, deletion, or insertion relative to SEQ ID NO:6.

[0065] In some embodiments, the substitutions disclosed herein are conservative substitutions.

[0066] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced by another amino acid residue from the same side chain family. In another embodiment, an amino acid string may be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0067] Non-limiting examples of conservative amino acid substitutions are provided in Tables AB, where a similarity score of 0 or higher (see Table A) indicates a conservative substitution between two amino acids.

[0068] Table A. Amino acid similarity matrix

[0069] C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0070] Table B. Conservative amino acid substitutions

[0071]

[0072] Examples of antibodies and fragments include those having a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 7 and 9-14, or a peptide having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NOs: 7 and 9-14, while retaining the CDRs. Examples of such antibodies and fragments include those having a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 8 and 15-18, or a peptide having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NOs: 8 and 15-18, while retaining the CDRs.

[0073] Examples of antibodies and fragments include those having a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 9-14, or a peptide having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NOs: 9-14, while retaining the CDRs. Examples of such antibodies and fragments include those having a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 15-18, or a peptide having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with an amino acid sequence selected from SEQ ID NOs: 15-18, while retaining the CDRs.

[0074] Examples of antibodies and fragments include those having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11, while retaining the CDRs. Examples of such antibodies and fragments include those having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, or a peptide having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 15, while retaining the CDRs. An exemplary antibody is Hu02.

[0075] Examples of antibodies and fragments include those having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10, or a peptide having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, while retaining the CDRs. Examples of such antibodies and fragments include those having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16, or a peptide having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 16, while retaining the CDRs. An exemplary antibody is Hu06.

[0076] In some embodiments, anti-TIGIT antibodies or fragments that specifically bind to TIGIT competitively with any of the anti-TIGIT antibodies or fragments described herein are also provided. In some embodiments, an ELISA assay can be used to determine competitive binding. In some embodiments, the K of binding between the competing anti-TIGIT antibody or fragment and TIGIT is d About 10 - 5 M to about 10 -12 M (such as about 10 -7 M to about 10 -12 M, or about 10 -8 M to about 10 -12 M). In some embodiments, the competing anti-TIGIT antibody or fragment is chimeric, human, partially humanized, or fully humanized.

[0077] It will also be appreciated by those skilled in the art that the antibodies disclosed herein can be modified so that they differ in amino acid sequence from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence derived from a given protein can be similar, e.g., having a certain percentage of identity with the starting sequence, e.g., it can have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or a range of between any two of these values ​​with the starting sequence.

[0078] In some embodiments, the antibody comprises an amino acid sequence or one or more moieties that are not normally associated with an antibody. Exemplary modifications are described in more detail herein. For example, the antibodies disclosed herein may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0079] Antibodies, variants or derivatives thereof disclosed herein include modified derivatives, i.e., derivatives in which any type of molecule is covalently attached to the antibody such that the covalent attachment does not prevent the antibody from binding to the epitope. For example, but not limited to, the antibody may be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, etc. Any of a number of chemical modifications may be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the antibody may contain one or more non-classical amino acids.

[0080] In some embodiments, the antibody may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a pharmaceutical agent, or PEG.

[0081] The antibody may be conjugated or fused to a therapeutic agent which may include a detectable label, such as a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photosensitive therapeutic or diagnostic agent, a cytotoxic agent (which may be a drug or a toxin), an ultrasound enhancing agent, a non-radioactive label, combinations thereof, and other such agents known in the art.

[0082] The antibody can be detectably labeled by coupling with a chemiluminescent compound. The presence of the chemiluminescent labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0083] Antibodies can also be used with fluorescent emitting metals such as 152Eu or other lanthanides can be detectably labeled. These metals can be linked to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). The techniques for conjugating various moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-256 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-653 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press, pp. 303-316 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. (Vol. 52: pp. 119-158 (1982)).

[0084] Bifunctional molecules and combination therapies

[0085] TIGIT is an immune checkpoint. As an immune checkpoint inhibitor, an antibody or antigen binding fragment specific for TIGIT can be combined with a second antigen binding fragment specific for tumor cells or immune cells to produce a bispecific antibody. In some embodiments, the antibodies or antigen binding fragments disclosed herein can be used in combination with a second agent, such as a second antibody or antigen binding fragment specific for tumor cells or immune cells.

[0086] In some embodiments, the immune cell is selected from T cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, phagocytes, natural killer cells, eosinophils, basophils and mast cells. Molecules that can be targeted on immune cells include, for example, CD33, CD47, CD73, Her2, EGFR, CEA VEGF, CD3, CD16, CD19, CD28, CD47, CD64, CD155, CD112, CD113, PVRL3 and PVRIG. Other examples include PD-1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 or CD40L, LIGHT, ICOS / ICOSL, GITR / GITRL, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), and killer cell immunoglobulin-like receptors (KIRs).

[0087] "Tumor antigen" refers to an antigenic substance produced in tumor cells, that is, it triggers an immune response in the host. Tumor antigens can be used to identify tumor cells and are potential candidates for cancer treatment. Normal proteins in the body are not antigenic. However, certain proteins are produced or overexpressed during tumorigenesis and therefore appear to be "foreign" to the body. This can include normal proteins that are well isolated from the immune system, proteins that are usually produced in very small amounts, proteins that are usually only produced at certain stages of development, or proteins whose structure is modified due to mutations.

[0088] A large number of tumor antigens are known in the art, and new tumor antigens can be easily identified by screening. Non-limiting examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tenascin.

[0089] Specific examples of bispecifics include, but are not limited to, TIGIT / LAG3, TIGIT / CD47, TIGIT / PD-1, TIGIT / PD-L1, TIGIT / CD155, TIGIT / CD112, TIGIT / CD113, TIGIT / PVRL3, TIGIT / PVRIG, and TIGIT / CD3.

[0090] Different forms of bispecific antibodies are also provided. In some embodiments, each anti-TIGIT fragment and the second fragment are each independently selected from a Fab fragment, a single-chain variable fragment (scFv) or a single domain antibody. In some embodiments, the bispecific antibody also includes an Fc fragment.

[0091] Also provided are bifunctional molecules including not only antibodies but also antigen binding fragments. As tumor antigen targeting molecules, antibodies or antigen binding fragments specific for TIGIT, such as those described herein, can be optionally combined with immunocytokines or ligands through peptide linkers. The connected immunocytokines or ligands include but are not limited to IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. This bifunctional molecule can combine the immune checkpoint blocking effect with local immunomodulation at the tumor site.

[0092] Polynucleotide encoding antibody and method for preparing antibody

[0093] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding antibodies, variants or derivatives thereof of the present disclosure. The polynucleotides of the present disclosure can encode the entire heavy chain and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules. In addition, the polynucleotides of the present disclosure can encode portions of the heavy chain and light chain variable regions of antigen-binding polypeptides, variants or derivatives thereof on the same polynucleotide molecule or on different polynucleotide molecules.

[0094] Methods for preparing antibodies are well known in the art and are described herein. In certain embodiments, the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human. Fully human antibodies can be prepared using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic attack, but whose endogenous loci have lost function.

[0095] In certain embodiments, the prepared antibodies do not induce a harmful immune response in the animal to be treated (e.g., in humans). In one embodiment, the disclosed antigen-binding polypeptides, variants or derivatives thereof are modified using techniques recognized in the art to reduce their immunogenicity. For example, antibodies may be humanized, primatized, deimmunized, or chimeric antibodies may be prepared. These types of antibodies are derived from non-human antibodies, typically mouse antibodies or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody, but have lower immunogenicity in humans. This can be achieved by various methods, including (a) transplanting the entire non-human variable domain onto a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementary determining regions (CDRs) into a human framework and constant region, retaining or not retaining key framework residues; or (c) transplanting the entire non-human variable domain, but "masking" them with human-like segments by replacing surface residues.

[0096] Deimmunization can also be used to reduce the immunogenicity of antibodies. As used herein, the term "deimmunization" includes changing antibodies to modify T cell epitopes (see, for example, International Application Publication Nos.: WO / 9852976A1 and WO / 0034317A2). For example, the variable heavy chain sequence and variable light chain sequence from the starting antibody are analyzed, and a human T cell epitope "atlas" from each V region is created, which shows the epitope position associated with the complementary determining region (CDR) and other key residues in the sequence. Analyze a single T cell epitope from the T cell epitope atlas to identify the replacement amino acid substitution with a low risk of final antibody activity change. A series of variable heavy chain and variable light chain sequences for selection are designed, including a combination of amino acid substitutions, and these sequences are subsequently incorporated into a series of binding polypeptides. Typically, 12 to 24 variant antibodies are produced and tested for their combination and / or function. The complete heavy and light chain genes comprising the modified variable region and human constant region are then cloned into an expression vector, and then the plasmid is introduced into a cell line to produce a complete antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

[0097] The binding specificity of the antigen-binding polypeptides of the present disclosure can be determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0098] Examples of techniques that can be used to produce single-chain Fvs (scFvs) and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, Vol. 203: pp. 46-88 (1991); Shu et al., Proc. Natl. Sci. USA, Vol. 90: pp. 1995-1999 (1993); and Skerra et al., Science, Vol. 240: pp. 1038-1040 (1998). For some uses, including use of antibodies in humans and in vitro detection assays, it may be preferred to use chimeric, humanized, or human antibodies. Chimeric antibodies are molecules in which different portions of an antibody are derived from different animal species, such as antibodies having variable regions derived from a mouse monoclonal antibody and human immunoglobulin constant regions. Methods for producing chimeric antibodies are known in the art.

[0099] Humanized antibodies are antibody molecules derived from non-human species antibodies that bind to the desired antigens with one or more complementary determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. Typically, framework residues in human framework regions will be replaced by corresponding residues from CDR donor antibodies to change, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, by simulating the interaction of CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions.

[0100] DNA encoding the desired monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). Hybridoma cells isolated and subcloned are used as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into prokaryotic or eukaryotic host cells, such as Escherichia coli (E. coli) cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not originally produce immunoglobulins.

[0101] In addition, using conventional recombinant DNA technology, one or more CDRs of the antigen-binding polypeptides of the present disclosure can be inserted into the framework region, for example, into the human framework region, to humanize the non-human antibody. The framework region can be a naturally occurring or shared framework region, and preferably a human framework region (see, for example, Chothia et al., J. Mol. Biol. Vol. 278: pp. 457-479 (1998), for a list of human framework regions). Preferably, the polynucleotides generated by the combination of the framework region and the CDR encode antibodies that specifically bind to at least one epitope of the desired polypeptide (e.g., LIGHT). Preferably, one or more amino acid substitutions can be made in the framework region, and preferably, these amino acid substitutions improve the binding of the antibody to its antigen. In addition, such methods can be used to amino acid substitute or delete one or more variable region cysteine ​​residues that participate in intrachain disulfide bonds to produce antibody molecules lacking one or more intrachain disulfide bonds. Other changes to the polynucleotides are included in the present disclosure and are within the technical scope of the art.

[0102] treat

[0103] As described herein, the antibodies, variants or derivatives of the present disclosure may be used in certain therapeutic and diagnostic methods.

[0104] The present disclosure also relates to antibody-based therapies, which involve administering the antibodies of the present disclosure to patients such as animals, mammals and humans for the treatment of one or more diseases or conditions described herein. The therapeutic compounds of the present disclosure include, but are not limited to, antibodies of the present disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the present disclosure (including variants and derivatives thereof as described herein).

[0105] In some embodiments, a method for treating cancer in a patient in need is provided. In one embodiment, the method requires administering an effective amount of an antibody of the present disclosure to the patient. In some embodiments, at least one cancer cell (e.g., stromal cell) in the patient overexpresses TIGIT or is induced to express TIGIT.

[0106] Non-limiting examples of cancer include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0107] The present disclosure also provides cell therapy, and more specifically chimeric antigen receptor (CAR) T cell (CAR-T) and NK cell (CAR-NK) therapy. Suitable T cells or NK cells can be used to contact them with the anti-TIGIT antibodies of the present disclosure (or alternatively, engineered to express the anti-TIGIT antibodies of the present disclosure). After such contact or engineering, T cells or NK cells can then be introduced into cancer patients in need of treatment. Cancer patients may suffer from any type of cancer disclosed herein. T cells can be, for example, tumor infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto.

[0108] In some embodiments, T cells or NK cells are separated from cancer patients themselves. In some embodiments, T cells or NK cells are provided by donors or cell banks. When T cells or NK cells are isolated from cancer patients, undesirable immune responses can be minimized.

[0109] Other diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed and / or prognosed with the antibodies of the present disclosure, or variants or derivatives thereof, include, but are not limited to, progression and / or metastasis of malignancies and related conditions such as leukemias (including acute leukemias (e.g., acute lymphocytic leukemia, acute myeloid leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia)) and chronic leukemias (e.g., chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, including, but not limited to, sarcomas and carcinomas such as fibrosarcomas, myxosarcoma, liposarcoma, tumors, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangioma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0110] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody used, its variant or derivative, the patient's age, weight, general health, sex and diet, administration time, excretion rate, drug combination and the severity of the specific disease being treated. The judgment of these factors by the medical caregiver is within the ordinary skill of the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0111] The method of administration of antibodies and variants includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Antigen-binding polypeptides or compositions can be applied by any convenient route, such as by infusion or push injection, absorbed by epithelial or mucocutaneous lining (e.g., oral mucosa, rectum and intestinal mucosa, etc.), and can be applied together with other bioactive agents. Therefore, pharmaceutical compositions containing antigen-binding polypeptides of the present disclosure can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneal, locally (such as by powder, ointment, drops or transdermal patch), orally or as oral spray or nasal spray.

[0112] The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intraarticular injection and infusion.

[0113] Administration may be systemic or local. In addition, it may be desirable to introduce the antibodies of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injections; intraventricular injections may be facilitated, for example, by an intraventricular catheter connected to a reservoir such as an Ommaya reservoir. Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0114] It may be desirable to administer the antigen-binding polypeptides or compositions of the present disclosure locally to the area in need of treatment; this may be accomplished, for example but not limited to, by local infusion during surgery, topical application (e.g., in conjunction with a wound dressing after surgery), by injection, by catheter, by suppository, or by implant, the implant being a porous, non-porous, or gel-like material, including membranes such as sialic acid membranes or fibers. Preferably, when administering proteins of the present disclosure (including antibodies), care must be taken to use materials to which the proteins are not absorbed.

[0115] In another embodiment, the compositions of the present disclosure are administered in combination with an anti-tumor agent, an anti-viral agent, an anti-bacterial agent, or an antibiotic agent or an anti-fungal agent. Any of these agents known in the art may be administered in the compositions of the present disclosure.

[0116] In another embodiment, the composition of the present disclosure is administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that can be administered with the composition of the present disclosure include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon α-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorethoxyquin, and testolactone); nitrogen mustard derivatives (e.g., melphalan, chlorambucil, mechlorethamine (nitrogen mustard), and thiotepa); steroids and combinations (e.g., betamethasone sodium phosphate); and other substances (e.g., dicarbohydrine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0117] In other embodiments, the compositions of the present disclosure are administered in combination with cytokines. Cytokines that can be administered with the compositions of the present disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0118] In additional embodiments, the compositions of the present disclosure are administered in combination with other therapeutic or preventive regimens (eg, radiation therapy).

[0119] In some embodiments, the anti-TIGIT antibodies of the present disclosure can be used with immune checkpoint inhibitors. Immune checkpoints are molecules in the immune system that either upregulate signals (co-stimulatory molecules) or downregulate signals. Many cancers protect themselves from the immune system by inhibiting T cell signals. Immune checkpoint inhibitors can help cells stop this protective mechanism. Immune checkpoint inhibitors can target any one or more of the following checkpoint molecules: PD-1, PD-L1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137) or BTLA (also known as CD272).

[0120] Programmed T cell death 1 (PD-1) is a transmembrane protein found on the surface of T cells. When it binds to programmed T cell death ligand 1 (PD-L1) on tumor cells, it leads to the inhibition of T cell activity and the reduction of T cell-mediated cytotoxicity. Therefore, PD-1 and PD-L1 are immune downregulators or immune checkpoint "off switches". Exemplary PD-1 inhibitors include but are not limited to nivolumab (Opdivo) (BMS-936558), pembrolizumab (Keytruda), pidilizumab, AMP-224, MEDI0680 (AMP-514), PDR001, MPDL3280A, MEDI4736, BMS-936559 and MSB0010718C.

[0121] Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene in humans. Non-limiting examples of PD-L1 inhibitors include atezolizumab (Tecentriq), durvalumab (MEDI4736), avelumab (MSB0010718C), MPDL3280A, BMS935559 (MDX-1105), and AMP-224.

[0122] CTLA-4 is a protein receptor that downregulates the immune system. Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerly known as ticilimumab, CP-675,206).

[0123] Lymphocyte activation gene 3 (LAG-3) is an immune checkpoint receptor on the cell surface that inhibits immune responses through effects on Tregs and direct effects on CD8+ T cells. LAG-3 inhibitors include, but are not limited to, LAG525 and BMS-986016.

[0124] CD28 is constitutively expressed on nearly all human CD4+ T cells and on approximately half of all CD8 T cells, which promotes T cell expansion. Non-limiting examples of CD28 inhibitors include TGN1412.

[0125] CD122 increases the proliferation of CD8+ effector T cells. Non-limiting examples include NKTR-214.

[0126] 4-1BB (also referred to as CD137) participates in T cell proliferation. It is also known that CD137-mediated signaling protects T cells, particularly CD8+T cells from activation-induced cell death. PF-05082566, Urecirumab (BMS-663513) and lipocalin are exemplary CD137 inhibitors.

[0127] For any of the above combination therapies, the anti-TIGIT antibody can be administered simultaneously with or separately from the other anti-cancer agent. When administered alone, the anti-TIGIT antibody can be administered before or after the other anti-cancer agent.

[0128] In one embodiment, a method of treating or inhibiting an infection in a patient in need thereof is provided, the method comprising administering to the patient an effective amount of an antibody or fragment thereof of the present disclosure. In some embodiments, the infection is a viral infection (such as HIV infection), a bacterial infection, a fungal infection, or a parasitic infection.

[0129] Infection is the invasion of organisms' body tissues by pathogenic agents, their reproduction, and the response of host tissues to these organisms and toxins they produce. Infection can be caused by infectious agents such as viruses, viroids, prions, bacteria, nematodes (such as parasitic roundworms and pinworms), arthropods (such as ticks, mites, fleas, and lice), fungi such as ringworms, and other large parasites (such as tapeworms and other worms). In one aspect, the infectious agent is a bacterium, such as a gram-negative bacterium. In one aspect, the infectious agent is a virus, such as a DNA virus, an RNA virus, and a retrovirus. Non-limiting examples of viruses include adenovirus, coxsackievirus, Epstein-Barr virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpes virus type 8, HIV, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, polio virus, rabies virus, respiratory syncytial virus, rubella virus, and varicella-zoster virus.

[0130] The antibodies disclosed herein can also be used to treat infectious diseases caused by microorganisms, or to kill microorganisms by targeting microorganisms and immune cells to achieve elimination of microorganisms. In one aspect, the microorganism is a virus, including RNA viruses and DNA viruses, gram-positive bacteria, gram-negative bacteria, protozoa or fungi.

[0131] Diagnostic Methods

[0132] Overexpression of TIGIT is observed in certain tumor samples, and patients with cells that overexpress TIGIT may respond to treatment with the anti-TIGIT antibodies of the present disclosure. Therefore, the antibodies of the present disclosure may also be used for diagnostic and prognostic purposes.

[0133] Preferably, the sample including the cells can be obtained from a patient, which can be a cancer patient or a patient in need of diagnosis. The cells can be cells of tumor tissue or tumor mass, blood sample, urine sample or any sample from the patient. After the sample is optionally pretreated, the sample can be incubated with the antibody of the present disclosure under conditions that allow the antibody to interact with the TIGIT protein that may be present in the sample. The presence of TIGIT protein in the sample can be detected using methods such as ELISA using anti-TIGIT antibodies.

[0134] The presence of TIGIT protein in a sample (optionally with a specific amount or concentration) can be used for the diagnosis of cancer, as an indication that the patient is suitable for antibody treatment, or as an indication that the patient has (or has not) responded to cancer treatment. For prognostic methods, one, two or more tests can be performed at certain stages after the start of cancer treatment to indicate the progress of treatment.

[0135] Composition

[0136] The present disclosure also provides a pharmaceutical composition. Such a composition comprises an effective amount of an anti-TIGIT antibody or fragment thereof disclosed herein, and a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0137] In some embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans. A "pharmaceutically acceptable carrier" is generally a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0138] The term "carrier" refers to a diluent, adjuvant, excipient or solvent used together with the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oil, including sterile liquids from petroleum, animal, plant or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and glucose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffers such as acetate, citrate or phosphate. Antimicrobial agents such as benzyl alcohol or methyl paraben are also contemplated; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for adjusting tension such as sodium chloride or glucose. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition can be formulated into suppositories with conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a therapeutically effective amount of an antigen-binding polypeptide, preferably in a purified form, and a suitable amount of a carrier so as to provide a form for appropriate administration to a patient. The formulation should be suitable for the mode of administration. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0139] In some embodiments, the composition is formulated as a pharmaceutical composition suitable for intravenous administration to a human being according to conventional procedures. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Typically, the composition is provided separately or mixed together in a unit dose form, for example, as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or a pouch indicating the amount of the active agent. When the composition is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline may be used for distribution. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that each component may be mixed before administration.

[0140] The compounds of the present disclosure may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0141] Example

[0142] Example 1: Generation of mouse monoclonal antibodies against human TIGIT

[0143] This example describes the generation of anti-human TIGIT mouse monoclonal antibodies using hybridoma technology.

[0144] Antigens: human TIGIT-Fc protein and CHOK1 cell line overexpressing human TIGIT (TIGIT-CHOK1 cell line).

[0145] Immunization: To generate mouse monoclonal antibodies against human TIGIT, Balb / c mice, SJL mice, and Wistar rats were first immunized with TIGIT-Fc protein. Mice and rats immunized were boosted with TIGIT-Fc fusion protein, CHO-K1 / TIGIT stable cells, and TIGIT-Fc protein, respectively. In order to select mice or rats that produce antibodies that bind to TIGIT protein, the antibody titer of the serum of immunized mice was evaluated by ELISA. Briefly, microtiter plates were coated with ELISA coating buffer of 0.5 μg / ml human TIGIT protein at 100 μl / well, overnight at 4°C, and then blocked with 150 μl / well of 1% BSA. Dilutions of serum from immunized mice were added to each well and incubated at 37°C for 1-2 hours. The plates were washed with PBS / Tween and then incubated with anti-mouse IgG antibodies conjugated with horseradish peroxidase (HRP) or anti-rat IgG antibodies conjugated with HRP. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. After 3 rounds of immunization, immune responses were tested by serum ELISA for rhTIGIT protein and FACS for CHO-K1 / TIGIT stable cell lines, while the CHO-K1 parental cell line was used as a negative control. After 3 rounds of immunization, mice with sufficient anti-TIGIT IgG titers were boosted with 25 μg human TIGIT-Fc protein. The resulting mice were used for fusion. Hybridoma supernatants were tested for anti-TIGIT IgG by ELISA.

[0146] Cell fusion and hybridoma screening: Fusion was performed by electrofusion. The fused cells were plated in 50 plates for each fusion. The supernatants were screened by ELISA against recombinant human (rh) TIGIT Fc protein and counter screening antigen. The supernatants of positive clones were then screened for their function in blocking the binding of TIGIT to its ligand hCD155 by a cell-based receptor blocking assay.

[0147] Subcloning and screening: Positive primary clones from each fusion were subcloned by limiting dilution to ensure that the subclones were derived from a single parental cell. Supernatants from the subclones were screened by cell-based receptor blocking assays and affinity ranking.

[0148] Hybridoma clone 76D12B10 was selected for further analysis. The amino acid sequence of the variable region of 76D12B10 is provided in Table 1 below.

[0149] Table 1. Sequences of the variable regions of 76D12B10

[0150]

[0151] Table 1A. CDR sequences of 76D12B10

[0152] 76D12B10 sequence SEQ ID NO: CDRH1 SGD 1 CDRH2 FIRDSGSTVYNPSLKG 2 CDRH3 GLFWYFD 3 CDRL1 RTSENIFSYLA 4 CDRL2 NTKTLAE 5 CDRL3 QHHYGNPLT 6

[0153] Example 2: Antigen Binding Properties of Anti-TIGIT Mouse Monoclonal Antibodies

[0154] This example tests the binding properties of anti-TIGIT mouse antibodies to TIGIT protein.

[0155] To evaluate the binding activity of 76D12B10, the chimeric mAb was subjected to ELISA testing. Briefly, microtiter plates were coated with 0.5 μg / ml of human TIGIT-His protein in PBS at 100 μl / well, overnight at 4°C, and then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of 76D12B10 antibody starting at 10 μg / ml were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG antibody conjugated with horseradish peroxidase (HRP) at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. Figure 1 As shown, 76D12B10 binds to human TIGIT with high affinity (EC 50 =8.566ng / ml).

[0156] Interspecies activity

[0157] ELISA tests were performed to evaluate the binding of chimeric antibodies to human, mouse, rat and cynomolgus monkey TIGIT, respectively. Briefly, microtiter plates were coated with 0.5 μg / ml of human, mouse, rat and cynomolgus monkey TIGIT proteins in PBS at 100 μl / well, overnight at 4°C, and then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of chimeric antibodies or biotinylated chimeric antibodies starting at 10 μg / ml were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG Fab antibodies conjugated with horseradish peroxidase (HRP) or streptavidin-HRP at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. The 76D12B10 antibody binds to cynomolgus monkey TIGIT with high affinity (EC 50 =8.298 ng / ml), but did not bind to rat or mouse TIGIT ( Figure 2 and Table 2 ).

[0158] Table 2. Interspecies activity of 76D12B10

[0159] people Cynomolgus monkey Rat Mouse <![CDATA[EC of 76D12B10 50 > 8.566ng / ml 8.298ng / ml No combination No combination

[0160] Biacore analysis of TIGIT antibodies

[0161] Using the capture method with BIACORE TM The binding of 76D12B10 antibody to recombinant TIGIT protein (human TIGIT-his tag) was tested. 76D12B10 mAb was captured using a CM5 chip. Serial dilutions of human TIGIT-his tag protein were injected onto the captured antibody at a flow rate of 30 μl / min for 1 minute. Antigen dissociation was allowed for 300 seconds. All experiments were performed on Biacore T200. Data analysis was performed using Biacore T200 evaluation software. The results are shown in Figure 3 and as shown in Table 3 below.

[0162] Table 3. Binding of antibodies to recombinant TIGIT protein

[0163]

[0164]

[0165] Example 3. Humanization of mouse antibodies

[0166] The 76D12B10 variable region genes were used to generate humanized mAbs (with IgG1 N297A Fc fragment). In the first step of the process, the amino acid sequences of the VH and VL or VK of 76D12B10 were compared with the available human Ig gene sequence database to find the most matching human germline Ig gene sequence overall. For the light chain of 76D12B10, the closest human match was the A20 / JK2 gene, while for the heavy chain, the closest human match was the VH4-B / JH6 gene.

[0167] Then design the humanized variable domain sequence of 76D12B10, wherein CDRL1, L2 and L3 are transplanted to the framework sequence of A20 / JK2 gene, and CDRH1, H2 and H3 are transplanted to the framework sequence of VH4-B / JH6 gene.Then generate 3D model to determine whether there is any framework position, wherein replacing mouse amino acid with human amino acid may affect binding and / or CDR conformation.For heavy chain, V24A, S30T, G45K, I49M, V68I, V72H and Y95F in framework participate in back mutation.For light chain, V43S, I48V and Y49H in framework participate in back mutation (Table 4 and Table 5).

[0168] Table 4. Humanized antibody sequences (underlined CDRs; bold / italics indicate back mutations)

[0169]

[0170]

[0171] Table 5. VH and VL pairings of humanized antibodies

[0172]

[0173] Example 4: Antigen binding properties of humanized antibodies and binding to recombinant human TIGIT

[0174] To evaluate antigen binding activity, humanized antibodies were subjected to ELISA tests. Briefly, microtiter plates were coated with 0.5 μg / ml of human TIGIT-his protein in PBS at 100 μl / well, overnight at 4°C, and then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of humanized antibodies starting at 3 μg / ml were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG antibodies conjugated with horseradish peroxidase (HRP) at 37°C for 30 minutes. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. Figure 4As shown, all humanized antibodies showed comparable binding efficacy to human TIGIT as chimeric antibodies. Biacore affinity ranking of humanized antibodies

[0175] In order to explore the binding kinetics of humanized antibodies, affinity ranking was performed using Biacore in this example. As shown in Table 6, all humanized antibodies from Hu02 to Hu20 showed excellent affinity comparable to chimeric antibodies.

[0176] Table 6. Affinity ranking results of humanized antibodies

[0177]

[0178] Binding to recombinant human TIGIT

[0179] To evaluate antigen binding activity, ELISA tests were performed on Hu02 and Hu06. Briefly, microtiter plates were coated with 0.5 μg / ml of human TIGIT-his protein in PBS at 100 μl / well, overnight at 4°C, and then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of humanized antibodies starting at 3 μg / ml were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG antibodies conjugated with horseradish peroxidase (HRP) at 37°C for 0.5 hours. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. Figure 5 As shown, Hu02 and Hu06 showed comparable binding efficacy to human TIGIT as the chimeric antibodies. Biacore full kinetic affinity for humanized antibodies

[0180] Using the capture method with BIACORE TM The humanized antibodies were tested for binding to recombinant TIGIT protein (human TIGIT-his tag). Hu02 and Hu06 mAbs were captured using a protein A chip. Serial dilutions of human TIGIT-his tag protein were injected onto the captured antibodies at a flow rate of 30 μl / min for 2 minutes. Antigen dissociation was allowed for 1800 seconds. All experiments were performed on a Biacore T200. Data analysis was performed using Biacore T200 evaluation software, as shown in Figure 2. Figure 6 And as shown in Table 7.

[0181] Table 7. Affinity testing by Biacore

[0182]

[0183] Example 5: Functional properties of anti-TIGIT humanized antibodies

[0184] Blockade of human TIGIT protein binding to CD155

[0185] To evaluate the blocking effect of Hu02 and Hu06 on the binding of recombinant human TIGIT to its receptor CD155, an ELISA-based receptor blocking assay was used.

[0186] Briefly, microtiter plates were coated with 5 μg / ml human CD155-hFc protein in PBS at 100 μl / well, overnight at 4°C, and then blocked with 150 μl / well 1% BSA. 50 μl of biotinylated human TIGIT protein and 50 μl of three-fold dilutions of Hu02 and Hu06 antibodies starting at 10 μg / ml were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with streptavidin-HRP at 37°C for 10 minutes. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. Figure 7 As shown, Hu02 and Hu06 effectively inhibited the binding of human TIGIT protein to human CD155 (Hu02 IC 50 =1129ng / ml, Hu06 IC 50 =866.0 ng / ml), similar to the 76D12B10 chimera (IC 50 =1112ng / ml).

[0187] In vitro effects of anti-TIGIT antibodies on cell-based functional assays

[0188] To evaluate the functional activity of anti-TIGIT antibodies in blocking the effects of TIGIT signaling on T cell activation, a cell-based in vitro functional assay was used.

[0189] Jurkat T NFAT cells were engineered to express human TIGIT and CD226, and they responded to TCR activation and CD226 co-stimulation. Raji cells were engineered to express human CD155. Superantigens (staphylococcal enterotoxins) were designed to activate the T cell receptor (TCR) complex in an antigen-dependent manner. When the two cell types were co-cultured, TIGIT inhibited CD226 activation and NFAT luciferase production. Addition of anti-TIGIT antibodies blocked the interaction of TIGIT with CD155 or inhibited the ability of TIGIT to prevent CD226 homodimerization, resulting in luciferase production. Figure 8 As shown, with 76D12B10 chimera (EC 50 =1.237 nM), Hu02 and Hu06 effectively blocked the interaction between TIGIT and CD155 (Hu02 EC 50 =1.757nM, Hu06EC50 =1.671nM).

[0190] Example 6: Efficacy in MC38 tumor mouse model

[0191] This example uses a tumor mouse model to test the in vivo efficacy of functional molecules.

[0192] MC38 cells resuspended in PBS were plated at a volume of 0.1 mL at 5 × 10 5 The concentration of 10 cells was subcutaneously administered into the right skin of B-hTIGIT humanized mice. When the average tumor volume reached approximately 100 mm 3 At 1:10, animals were randomly assigned to experimental groups according to tumor volume, with 6 animals in each group. Total human IgG, Hu02-mIgG2a (Hu02-mouse IgG2a Fc) and tirilimumab-mIgG2a (tirilimumab-mouse IgG2a Fc) were administered twice a week by intraperitoneal injection. The dose was calculated based on the body weight of the experimental animals at 3 mg / kg. Mouse body weight and tumor size were tested twice a week.

[0193] The results are as follows Fig. 9 Compared with the tirilimumab-mIgG2a group, Hu02-mIgG2a showed significantly higher tumor growth inhibition at a dose level of 3 mg / kg on day 11 after treatment (p < 0.05).

[0194] Example 7: Comparison of TIGIT antibodies

[0195] Binding to recombinant human TIGIT

[0196] To evaluate antigen binding activity, ELISA tests were performed on LP010-02 (Hu02-human IgG1 Fc) and the control antibody Tirilimumab (RG6058, Roche) containing the same IgG1 Fc. Briefly, microtiter plates were coated with 0.5 μg / ml of human TIGIT-his protein in PBS at 100 μl / well, overnight at 4°C, and then blocked with 150 μl / well of 1% BSA. Three-fold dilutions of humanized antibodies starting from 3 μg / ml were added to each well and incubated at 37°C for 1 hour. The plates were washed with PBS / Tween and then incubated with goat anti-human IgG antibodies conjugated with horseradish peroxidase (HRP) at 37°C for 0.5 hours. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. As Fig.10 As shown, LP010-02 and tirilimumab showed comparable binding efficacy to human TIGIT. Blockade of human TIGIT protein binding to CD155

[0197] To evaluate the blocking effect of LP010-02 and teiritumab on the binding of recombinant human TIGIT to its receptor CD155, an ELISA-based receptor blocking assay was used.

[0198] Briefly, microtiter plates were coated with 5 μg / ml human CD155-hFc protein in PBS at 100 μl / well at 4°C overnight and then blocked with 150 μl / well 1% BSA. 50 μl of biotinylated human TIGIT protein and 50 μl of 3-fold dilutions of LP010-02 and Tirelliumab antibodies starting at 10 μg / ml were added to each well. The plates were washed with PBS / Tween and then incubated with streptavidin-HRP at 37°C for 10 minutes. After washing, the plates were developed with TMB substrate and analyzed by spectrophotometer at OD 450nm. Fig.11 As shown in Figure 2, LP010-02 and tirilimumab effectively inhibited the binding of human TIGIT protein to human CD155 (LP010-02 IC 50 =531.2ng / ml, Tirelizumab IC 50 =531.3ng / ml).

[0199] Binding to mammalian cells overexpressing human TIGIT

[0200] To evaluate the antigen binding properties, the binding of humanized antibodies to mammalian cells overexpressing human TIGIT was analyzed by FACS. Briefly, human TIGIT cells were first incubated with 3-fold serial dilutions of humanized antibodies starting from 5 μg / ml at 4°C for 40 min. After washing with PBS, Alexa Fluor 647AffiniPure goat anti-human IgG (H+L) antibody was added to each well and incubated at 4°C for 30 minutes. The MFI of 647 was evaluated by FACSCanto. Fig.12 As shown, LP010-02 showed better binding efficacy to human TIGIT expressed on mammalian cells than tirelizumab (LP010-02 EC 50 =32.81ng / ml, Tirelizumab EC 50 =36.82ng / ml).

[0201] Example 8: Functional assay of TIGIT / CD155 blockade

[0202] In this example, a bioluminescent cell-based assay was used to measure the activity of functional molecules in blocking TIGIT / CD155 interaction.

[0203] In this assay, when TIGIT-CD226-NFAT effector cells and Raji-CD155 target cells are co-cultured, TIGIT can bind to CD155 with higher affinity or disrupt CD226 homodimerization and inhibit CD226 signal transduction in the presence of superantigens, thereby inhibiting the NFAT reporter gene activated by superantigens. Addition of anti-TIGIT antibodies (LP010-02, tirelizumab and 22G2, containing the same human IgG1 Fc) that block TIGIT / CD155 interactions leads to the restoration of NFAT-mediated signals. 22G2 is an anti-TIGIT antibody developed by Bristol-Myers Squibb (see, e.g., U.S. Patent Application Publication No.: US20160176963).

[0204] like Fig.13 As shown, LP010-02 blocked the interaction of TIGIT and CD155 with higher activity than tisleliumab and 22G2.

[0205] Example 9: Efficacy of combined treatment with PD-1 antibody

[0206] This example uses a tumor mouse model to test the in vivo efficacy of the combination of LP010-02 (Hu02-WT human IgG1 Fc) and the anti-PD-1 antibody HX008.

[0207] CT26 cells were cultured at 5 × 10 5 The concentration of 10 cells was subcutaneously administered into the right skin of BALB / c-hPD1-hTIGIT humanized mice. When the average tumor volume reached approximately 85 mm 3 At 14:00, animals were randomly assigned to experimental groups according to tumor volume, with 8 animals in each group. HX008, LP010-02, and the combination of LP010-02 and HX008 were administered once a week by intraperitoneal injection. The doses of HX008 and LP010-02 were calculated at 1 mg / kg and 2 mg / kg, respectively, based on the body weight of the experimental animals. The body weight and tumor size of the mice were tested twice or three times a week.

[0208] The results are as follows Fig.14 In the CT26 mouse model, the combination of HX008 and LP010-02 significantly inhibited tumor growth, with greater efficacy than either HX008 or LP010-02 alone.

[0209] Example 10: Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of LP010-02

[0210] This example compares the ADCC of LP010-02 and tirelimumab in vitro.

[0211] In this experiment, Jurkat cells overexpressing TIGIT were used as target cells, PBMCs were used as effector cells, tirilimumab was used as a control, and the test article was LP010-02. A dose-response experiment was performed at a ratio of 50:1 between effector cells and target cells to evaluate the in vitro ADCC of LP010-02.

[0212] like Fig.15 As shown, LP010-02 has higher ADCC activity than tirelimumab.

[0213] Example 11: Complement-dependent cytotoxicity (CDC) activity of LP010-02

[0214] This example compares the CDC of LP010-02 and tirelimumab in vitro.

[0215] In this experiment, Jurkat cells overexpressing TIGIT were used as target cells, complement protein NHSC was used as effector molecule (NHSC concentration was set to 5%), and tirilimumab was used as control. A dose response experiment was performed to evaluate the in vitro CDC activity of the test article LP010-02.

[0216] The results are as follows Fig.16 As shown in the figure, LP010-02 has higher in vitro CDC activity than tirelimumab.

[0217] ***

[0218] The scope of the present disclosure is not limited by the specific embodiments, which are intended to be a single illustration of the various aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It is apparent to those skilled in the art that various modifications and variations may be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they fall within the scope of the appended claims and their equivalents.

[0219] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. SEQUENCE LISTING <110> Lepu Biotech Co., Ltd. <120> Anti-TIGIT antibodies and their uses <130> 02F196982CNP <150> PCT / CN2020 / 127710 <151> 2020-11-10 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 1 Ser Gly Tyr Asp Trp His 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 2 Phe Ile Arg Asp Ser Gly Ser Thr Val Tyr Asn Pro Ser Leu Lys Gly 1 5 10 15 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 3 Gly Leu Phe Trp Tyr Phe Asp Val 1 5 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Arg Thr Ser Glu Asn Ile Phe Ser Tyr Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Asn Thr Lys Thr Leu Ala Glu 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 6 Gln His His Tyr Gly Asn Pro Leu Thr 1 5 <210> 7 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Met Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Ala Thr Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Asp Trp His Trp Ile Arg His Phe Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Phe Ile Arg Asp Ser Gly Ser Thr Val Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Ile Ser Ile Thr His Asp Thr Ser Lys Asn Arg Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Leu Phe Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Asn Ser Pro Gln Leu Leu Val 35 40 45 His Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Asn Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Asn Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 9 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 9 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Asp Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Arg Asp Ser Gly Ser Thr Val Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Phe Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 10 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Asp Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Arg Asp Ser Gly Ser Thr Val Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Val Thr Ile Ser His Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Phe Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 11 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Asp Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Arg Asp Ser Gly Ser Thr Val Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Ile Thr Ile Ser His Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Phe Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 12 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Ser Ser Gly 20 25 30 Tyr Asp Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Phe Ile Arg Asp Ser Gly Ser Thr Val Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Ile Thr Ile Ser His Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Phe Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 13 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 13 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Asp Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Met Gly Phe Ile Arg Asp Ser Gly Ser Thr Val Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Ile Thr Ile Ser His Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Phe Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 14 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 14 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ala Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Asp Trp His Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp 35 40 45 Met Gly Phe Ile Arg Asp Ser Gly Ser Thr Val Tyr Asn Pro Ser Leu 50 55 60 Lys Gly Arg Ile Thr Ile Ser His Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Leu Phe Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 15 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Asn Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 16 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile 35 40 45 His Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Asn Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 17 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Val 35 40 45 His Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Asn Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Phe Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 His Asn Thr Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Asn Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105

Claims

1. An antibody or a fragment thereof, wherein the antibody or a fragment thereof is specific to a T cell immune receptor (TIGIT) protein having an Ig and ITIM domain, and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 1, a VH CDR2 having an amino acid sequence of SEQ ID NO: 2, and a VH CDR3 having an amino acid sequence of SEQ ID NO: 3, and the light chain variable region comprises a VL CDR1 having an amino acid sequence of SEQ ID NO: 4, a VL CDR2 having an amino acid sequence of SEQ ID NO: 5, and a VLCDR3 having an amino acid sequence of SEQ ID NO:

6. 2 . The antibody or fragment thereof according to claim 1 , further comprising an Fc fragment. The antibody or fragment thereof according to claim 2 , wherein the Fc fragment is a human IgG1 Fc fragment.

4. The antibody or fragment thereof according to any one of the preceding claims, which is humanized.

5. The antibody or fragment thereof according to any one of the preceding claims 1 to 3, wherein the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-18. 6 . The antibody or fragment thereof according to claim 5 , wherein the VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO:

15.

7. The antibody or fragment thereof according to claim 5, wherein the VH comprises the amino acid sequence of SEQ ID NO: 10, and the VL comprises the amino acid sequence of SEQ ID NO:

16.

8. A composition comprising the antibody or fragment thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

9. One or more polynucleotides encoding the antibody or fragment thereof according to any one of claims 1 to 7.

10. An isolated cell comprising one or more polynucleotides encoding the antibody or fragment thereof according to any one of claims 1 to 7.

11. A method for detecting the expression of TIGIT in a sample for non-diagnostic purposes, the method comprising contacting the sample with an antibody or a fragment thereof according to any one of claims 1 to 7 under conditions such that the antibody or fragment thereof binds to TIGIT, and detecting the binding indicative of the expression of TIGIT in the sample.

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