Anti-TIGIT antibodies, pharmaceutical compositions thereof, and uses thereof

Abstract

The present invention belongs to the pharmaceutical field and relates to an anti-TIGIT antibody, a pharmaceutical composition thereof, and uses thereof. Specifically, the present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region of the antibody comprises HCDR1 to HCDR3, the amino acid sequences of which are set forth in SEQ ID NOS: 3 to 5, respectively, and the light chain variable region of the antibody comprises LCDR1 to LCDR3, the amino acid sequences of which are set forth in SEQ ID NOS: 8 to 10, respectively. The antibody of the present invention can effectively bind to TIGIT and may be used for the prevention and treatment of tumors.

Description

[Technical Field]

[0001] The present invention relates to an anti-TIGIT antibody, its pharmaceutical composition and use, particularly to an anti-TIGIT monoclonal antibody. [Background technology]

[0002] TIGIT (also known as T cell Ig and ITIM domain: WUCAM, Vstm3, and VSIG9) is a member of the poliovirus receptor (PVR) / Nectin family. It consists of an extracellular immunoglobulin variable region (IgV) domain, a type I transmembrane domain, and an intracellular domain with typical immunoreceptor tyrosine-based inhibitory motifs (ITIM) and immunoglobulin tail tyrosine-based (ITT) motifs. TIGIT is highly expressed on lymphoid cells, particularly effector and regulatory CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells, and natural killer (NK) cells (Yu X, Harden K, Gonzalez LC, et al. The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells[J]. Nature Immunology, 2009, 10(1): 48).

[0003] CD155 (also known as PVR, Necl5, or Tage4), CD112 (also known as PVRL2 / nectin 2), and CD113 (also known as PVRL3) are ligands bound by TIGIT (Martinet L, Smyth M J. Balancing natural killer cell activation through paired receptors[J]. Nature Reviews Immunology, 2015, 15(4): 243-254.), and among them, CD155 is a high-affinity ligand for TIGIT. In NK cells, binding of TIGIT to its ligands CD155 and CD112 suppresses NK cell killing of TIGIT-high expressing cells (Stanietsky N, Simic H, Arapovic J, et al. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity[J]. Proceedings of the National Academy of Sciences, 2009, 106(42): 17858-17863). One report showed that simultaneous blockade of PD-1 and TIGIT enhanced the killing activity of CD8+ T cells (Johnston RJ, Comps-Agrar L, Hackney J, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function[J]. Cancer cell, 2014, 26(6): 923-937).Recent research has demonstrated that TIGIT acts as an immune checkpoint for NK cells, and that the inhibitory receptor TIGIT leads to NK cell exhaustion during tumor progression. Anti-TIGIT monoclonal antibodies can reverse NK cell exhaustion and have been shown to be useful in immunotherapy of various tumors, including non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, pancreatic cancer, cervical cancer, multiple myeloma, non-Hodgkin's lymphoma, B-cell lymphoma, and plasmacytoma (Zhang Q, Bi J, Zheng X, et al. Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity[J]. Nature Immunology, 2018, 19(7): 723-732).

[0004] The expression levels of TIGIT and CD155 in hepatocellular carcinoma (HCC) tissue are upregulated as the differentiation stage progresses. After HCC surgery, the frequency of TIGIT+ CD4+ T cells and TIGIT+ Treg cells in the peripheral blood of patients decreases. Increased TIGIT expression is positively correlated with AFP levels. These results suggest that the co-inhibitory receptor TIGIT may be involved in the pathogenesis of HCC and may represent a novel target for HCC diagnosis and treatment (Duan Xiangguo, Liu Juanxi, Cui Jianjian et al. Expression of TIGIT / CD155 and correlations with clinical pathological features in human hepatocellular carcinoma. [J] . Mol Med Rep, 2019, 20: 3773-3781.).

[0005] Other studies have shown that TIGIT blockade, alone or in combination with PD-1 blockade, plus CD96 blockade, significantly reduces B16 melanoma growth in wild-type and Cd155- / - mouse models (Li XY, Das I, Lepletier A, et al. . Cd155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms. J Clin Invest 2018;128:2613-25). CD112R blockade, alone or in combination with TIGIT blockade and / or PD-1 blockade, can increase the cytokine production of TILs in ovarian, endometrial, and lung tumors (Whelan S, Ophir E, Kotturi MF, et al. . PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+ T-cell Function. Cancer Immunol Res 2019;7:257-68).

[0006] Anti-TIGIT antibodies are novel immune checkpoint antibodies with broad potential for use in tumor immunotherapy. Tiragolumab, developed by Roche, is currently in phase 3 clinical trials. Furthermore, a phase 2 clinical trial of the TIGIT monoclonal antibody tiragolumab in combination with the PD-L1 inhibitor Tecentriq (atezolizumab) as first-line treatment for patients with PD-L1-positive metastatic non-small cell lung cancer (NSCLC) demonstrated that the combination of tiragolumab and Tecentriq was well tolerated and significantly reduced the risk of disease progression by 43% (Exit C. Roche to present first clinical data on novel anti-TIGIT cancer immunotherapy tiragolumab at ASCO[J]).

[0007] However, existing anti-human TIGIT antibody drugs have low affinity, and anti-TIGIT antibodies with high affinity are still lacking.

[0008] Therefore, it is of great significance to develop an antibody drug for the treatment of autoimmune diseases that has high affinity for TIGIT and that has high therapeutic efficacy and low toxic reactions and side effects. Summary of the Invention

[0009] Through intensive research and creative efforts, the inventors have used a mammalian cell expression system to express recombinant human TIGIT as an antigen, immunized mice, and fused the mouse spleen cells with myeloma cells to obtain hybridoma cells. After screening a large number of samples, the inventors obtained the hybridoma cell line LT019 (Deposit No. CCTCC NO: C2020208).

[0010] The present inventors surprisingly discovered that the hybridoma cell line LTO19 can secrete and produce a specific monoclonal antibody (designated 26B12) that specifically binds to human TIGIT, and that this monoclonal antibody can highly effectively bind to TIGIT, reduce the immune cell suppression effect of TIGIT, promote T cell activity, reverse NK cell exhaustion, and enhance the tumor-killing effect of immune cells. Furthermore, the present inventors creatively constructed humanized anti-human TIGIT antibodies (designated 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4).

[0011] The present inventors further surprisingly discovered that the antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 of the present invention have the activity of binding to TIGIT and with very strong affinity, and that 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 can effectively reduce the activity of TIGIT. The antibodies of the present invention may be used in the treatment and / or prevention of diseases such as tumors (e.g., liver cancer, kidney cancer, brain tumor, urothelial cancer, bone tumor, bile duct cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, colorectal cancer, malignant melanoma, pancreatic cancer, cervical tumor, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, ovarian cancer, plasmacytoma, endometrial cancer, prostate cancer, and testicular cancer). To this end, the following inventions are provided.

[0012] One aspect of the present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof, wherein: The heavy chain variable region of the antibody comprises HCDR1 to HCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 3 to 5, respectively, and the light chain variable region of the antibody comprises LCDR1 to LCDR3 whose amino acid sequences are set forth in SEQ ID NOs: 8 to 10, respectively.

[0013] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof has an amino acid sequence of a heavy chain variable region of the antibody selected from SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17; The amino acid sequence of the light chain variable region of the antibody is selected from SEQ ID NO:6, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:25.

[0014] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof is the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 19; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 19; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 21; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 21; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 11 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 25; or The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 25.

[0015] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof is an antibody that comprises non-CDR regions, and the non-CDR regions are derived from a non-murine species, such as a human antibody.

[0016] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof has a heavy chain constant region that is an Ig gamma-1 chain C region (e.g., NCBI ACCESSION: P01857) or an Ig gamma-4 chain C region (e.g., NCBI ACCESSION: P01861.1), and a light chain constant region that is an Ig kappa chain C region (e.g., NCBI ACCESSION: P01834).

[0017] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody, or bibody.

[0018] In one or more embodiments of the invention, the anti-TIGIT antibody or antigen-binding fragment thereof has a K D and preferably binds to TIGIT-mFc at the K D is measured using a Fortebio molecular interaction analyzer.

[0019] In one or more embodiments of the invention, the anti-TIGIT antibody or antigen-binding fragment thereof has an EC2 of less than 1.5 nM, less than 1.2 nM, or less than 1 nM. 50 and preferably, the EC 50 is measured by a flow cytometer.

[0020] In some embodiments of the present invention, the anti-TIGIT antibody is a monoclonal antibody.

[0021] In some embodiments of the present invention, the anti-TIGIT antibody is a humanized antibody, a chimeric antibody, or a multispecific antibody (eg, a bispecific antibody).

[0022] In some embodiments of the invention, the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, a complementarity-determining region fragment, a single-chain antibody (e.g., scFv), a humanized antibody, a chimeric antibody, or a bispecific antibody.

[0023] In one or more embodiments of the present invention, the anti-TIGIT antibody or antigen-binding fragment thereof is an antibody produced by hybridoma cell line LT019, which is deposited at the China Center for Type Culture Collection (CCTCC) under the deposit number CCTCC NO: C2020208.

[0024] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the TIGIT antibody or antigen-binding fragment thereof described in any one of the present invention.

[0025] A further aspect of the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.

[0026] A further aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention.

[0027] A further aspect of the present invention relates to hybridoma cell line LT019, which has been deposited at the China Typical Culture Collection Center (CCTCC) and has deposit number CCTCC NO: C2020208.

[0028] A further aspect of the present invention relates to a conjugate comprising an antibody and a coupling moiety, wherein the antibody is an anti-TIGIT antibody or an antigen-binding fragment thereof described in any one of the aspects of the present invention, and the coupling moiety is a detectable label, preferably a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0029] A further aspect of the present invention relates to a kit comprising an anti-TIGIT antibody or antigen-binding fragment thereof according to any one of the present invention, or a conjugate of the present invention, Preferably, the kit further comprises a secondary antibody that specifically recognizes the antibody, and optionally, the secondary antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0030] A further aspect of the present invention relates to the use of an antibody according to any one of the present invention or a conjugate of the present invention in the preparation of a kit for use in detecting the presence or level of TIGIT in a sample.

[0031] A further aspect of the present invention relates to a pharmaceutical composition comprising an anti-TIGIT antibody or antigen-binding fragment thereof described in any one of the present invention or a conjugate of the present invention, and optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable vector and / or excipient.

[0032] In one or more embodiments of the invention, the pharmaceutical composition further comprises one or more anti-PD-1 antibodies or one or more anti-PD-L1 antibodies.

[0033] In one or more embodiments of the present invention, the mass ratio of the anti-TIGIT antibody or antigen-binding fragment thereof to the anti-PD-1 antibody or anti-PD-L1 antibody in the pharmaceutical composition, calculated based on the mass of the antibodies, is 1:5 to 5:1, for example, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1.

[0034] A further aspect of the invention relates to a bispecific antibody comprising a first protein functional domain and a second protein functional domain, wherein: the first protein functional region targets TIGIT; the second protein functional region targets a target different from TIGIT (e.g., PD-1); wherein the first protein functional domain is the antibody or antigen-binding fragment according to any one of the present invention; Preferably, the bispecific antibody is in IgG-scFv mode, Preferably, the first protein functional domain is an antibody according to any one of the present invention and is in the immunoglobulin format, and the second protein functional domain is a single chain antibody; or Preferably, the first protein functional domain is a single chain antibody and the second protein functional domain is an immunoglobulin-type antibody.

[0035] In some embodiments of the invention, the bispecific antibody is an antibody according to any one of the invention in the form of an immunoglobulin.

[0036] In some embodiments of the present invention, the bispecific antibody is such that the single-chain antibody is in the format of heavy chain variable region-linker-light chain variable region.

[0037] In some embodiments of the present invention, the bispecific antibody has the first protein functional domain and the second protein functional domain linked directly or via a linker, which may be the same as or different from the linker in the previous single-chain antibody, and any linker commonly used in the art may be used.

[0038] In some embodiments of the present invention, the bispecific antibody has one, two, or more than two first protein functional domains and two second protein functional domains, independently of each other.

[0039] In some embodiments of the invention, the bispecific antibody is one in which the single-chain antibodies are linked to the C-terminus of each of two heavy chains of an immunoglobulin-format antibody, preferably each heavy chain is linked to one single-chain antibody.

[0040] A further aspect of the invention relates to a combination product comprising a first product and a second product packaged independently of one another, wherein: the first product comprises the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of the present invention, the conjugate of the present invention, or the pharmaceutical composition according to any one of the present invention; the second product comprises at least one anti-PD-1 antibody or at least one anti-PD-L1 antibody; Preferably, the first product and the second product further independently comprise one or more pharmaceutically acceptable adjuvants (e.g., vectors and / or excipients); Preferably, the combination product further comprises a product insert.

[0041] In one or more embodiments of the invention, the mass ratio of the anti-TIGIT antibody or antigen-binding fragment thereof to the anti-PD-1 antibody or anti-PD-L1 antibody in the combination product is (1:5) to (5:1), calculated in terms of antibody mass, such as 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, or 5:1.

[0042] A further aspect of the present invention relates to the use of an antibody or antigen-binding fragment thereof according to any one of the present invention, a conjugate of the present invention, a bispecific antibody according to any one of the present invention, a pharmaceutical composition according to any one of the present invention or a combination product according to any one of the present invention in the preparation of a medicament for treating and / or preventing a tumor, preferably said tumor being one or more types selected from liver cancer, kidney cancer, brain tumor, urothelial carcinoma, bone tumor, cholangiocarcinoma, non-small cell lung cancer, small cell lung cancer, breast cancer, colorectal cancer, malignant melanoma, pancreatic cancer, cervical tumor, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, ovarian cancer, plasmacytoma, endometrial cancer, prostate cancer and testicular cancer.

[0043] The antibody or antigen-binding fragment thereof according to any one of the aspects of the present invention, the conjugate of the present invention, the bispecific antibody according to any one of the aspects of the present invention, the pharmaceutical composition according to any one of the aspects of the present invention or the combination product according to any one of the aspects of the present invention is used for treating and / or preventing tumors, preferably the tumors are one or more types selected from liver cancer, kidney cancer, brain tumor, urothelial carcinoma, bone tumor, cholangiocarcinoma, non-small cell lung cancer, small cell lung cancer, breast cancer, colorectal cancer, malignant melanoma, pancreatic cancer, cervical tumor, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, ovarian cancer, plasmacytoma, endometrial cancer, prostate cancer and testicular cancer.

[0044] A further aspect of the invention relates to a method for treating and / or preventing tumors, comprising the step of administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of the invention, the conjugate of the invention, the bispecific antibody according to any one of the invention, the pharmaceutical composition according to any one of the invention or the combination product according to any one of the invention, wherein preferably the tumor is one or more selected from liver cancer, kidney cancer, brain tumor, urothelial carcinoma, bone tumor, cholangiocarcinoma, non-small cell lung cancer, small cell lung cancer, breast cancer, colorectal cancer, malignant melanoma, pancreatic cancer, cervical tumor, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, ovarian cancer, plasmacytoma, endometrial cancer, prostate cancer and testicular cancer.

[0045] In some embodiments of the present invention, the liver cancer is hepatocellular carcinoma.

[0046] The variable regions of the light and heavy chains determine antigen binding, and each chain variable region contains three highly variable regions called complementarity-determining regions (CDRs). (The CDRs of the heavy chain (H) contain HCDR1, HCDR2, and HCDR3, while the CDRs of the light chain (L) contain LCDR1, LCDR2, and LCDR3. These CDRs were named by Kabat et al., Bethesda, Md., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242.)

[0047] Preferably, the CDRs may be defined by the IMGT numbering system. See Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0048] By technical means well known to those skilled in the art, the amino acid sequences of the CDR regions of the monoclonal antibody sequence are analyzed based on the IMGT definition, for example, in the VBASE2 database.

[0049] Antibodies 26B12, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 according to the present invention have the same CDRs.

[0050] The amino acid sequences of the three CDR regions of the heavy chain variable region are as follows: HCDR1: GHSFTSDYA (SEQ ID NO: 3) HCDR2: ISYSDST (SEQ ID NO: 4) HCDR3: ARLDYGNYGGAMDY (SEQ ID NO: 5) The amino acid sequences of the three CDR regions of the light chain variable region are as follows: LCDR1:QHVSTA (SEQ ID NO: 8) LCDR2:SAS (SEQ ID NO: 9) LCDR3:QQHYITPWT (SEQ ID NO: 10)

[0051] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunological laboratory procedures used herein are all common procedures widely used in the relevant technical fields. At the same time, in order to better understand the present invention, the following definitions and explanations of relevant terms are provided.

[0052] As used herein, reference to the amino acid sequence of TIGIT (NCBI GenBank ID: NP_776160.2) refers to the full-length TIGIT protein, the extracellular immunoglobulin variable region (IgV) domain, or a fragment containing the extracellular immunoglobulin variable region (IgV) domain, and further includes fusion proteins of TIGIT, such as a fragment fused to an Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of the TIGIT protein can be naturally produced or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "TIGIT protein" or "TIGIT" is intended to encompass all such sequences, including the sequences shown and natural or artificial variants thereof. Furthermore, when a sequence fragment of the TIGIT protein is described, it not only includes the sequence fragment but also the corresponding sequence fragment in the natural or artificial variant.

[0053] As used herein, the term EC 50 This refers to the concentration for 50% of maximal effect, which is the concentration that can produce 50% of the maximum effect.

[0054] As used herein, the term "antibody" refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains are classified as kappa and lambda types. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon types, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are connected through a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 3 or more amino acids. Each heavy chain comprises a heavy chain variable region (V H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains (C H1 , C H2 and C H3 Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L The light chain constant region consists of one domain, C L The antibody constant region mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The regions can be further subdivided into regions of high variability (called complementarity determining regions (CDRs)) interspersed with conserved regions called framework regions (FRs). H and V L Each heavy / light chain pair consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. H and V L), each forming an antigen-binding site. The assignment of amino acids to each region or domain is defined according to the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al. Nature 1989; 342:878-883, or the IMGT numbering system. See the definitions in Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF, and MhcSF." Nucleic acids research 2009; 38(suppl_1): D301-D307. The term "antibody" is not limited by any particular method of producing the antibody. For example, this includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies, among others. The antibody may be of different isotypes, such as IgG (e.g., subclasses IgG1, IgG2, IgG3, or IgG4), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0055] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide, including a fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety and for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability on the polypeptide.

[0056] As used herein, the term "Fd fragment" refers to a V H and C H The term "Fv fragment" refers to an antibody fragment consisting of a single V domain of an antibody. L and V H The term "dAb fragment" refers to an antibody fragment consisting of the V domain. H The term "Fab fragment" refers to an antibody fragment consisting of the V domain (Ward et al., Nature 341:544-546 (1989)). L , V H , C L and C H The term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region.

[0057] In some cases, the antigen-binding fragment of an antibody comprises a V L and V HSingle-chain antibodies (e.g., scFvs) are monovalent molecules that are paired with a linker that allows the domains to be produced as a single polypeptide chain (see, e.g., Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules include NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L The linker may have the general structure -COOH. Suitable prior art linkers consist of repeats of the GGGGS amino acid sequence or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers for use in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31: 94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56; and Roovers et al. (2001), Cancer Immunol.

[0058] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., V H and V L A bivalent antibody is one in which the domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain to produce two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0059] In other cases, the antigen-binding fragment of an antibody is a "bispecific antibody," which refers to a complex formed by coupling a first antibody (fragment) with a second antibody (fragment) or antibody analog via coupling arms, and coupling methods include, but are not limited to, chemical reaction, gene fusion, and enzyme catalysis. The antigen-binding fragment of an antibody may also be a "multispecific antibody," including, for example, triabodies, which are antibodies with three different antigen-binding specificities, and tetrabodies, which are antibodies with four different antigen-binding specificities. For example, a designed ankyrin repeat protein (DARPin) may be linked to an IgG antibody, scFv-Fc antibody fragment, or a combination thereof, as shown, for example, in CN104341529A. An anti-IL-17a fusion molecule may be linked to an anti-IL-6R antibody, as shown, for example, in WO2015141862A1.

[0060] Antigen-binding fragments of an antibody (e.g., the above-described antibody fragments) can be obtained from a given antibody (e.g., monoclonal antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage), and the antigen-binding fragments can be specifically screened for using the same methods as those used for whole antibodies.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody or antibody fragment derived from a series of highly homologous antibody molecules, i.e., a series of antibody molecules that are completely identical except for spontaneous mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Compared to monoclonal antibodies, polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies are generally obtained using the hybridoma technology first described by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but they may also be obtained using recombinant DNA technology (see, e.g., U.S. Patent 4,816,567).

[0062] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR regions of a human immunoglobulin (receptor antibody) with the CDR regions of a non-human antibody (donor antibody), where the donor antibody may be a non-human antibody (e.g., mouse, rat, or rabbit) with the desired specificity, affinity, or reactivity. In addition, to further improve or optimize the performance of the antibody, some amino acid residues in the framework region (FR) of the receptor antibody may be replaced with corresponding amino acid residues of a non-human antibody or with amino acid residues of another antibody. For further details on humanized antibodies, see, e.g., Jones et al., Nature 1986; 321:522-525; Reichmann et al., Nature 1988; 332:323-329; Presta, Curr. Op. Struct. Biol., 1992; 2:593-596; and Clark M. Antibody humanization: a case of the 'Emperor's new clothes'? [J]. Immunol. Today, 2000; 21(8): 397-402.

[0063] As used herein, the term "isolated" or "isolated" refers to being obtained by artificial means from a natural state. When an "isolated" substance or component exists in nature, the natural environment in which it exists may have been altered, or the substance may have been isolated from its natural environment, or both. For example, a non-isolated polynucleotide or polypeptide may naturally occur in the living body of an animal, but a highly purified version of the same polynucleotide or polypeptide isolated from such a natural state is referred to as isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic materials, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0064] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A vector that allows for the expression of a protein encoded by the inserted polynucleotide is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, allowing the genetic material elements carried by the vector to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, Cox plasmids, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages (e.g., λ bacteriophage or M13 bacteriophage), and animal viruses. Animal viruses used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papilloma baculoviruses (e.g., SV40). Vectors may contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain an origin of replication.

[0065] As used herein, the term "host cell" refers to cells useful for introducing vectors, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells. As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., a reaction between an antibody and an antigen to which it is directed. In one embodiment, an antibody that specifically binds to an antigen (or has specificity for an antigen) is one in which the antibody binds to an antigen within about 10 -5 Less than M, e.g., about 10-6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Less than M or 10 -10 Affinity (K D ) to bind to the antigen.

[0066] As used herein, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies have a dissociation equilibrium constant of about 10 -5 Less than M, e.g., about 10 -6 Under M, 10 -7 Less than 10 -8 Under M, 10 -9 Less than M or 10 -10 The dissociation equilibrium constant (K D ) binds to an antigen (e.g., TIGIT protein). D may be measured by methods well known to those skilled in the art, such as measurement with a Fortebio molecular interaction measurement device.

[0067] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and can be used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and can be used interchangeably; and the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. In the present invention, amino acids are generally represented by one- or three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0068] As used herein, the term "pharmaceutically acceptable vector and / or excipient" refers to a vector and / or excipient that is pharmacologically and / or physiologically compatible with the active ingredient in a subject, as known in the art (see, e.g., Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0069] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor), and an effective amount for treating a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient who has the disease.

[0070] As used herein, reference to the amino acid sequence of the TIGIT protein (NCBI GenBank: NP_776160.2) includes the full-length TIGIT protein, the extracellular fragment of TIGIT, the TIGIT ECD, or a fragment containing the TIGIT ECD, and further includes a fusion protein of the full-length TIGIT protein or a fusion protein of the TIGIT ECD, such as a fragment fused with an Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of the TIGIT protein can be naturally produced or artificially introduced without affecting its biological function. Therefore, in the present invention, the term "TIGIT protein" includes all such sequences, including natural or artificial variants thereof. Furthermore, when a sequence fragment of the TIGIT protein is described, it also includes the corresponding sequence fragment in the natural or artificial variant.

[0071] As used herein, the terms "hybridoma" and "hybridoma cell line" are used interchangeably, and references to the terms "hybridoma" and "hybridoma cell line" further include subclones and progeny of hybridomas.

[0072] In the present invention, unless otherwise specified, the terms "first" (e.g., first product) and "second" (e.g., second product) are used for the purpose of indicating distinction or clarifying expression, and do not have a typical sequential meaning. [Effects of the Invention]

[0073] The monoclonal antibody of the present invention specifically binds to TIGIT with strong affinity, reduces the immune cell suppression effect of TIGIT, promotes T cell activity, reverses NK cell exhaustion, and enhances the antitumor killing effect of immune cells. It can be used to prepare drugs that inhibit TIGIT and to prepare drugs for treating or preventing diseases such as tumors (e.g., liver cancer, kidney cancer, brain tumor, urothelial carcinoma, bone tumor, bile duct cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, colorectal cancer, malignant melanoma, pancreatic cancer, cervical cancer, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, ovarian cancer, plasmacytoma, endometrial cancer, prostate cancer, and testicular cancer), and its application has good prospects and market value. [Brief explanation of the drawings]

[0074]

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[0075] Deposit of biological material: The hybridoma cell line LT019 has been deposited at the China Center for Typical Culture Collection (CCTCC) since October 23, 2020, with the deposit number CTCCC NO: C2020208. The deposit address is Wuhan University, Wuhan, China, with the postal code 430072.

[0076] The present invention relates to the following sequences 1 to 26. 1. Amino acid sequence of 26B12VH EVQLQESGPGLVKPSQSLSLTCTVT GHSFTSDYA WNWIRQFPGNRLEWMGY ISYSDST NYNPSLKSRISITRDTSKNQFFLQMNSVTTEDTATYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 1) 2. Nucleic acid sequence of 26B12VH GAGGTGCAGCTGCAGGAGTCTGGACCTGGCCTGGTGAAACCCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACT GGCCACTCATTCACCAGTGATTATGCC TGGAACTGGATCCGGCAGTTTCCAGGAAACAGACTGGAGTGGATGGGCTAC ATAAGCTACAGTGATAGCACT AACTACAACCCATCTCTCAAAGGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCTTGCAGATGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGT GCAAGATTGGACTATGGTAACTACGGTGGGGCTATGGACTAC TGGGGTCAAGGGACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 2) 3. HCDR1: GHSFTSDYA (SEQ ID NO: 3) 4. HCDR2: ISYSDST (SEQ ID NO: 4) 5. HCDR3: ARLDYGNYGGAMDY (SEQ ID NO: 5)

[0077] 6. Amino acid sequence of 26B12VL DIVLTQSHEFMSTSLRDRVSITCKSS QHVSTA VAWYQQKPGQSPKLLIY SAS YRYTGVPDRFTGSGSGTDFTFTISSVKAEDLAVYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO: 6) 7. Nucleic acid sequence of 26B12VL GATATTGTGCTAACTCAGTCTCACGAATTCATGTCCACCTCATTACGAGACAGGGTCAGCATCACCTGCAAATCCAGT CAACATGTGAGTACTGCT GTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTAC TCGGCATCC TACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCATGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGT CAGCAACATTATATTACTCCGTGGACG TTCGGTGGAGGCACCAAGCTGGAAATAAAA (SEQ ID NO: 7) 8. LCDR1:QHVSTA (SEQ ID NO:8) 9. LCDR2:SAS (SEQ ID NO: 9) 10. LCDR3: QQHYITPWT (SEQ ID NO: 10)

[0078] 11. Amino acid sequence of 26B12H1 DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WNWIRQFPGKGLEWIGY ISYSDST NYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 11) 12. Nucleic acid sequence of 26B12H1 GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCTGTCTCTGACCTGTACAGTGTCT GGCCACAGCTTCACATCCGACTACGCC TGGAACTGGATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATCGGCTAC ATCTCTTATAGCGACTCCACC AACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACATCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCACATACTATTGC GCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC TGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 12)

[0079] 13. Amino acid sequence of 26B12H2 DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WSWIRQPPGKGLEWIGY ISYSDST NYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 13) 14. Nucleic acid sequence of 26B12H2 GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCTGTCCCTGACCTGTACAGTGTCC GGCCACTCTTTTACAAGCGACTACGCC TGGTCTTGGATCAGGCAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTAC ATCTCCTATTCTGACAGCACC AACTATAATCCCTCCCTGAAGTCTCGGGTGACCATCTCTAGAGATACAAGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGC GCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC TGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 14)

[0080] 15. Amino acid sequence of 26B12H3 DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYA WSWIRQPPGKGLEWIGY ISYSDST NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 15) 16. Nucleic acid sequence of 26B12H3 GATGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCAAGCCAGACCCTGTCCCTGACCTGTACAGTGTCC GGCCACTCTTTTACAAGCGACTACGCC TGGTCTTGGATCAGACAGCCCCCTGGCAAGGGACTGGAGTGGATCGGCTAC ATCTCCTATTCTGACAGCACC AACTATAATCCCTCCCTGAAGTCTAGAGTGACCATCTCTGTGGATACAAGCAAGAACCAGTTCTCCCTGAAGCTGAGCTCCGTGACCGCAGCAGACACAGCCGTGTACTATTGC GCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC TGGGGCCAGGGCACCAGCGTGACAGTGTCTAGC (SEQ ID NO: 16)

[0081] 17. Amino acid sequence of 26B12H4 DVQLQESGPGLVKPSQTLSLTCTVS GHSFTSDYAWNWIRQFPGKGLEWMGY ISYSDST NYNPSLKSRITISRDTSKNQFFLQLNSVTAADTATYYC ARLDYGNYGGAMDY WGQGTSVTVSS (SEQ ID NO: 17) 18. Nucleic acid sequence of 26B12H4 GATGTGCAGCTGCAGGAGAGCGGCCCCGGACTGGTGAAGCCTTCCCAGACCCTGTCTCTGACCTGTACAGTGTCT GGCCACAGCTTCACATCCGACTACGCC TGGAACTGGATCAGGCAGTTTCCAGGCAAGGGCCTGGAGTGGATGGGCTAC ATCTCTTATAGCGACTCCACC AACTATAATCCCTCTCTGAAGAGCCGGATCACCATCAGCAGAGATACATCCAAGAACCAGTTCTTTCTGCAGCTGAACAGCGTGACAGCCGCCGACACCGCCACATACTATTGC GCCCGGCTGGACTACGGCAATTATGGCGGAGCCATGGATTAC TGGGGCCAGGGCACCTCCGTGACAGTGAGCTCC (SEQ ID NO: 18)

[0082] 19. Amino acid sequence of 26B12L1 DIQMTQSPKSLSTSVGDRVTITCRSS QHVSTA VAWYQQKPGKSPKLLIY SAS YRYSGVPDRFSGSGSGTDFTFTISSVQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO: 19) 20. Nucleic acid sequence of 26B12L1 GACATCCAGATGACCCAGTCCCCTAAGTCCCTGTCTACAAGCGTGGGCGATCGGTGACCATCACATGTAGAAGCTCC CAGCACGTGTCTACCGCA GTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTAT TCCGCCTCT TACAGGTATTCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACAATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGC CAGCAGCACTACATCACCCCATGGACCTTCGGCGGCGGCACAAAGCTGGAGATCAAG (SEQ ID NO: 20)

[0083] 21. Amino acid sequence of 26B12L2 DIQMTQSPSSLSASVGDRVTITCRSS QHVSTA LAWYQQKPGKSPKLLIY SAS SRYSGVPDRFSGSGSGTDFTFTISSLQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO: 21) 22. Nucleic acid sequence of 26B12L2 GACATCCAGATGACCCAGTCCCCTAGCTCCCTGTCTGCCAGCGTGGGCGATAGGGTGACCATCACATGTAGATCTAGC CAGCACGTGTCTACAGCC CTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTAC TCCGCCTCC TCTAGGTATTCTGGAGTGCCAGACCGGTTTTCCGGCTCTGGCAGCGGCACCGATTTCACCTTTACAATCAGCTCCCTGCAGCCAGAGGACTTCGCCACATACTATTGC CAGCAGCACTATATCACCCCATGGACC TTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 22)

[0084] 23. Amino acid sequence of 26B12L3 DIQMTQSPSSLSASVGDRVTITCRAS QHVSTA LAWYQQKPGKAPKLLIY SAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO: 23) 24. Nucleic acid sequence of 26B12L3 GACATCCAGATGACCCAGTCCCCTAGCTCCCTGAGCGCCTCCGTGGGCGATAGGGTGACCATCACATGTAGAGCCTCT CAGCACGTGAGCACAGCC CTGGCATGGTACCAGCAGAAGCCAGGCAAGGCCCCTAAGCTGCTGATCTATAGCGCCTCT AGCCTGCAGTCCGGAGTGCCATCTCGGTTCTCTGGCAGCGGCTCCGGAACCGACTTTACCCTGACAATCTCCTCTCTGCAGCCAGAGGATTTCGCCACATACTATTGC CAGCAGCACTACATCACCCCATGGACC TTCGGCGGCGGCACCAAGCTGGAGATCAAG (SEQ ID NO: 24)

[0085] 25. Amino acid sequence of 26B12L4 DIQMTQSPKSMSTSVGDRVTITCRSS QHVSTA VAWYQQKPGKSPKLLIY SAS YRYSGVPDRFSGSGSGTDFTFTISSVQPEDFATYYC QQHYITPWT FGGGTKLEIK (SEQ ID NO: 25) 26. Nucleic acid sequence of 26B12L4 GACATCCAGATGACCCAGTCCCCTAAGTCCATGTCTACAAGCGTGGGCGACAGGGTGACCATCACATGTAGAAGCTCC CAGCACGTGTCTACCGCA GTGGCATGGTACCAGCAGAAGCCAGGCAAGAGCCCTAAGCTGCTGATCTAT TCCGCCTCT TACAGGTATTCCGGAGTGCCAGACCGGTTTAGCGGCTCCGGCTCTGGCACCGATTTCACCTTTACAATCTCTAGCGTGCAGCCAGAGGACTTCGCCACATACTATTGC CAGCAGCACTACATCACCCCATGGACC TTCGGCGGCGGCACAAAGCTGGAGATCAAG (SEQ ID NO: 26) DETAILED DESCRIPTION OF THE INVENTION

[0086] Hereinafter, embodiments of the present invention will be described in detail with reference to examples. Those skilled in the art should understand that the following examples are merely illustrative of the present invention and do not limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, they will be performed according to techniques or conditions described in the literature in this field (see, for example, Sambrook J, et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press) or according to the product instructions. If the manufacturer of the reagents or equipment used is not specified, they may be commercially available general products. For example, 293T may be purchased from ATCC.

[0087] The BALB / c mice used in the following examples of the present invention were purchased from Guangdong Provincial Medical Experimental Animal Center.

[0088] In the following examples of the present invention, the positive control antibody RG6058 is used, and its sequence can be found in Sequence 34 and Sequence 36 of Chinese Patent Publication CN108290946A.

[0089] In the following examples of the present invention, the 293T-TIGIT cell line used was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-TIGIT cell line was generated by viral transfection of HEK293T cells, and the virus was prepared using 3rd Generation Lentiviral Systems. See, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was pCDH-CMV-PD-1FL-Puro (TIGIT has Genebank ID: NP_776160.2, and the vector pCDH-CMV-Puro was purchased from Youbao Bio and has the product number VT1480). [Example]

[0090] Example 1: Preparation of anti-TIGIT antibody 26B12 1. Preparation of hybridoma cell line LT019 The antigen used to prepare anti-TIGIT antibodies was human TIGIT-mFc (TIGIT Genbank ID: NP_776160.2). After immunization, splenocytes were collected from mice and fused with mouse myeloma cells to generate hybridoma cells. Using human TIGIT-mFc as the antigen, the hybridoma cells were screened by indirect ELISA to obtain hybridoma cells secreting antibodies that specifically bind to TIGIT. Stable hybridoma cell lines were isolated by limiting dilution. Each of the hybridoma cell lines was named LT019, and the monoclonal antibodies secreted by them were named 26B12.

[0091] The hybridoma cell line LT019 has been deposited at the China Center for Typical Culture Collection (CCTCC) since October 23, 2020, with the deposit number CTCCC NO: C2020208. Its former address is Wuhan University, Wuhan, China, with the postal code 430072.

[0092] 2. Preparation of anti-TIGIT antibody 26B12 The LT019 cell line was grown in CD medium (Chemical Defined Medium, containing 1% penicillin-streptomycin) and cultured at 37°C with 5% CO2. After 7 days, the cell culture supernatant was collected, centrifuged at high speed, vacuum filtered through a microporous membrane, and purified on a HiTrap protein A HP column to obtain antibody 26B12.

[0093] Example 2: Sequence analysis of anti-TIGIT antibody 26B12 mRNA was extracted from the LT019 cell line cultured in Example 1 using a cultured cell / bacteria total RNA extraction kit (Tiangen, product number DP430). cDNA was synthesized and PCR amplified according to the instructions attached to the Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR kit. TA cloning was performed directly on the PCR amplification product, and the specific procedure was performed by referring to the kit instructions for the pEASY-T1 Cloning Kit (Transgen CT101). The TA cloning product was directly sequenced, and the sequencing results were as follows: The nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 2, and the fragment length was 363 bp. The amino acid sequence it encoded was as shown in SEQ ID NO: 1 and was 121 amino acids in length. Of these, the sequence of heavy chain HCDR1 was as shown in SEQ ID NO:3, the sequence of HCDR2 was as shown in SEQ ID NO:4, and the sequence of HCDR3 was as shown in SEQ ID NO:5. The nucleic acid sequence of the light chain variable region was as shown in SEQ ID NO: 7 and was 321 bp in length. The amino acid sequence it encoded was as shown in SEQ ID NO: 6 and was 107 amino acids in length. Among them, the sequence of light chain LCDR1 is as shown in SEQ ID NO:8, the sequence of LCDR2 is as shown in SEQ ID NO:9, and the sequence of LCDR3 is as shown in SEQ ID NO:10.

[0094] Example 3: Design and preparation of the light and heavy chains of humanized anti-human TIGIT antibodies 1. Design of the light and heavy chains of the anti-human TIGIT humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 Based on the three-dimensional crystal structure of human TIGIT protein and the sequence of antibody 26B12 obtained from Example 2, an antibody model was simulated in a computer, and mutations were then designed based on the model to obtain the variable region sequences of antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 (the antibody constant region sequences were obtained from the NCBI database; the heavy chain constant regions were all from the Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant regions were from the Ig kappa chain C region, ACCESSION: P01834).

[0095] The designed variable region sequences are shown in Table A below.

[0096] [Table 1]

[0097] The nucleic acid sequences of the heavy chain variable regions of the above eight antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 were all 363 bp long and encoded amino acid sequences were all 121 aa long. The nucleic acid sequences of the light chain variable regions were all 321 bp long and encoded amino acid sequences were all 107 aa long. In addition, the above eight antibodies have the same HCDR1 to HCDR3 and LCDR1 to LCDR3, as follows: The sequence of HCDR1 was as shown in SEQ ID NO:3, the sequence of HCDR2 was as shown in SEQ ID NO:4, and the sequence of HCDR3 was as shown in SEQ ID NO:5. The sequence of LCDR1 was as shown in SEQ ID NO:8, the sequence of LCDR2 was as shown in SEQ ID NO:9, and the sequence of LCDR3 was as shown in SEQ ID NO:10.

[0098] 2. Preparation of humanized antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 The heavy chain constant region used in both cases was the Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region used in both cases was the Ig kappa chain C region, ACCESSION: P01834.

[0099] The 26B12H1L1 heavy chain cDNA and light chain cDNA, 26B12H4L1 heavy chain cDNA and light chain cDNA, 26B12H2L2 heavy chain cDNA and light chain cDNA, 26B12H3L2 heavy chain cDNA and light chain cDNA, 26B12H2L3 heavy chain cDNA and light chain cDNA, 26B12H3L3 heavy chain cDNA and light chain cDNA, 26B12H1L4 heavy chain cDNA and light chain cDNA, 26B12H2L4 heavy chain cDNA and light chain cDNA, and 26B12H4L4 heavy chain cDNA and light chain cDNA were cloned into pUC57simple (provided by GenScript) vectors, named pUC57simple-26B12H1, pUC57simple-26B12L1, and pUC57simple, respectively. -26B12H4, pUC57simple-26B12L1, pUC57simple-26B12H2, pUC57simple-26B12L2, pUC57simple-26B12H3, pUC57simple-26B12L2, pUC57simple-26B12H2, pUC57simple-26B12L3, pUC57simple-26B12H3, pUC57simple-26B12L3, pUC57simple-26B12H1, pUC57simple-26B12L4, pUC57simple-26B12H2, pUC57simple-26B12L4, and pUC57simple-26B12H4, pUC57simple-26B12L4 were obtained. According to the standard techniques described in "Molecular Cloning: A Laboratory Manual, 2nd Edition," the full-length synthetic heavy and light chain genes were digested with EcoRI and HindIII and subcloned into the expression vector pcDNA3.1 by digestion with restriction enzymes (EcoRI & HindIII). Expression plasmids pcDNA3.1-26B12H1, pcDNA3.1-26B12L1, pcDNA3.1-26B12H4, pcDNA3.1-126B12H2, pcDNA3.1-26B12L2, pcDNA3.1-26B12H3, pcDNA3.1-26B12L3, and pcDNA3.1-26B12L4 were obtained, and the heavy and light chain genes of the recombinant expression plasmids were subjected to sequencing analysis.Subsequently, the recombinant plasmids containing the corresponding light and heavy chains were subjected to gene combinations (pcDNA3.1-26B12H1 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H4 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H2 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H4 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H5 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H6 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H7 / pcDNA3.1-26B12L7, pcDNA3.1-26B12H8 / pcDNA3.1-26B12L8, pcDNA3.1-26B12H9 / pcDNA3.1-26B12L9, pcDNA3.1-26B12H10 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H11 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H12 / pcDNA3.1-26B12L2, pcDNA3.1-26B12H12 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H13 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H14 / pcDNA3.1-26B12L1, pcDNA3.1-26B12H15 / pcDNA3.1 The following vectors were designed: pcDNA3.1-26B12H2 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H3 / pcDNA3.1-26B12L3, pcDNA3.1-26B12H1 / pcDNA3.1-26B12L4, and pcDNA3.1-26B12H4 / pcDNA3.1-26B12L4. These vectors were co-transfected into 293F cells, and the culture medium was harvested and purified. After verifying the accuracy of the sequencing, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells to express the antibodies. After 7 days of incubation, the cell culture medium was harvested and affinity purified using a Protein A column to obtain the humanized antibodies.

[0100] Example 4: Measurement of antibody binding activity to antigen TIGIT-mFc by ELISA Experimental steps: A microplate was coated with 2 μg / mL goat anti-mouse IgG Fc and incubated at 4°C for 16 hours. After incubation, the goat anti-mouse IgG Fc-coated microplate was washed once with PBST and then blocked for 2 hours with 1% BSA in PBST as the blocking solution. After blocking, the microplate was washed three times with PBST. Then, 1 μg / mL of human TIGIT-mFc antigen was added and incubated at 37°C for 30 minutes. The plate was then washed three times with PBST. Gradient dilutions of antibodies in PBST were added to the wells of the microplate. The antibody dilution gradient is shown in Tables 1 and 2. The microplate containing the test antibody was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. After washing the plate, a working solution of HRP-conjugated goat anti-human IgG Fc secondary antibody diluted 1:5000 was added and incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST, and TMB (Neogen, 308177) was added and incubated for 4 minutes in the dark. The color reaction was terminated by adding stop solution. The microplate was immediately placed in a microplate reader and the OD of each well was read at a wavelength of 450 nm. Data analysis was performed using SoftMax Pro 6.2.1 software.

[0101] The results of antibody and antigen TIGIT-mFc binding are shown in Figures 1 and 2. The OD values ​​for each dosage are shown in Tables 1 and 2. Curve fitting was performed with the antibody concentration on the horizontal axis and the absorbance value on the vertical axis to determine the EC values ​​for antibody and antigen binding. 50 The results are shown in Tables 1 and 2 and Figures 1 and 2.

[0102] [Table 2]

[0103] [Table 3]

[0104] The results showed that antibodies 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 could all effectively bind to human TIGIT-mFc, the binding efficiency was dose-dependent and the binding activity was comparable to that of the positive control drug RG6058 for the same target, demonstrating that 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 have the function of effectively binding to TIGIT.

[0105] Example 5: Measurement of the activity of antibodies competing with CD155-hFc-Biotin for binding to TIGIT-mFc by competitive ELISA Experimental steps: TIGIT-mFc was coated onto a microplate at 2 μg / mL and incubated overnight at 4°C. After incubation, the antigen-coated microplate was washed once with PBST and then blocked for 2 hours using a 1% BSA solution in PBST as a microplate blocking solution. After blocking, the plate was washed three times with PBST. Antibodies gradient-diluted with PBST were added to the microplate. The antibody concentrations are shown in Tables 3 and 4. After incubation at room temperature for 10 minutes, an equal volume of 2 μg / mL (final concentration: 1 μg / mL) CD155-hFc-Biotin was added and mixed evenly with the antibody. The microplate was then incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. After washing, a 1:4000 diluted SA-HRP working solution was added and incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST, and then TMB (Neogen, 308177) was added to the plate for 5 minutes in the dark. The color reaction was terminated by adding stop solution. The microplate was immediately placed in a microplate reader, and the OD readings for each well were taken at 450 nm. Data analysis was performed using SoftMax Pro 6.2.1 software.

[0106] The activity results of antibodies competing with CD155-hFc-Biotin for binding to TIGIT-mFc are shown in Tables 3 and 4. Curve fitting was performed with the antibody concentration on the horizontal axis and the absorbance value on the vertical axis to determine the EC values ​​of antibodies competing with CD155-hFc-Biotin for binding to TIGIT-mFc. 50 The results are shown in Tables 3 and 4 below and Figures 3 and 4.

[0107] [Table 4]

[0108] [Table 5]

[0109] The results showed that under the same experimental conditions, 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4 and 26B12H4L4 could each compete with CD155-hFc-Biotin for binding to the antigen TIGIT-mFc, and their activity was comparable to that of the positive control drug RG6058 for the same target. This suggests that 26B12H1L1, 26B12H4L1, 26B12H2L2, 26B12H3L2, 26B12H2L3, 26B12H3L3, 26B12H1L4, and 26B12H4L4 have an effective function of competing with CD155-hFc-Biotin for binding to TIGIT-mFc.

[0110] Example 6: Measurement of kinetic parameters of binding of humanized antibodies 26B12H3L3, 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, 26B12H1L4, 26B12H4L1, and RG6058 to antigen TIGIT-mFc using a Fortebio molecular interaction analyzer The sample dilution buffer was PBS, 0.02% Tween-20, and 0.1% BSA, pH 7.4. TIGIT-mFc was immobilized on the AMC sensor at a concentration of 3 μg / mL for 50 s. The sensor was equilibrated with the buffer for 60 s. The immobilized TIGIT-mFc bound to the antibody at concentrations of 0.06-5 nM (3x dilution) for 120 s. The protein dissociated in the buffer for 300 s. The sensor was regenerated with 10 mM glycine at pH 1.7. The detection temperature was 37°C, the detection frequency was 0.3 Hz, and the sample plate vibration speed was 1000 rpm. The data were analyzed by fitting with a 1:1 model to obtain the affinity constant.

[0111] The results of measuring the affinity constants of the humanized antibodies and RG6058 (as a control antibody) to TIGIT are shown in Table 5, and the detection results are shown in Figures 5 to 13.

[0112] [Table 6]

[0113] The results show that the affinity constants of humanized antibodies 26B12H3L3, 26B12H1L1, 26B12H2L2, 26B12H2L3, 26B12H3L2, 26B12H4L4, 26B12H1L4, 26B12H4L1, and RG6058 to TIGIT-mFc are 9.64E-11 M, 1.64E-11 M, 8.40E-12 M, 4.85E-11 M, 5.40E-11 M, 3.69E-11 M, 4.63E-11 M, 8.57E-12 M, and 3.16E-11 M, respectively.

[0114] The results showed that the affinity of each TIGIT antibody for binding to TIGIT-mFc, from strongest to weakest, was 26B12H2L2, 26B12H4L1, 26B12H1L1, RG6058, 26B12H4L4, 26B12H1L4, 26B12H2L3, 26B12H3L2, and 26B12H3L3. Of these, the affinity of the humanized antibodies 26B12H2L2, 26B12H4L1, and 26B12H1L1 was stronger than that of the positive control drug RG6058, and the affinity of 26B12H4L4 was equivalent to that of the positive control drug RG6058.

[0115] Example 7: Detection of binding activity of humanized antibodies 26B12H2L2 and RG6058 to the 293T-TIGIT cell membrane surface antigen TIGIT by FACS Experimental Method: The TIGIT vector pLenti6.3 / V5-TIGITFL-BSD (vector pLenti6.3 was purchased from Invitrogen) was transfected into 293T cells, and a cell line stably expressing TIGIT, 293T-TIGIT cells, was obtained by screening.

[0116] 293T-TIGIT cells (DMEM + 10% FBS) were harvested and centrifuged for 5 minutes. The supernatant was removed and resuspended. The cell count and viability (P7, 95.79%) were calculated. The cells were diluted and placed in a 96-well clear V-bottom plate (30 μL per well). 200 μL of 1% PBSA was added to each tube. The cells were centrifuged for 5 minutes and the supernatant was removed. Based on the experimental design, 100 μL of antibody (final concentrations: 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM) was added to each well. Blank and isotype controls were also included. The cells were incubated on ice for 60 minutes. 200 μL of 1% PBSA was added to each tube, centrifuged for 5 minutes, the supernatant removed, and the tubes were washed twice. FITC goat anti-human IgG antibody (500-fold diluted with PBSA) was added to each sample and incubated on ice for 40 minutes in the dark. 200 μL of PBSA was added to each tube, centrifuged for 5 minutes, and the supernatant removed. The cells were resuspended in 200 μL of PBSA and transferred to flow cytometry tubes. The mean fluorescence intensity of the cells at each concentration was measured using a flow cytometer.

[0117] [Table 7]

[0118] The experimental results, as shown in Table 6 and Figure 14, show that the EC of the positive control antibody RG6058, which binds to the cell membrane surface antigen TIGIT, 50 The EC value of the humanized antibody 26B12H2L2, which binds to the cell membrane surface antigen TIGIT-, is 1.257 nM. 50 was 0.917nM.

[0119] The experimental results show that the binding ability of the humanized antibody 26B12H2L2 to the cell membrane surface antigen TIGIT is stronger than that of the positive control antibody RG6058.

[0120] Example 8: Detection of the competitive activity of humanized antibodies 26B12H2L2 and RG6058 with CD155 or CD112 for binding to the 293T-TIGIT cell membrane surface antigen TIGIT by FACS Experimental method: 293T-TIGIT cells were harvested, centrifuged for 5 minutes, the supernatant removed, and resuspended. The cell count and viability (94.95%) were calculated. The cells were diluted and placed in a 96-well clear V-bottom plate (30 μL per well). 200 μL of 1% PBSA was added to each well. The cells were centrifuged for 5 minutes, and the supernatant removed. Based on the experimental design, 100 μL of antibody (final concentrations: 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM) was added to each well. Blank and isotype controls were also included. The cells were incubated on ice for 30 minutes. CD155 (final concentration: 10 nM) or CD112 (final concentration: 30 nM) was added to each sample and incubated on ice in the dark for 60 min. Then, 200 μL of 1% PBSA was added to each tube, followed by centrifugation for 5 min, removal of the supernatant, and two washes. APC goat anti-mouse IgG (minimal x-reactivity) antibody (diluted 1:300 in PBSA) was added to each sample and incubated on ice in the dark for 40 min. Then, 200 μL of PBSA was added to each tube, followed by centrifugation for 5 min, removal of the supernatant, and the cells were resuspended in 200 μL of PBSA and transferred to flow cytometry tubes. The mean fluorescence intensity of the cells at each concentration was measured using a flow cytometer.

[0121] The experimental results are shown in Table 7 and Figure 15, and Table 8 and Figure 16, respectively.

[0122] [Table 8]

[0123] [Table 9]

[0124] Results show that the EC of the positive control antibody RG6058, which competes with CD155 for binding to TIGIT, 50 The EC of humanized antibody 26B12H2L2, which competes with CD155 for binding to TIGIT, was 1.212 nM.50 The EC value of the positive control antibody RG6058, which competes with CD112 for binding to TIGIT, was 1.049 nM. 50 The EC of humanized antibody 26B12H2L2, which competes with CD112 for binding to TIGIT, was 1.224 nM. 50 This shows that the concentration is 1.140 nM. The results showed that the ability of humanized antibody 26B12H2L2 to compete with CD155 or CD112 for binding to the cell membrane surface antigen TIGIT was stronger than that of the positive control antibody RG6058.

[0125] Example 9: Mixed lymphocyte reaction of Jurkat-TIGIT and HT1080-aCD3scFv cell lines with TIGIT antibody Experimental Method: The TIGIT vector plenti6.3 / V5-TIGITFL-BSD (vector pLenti6.3 purchased from Invitrogen) was transfected into Jurkat cells and screened to obtain the cell line Jurkat-TIGIT cells stably expressing TIGIT. The anti-CD3 antibody vector pCDH-aCD3scFv-puro (vector pCDH-CMV-MCS-EF1-Puro purchased from Youbao Bio) was transfected into HT-1080 cells and screened to obtain the cell line HT1080-aCD3scFv cells stably expressing anti-CD3scFv on the cell membrane.

[0126] Jurkat-TIGIT and HT1080-aCD3scFv cells were harvested during logarithmic growth and placed in a 96-well plate. 5W Jurkat-TIGIT cells were added to each well, and 1W HT1080-aCD3scFV cells were added to each well. Diluted antibodies (final concentrations of 10nM, 50nM, and 250nM) and anti-human CD28 antibody (3μg / mL) were added. The cells were then cultured in an incubator for 48 hours. The culture supernatants were then harvested and the IL-2 content was assayed using an IL-2 ELISA kit.

[0127] The experimental results are shown in FIG. The results show that both the humanized antibody 26B12H2L2 and the positive control antibody RG6058 can promote IL-2 secretion in the system, and that the IL-2 secretion-promoting level of the humanized antibody 26B12H2L2 at each concentration (10 nM, 50 nM, 250 nM) is equivalent to that of RG6058. The results demonstrate that the ability of humanized antibody 26B12H2L2 to induce cells to secrete IL-2 is comparable to that of the positive control antibody RG6058.

[0128] Example 10: Therapeutic effect of 26B12H2L2 inoculated into hTigit-BALB / c transgenic mice with CT26 mouse tumor xenografts hTigit-BALB / c transgenic mice (purchased from Jiangsu Jixi Yaokang Biotechnology Co., Ltd., in which the normal mouse TIGIT gene had been replaced with the human TIGIT gene) were inoculated with 500,000 CT26 cells (a mouse colon cancer cell line purchased from ATCC) in the dorsal region. Specifically, 100 μL of CT26 cells (50 million / mL) were inoculated into each mouse to establish a mouse tumor model. Each group consisted of eight mice, divided as follows:

[0129] Isotype control group G1: dosage 20 mg / kg, administration route intraperitoneal injection (ip), twice a week. Experimental group G2: the dosage was 4 mg / kg, and the administration route was intraperitoneal injection (ip), twice a week. Experimental group G3: the dosage was 20 mg / kg, and the administration route was intraperitoneal injection (ip), twice a week. Positive control group G4: the dosage was 20 mg / kg, and the administration method was intraperitoneal injection (ip), twice a week.

[0130] The specific plan is shown in Table 9.

[0131] [Table 10]

[0132] The experimental results are shown in FIG. The results show that 26B12H2L2 and RG6058 significantly reduced tumor volume in the hTIGIT-BALB / c transgenic mouse CT26 tumor model compared with the isotype control. The results showed that 26B12H2L2 had strong efficacy in the hTIGIT-BALB / c transgenic mouse CT26 tumor model, with efficacy comparable to that of RG6058, suggesting that 26B12H2L2 may be used in the treatment and / or prevention of tumors, especially colon cancer. At the same time, as shown in FIG. 19, 26B12H2L2 did not affect the body weight of hTIGIT-BALB / c transgenic mice, a CT26 tumor model, indicating that the 26B12H2L2 antibody did not cause toxic reactions or side effects in mice.

[0133] Although specific embodiments of the present invention have been described in detail, it is understood by those skilled in the art that, according to all the teachings disclosed, various modifications and substitutions to these details are possible, and all such modifications fall within the scope of protection of the present invention. The full scope of the present invention is defined by the appended claims and any equivalents thereof.

Claims

1. the heavy chain variable region comprises HCDR1 to HCDR3, whose amino acid sequences are set forth in SEQ ID NOS: 3 to 5, respectively, and the light chain variable region comprises LCDR1 to LCDR3, whose amino acid sequences are set forth in SEQ ID NOS: 8 to 10, respectively; An anti-TIGIT antibody or an antigen-binding fragment thereof.

2. the amino acid sequence of the heavy chain variable region of the antibody is selected from SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17; and the amino acid sequence of the light chain variable region of the antibody is selected from SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25; The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 1.

3. the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 11, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 19; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 19; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 21; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 21; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 23; the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 11 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 25; or The amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 17, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:

25. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 2.

4. The anti-TIGIT antibody or antigen-binding fragment thereof may be Fab, Fab', F(ab'), 2 , Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody or diabody, The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The region other than the CDR contained in the antibody is derived from a human antibody. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. the heavy chain constant region of the antibody is an Ig gamma-1 chain C region or an Ig gamma-4 chain C region, and the light chain constant region is an Ig kappa chain C region; The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The anti-TIGIT antibody is an antibody produced by the hybridoma cell line LT019, which has been deposited at the China Center for Type Culture Collection (CCTCC) under the deposit number CTCCC NO: C2020208. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A vector encoding the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 7. Isolated nucleic acid molecule.

9. The isolated nucleic acid molecule of claim 8. vector.

10. An isolated nucleic acid molecule according to claim 8, or a vector according to claim 9. host cell.

11. It has been deposited at the China Typical Culture Collection Center (CCTCC) and its deposit number is CTCCC NO: C2020208. Hybridoma cell line LT019.

12. A conjugate comprising an antibody and a coupling moiety, wherein the antibody is the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and the coupling moiety is a detectable label. Complex.

13. A kit comprising the anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the complex according to claim 12, The kit further comprises a secondary antibody that specifically recognizes the antibody, the secondary antibody further comprising a detectable label. kit.

14. In preparing a kit for use in detecting the presence or level of TIGIT in a sample, Use of the antibody according to any one of claims 1 to 7 or the complex according to claim 12.

15. A bispecific antibody comprising a first protein functional region and a second protein functional region, wherein: the first protein functional region targets TIGIT; the second protein functional region targets a target different from TIGIT; Among them, the first protein functional region is an antibody or antigen-binding fragment according to any one of claims 1 to 7, bispecific antibody. **Claim 16**: A bispecific antibody according to claim 15, characterized by one or more of the following: (1) A target different from TIGIT is PD-1; (2) The bispecific antibody is in the IgG-scFv mode; (3) The first protein functional region is an antibody according to any one of claims 1 to 7, and is in immunoglobulin form, and the second protein functional region is a single-chain antibody, and the single-chain antibody is linked to the C termini of the two heavy chains of the antibody in immunoglobulin form respectively; (4) The first protein functional region is a single-chain antibody, and the second protein functional region is an antibody in immunoglobulin form targeting a target different from TIGIT, and the single-chain antibody is linked to the C termini of the two heavy chains of the antibody in immunoglobulin form respectively; (5) The first protein functional region and the second protein functional region are directly linked or linked by a linking fragment; (6) The first protein functional region and the second protein functional region are one, two or more than two independently of each other. **Claim 17**: A pharmaceutical composition comprising an anti-TIGIT antibody or its antigen-binding fragment according to any one of claims 1 to 7 or a complex according to claim 12, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable vector and / or excipient, pharmaceutical composition. **Claim 18** The pharmaceutical composition according to claim 17, further comprising one or more anti-PD-1 antibodies, or one or more anti-PD-L1 antibodies. **Claim 19**: The pharmaceutical composition according to claim 18, wherein the mass ratio of the anti-TIGIT antibody or its antigen-binding fragment to the anti-PD-1 antibody or anti-PD-L1 antibody is (1:5) to (5:1) calculated by the mass of the antibody. **Claim 20** A combination product comprising a first product and a second product packaged independently of each other, wherein the first product comprises an anti-TIGIT antibody or its antigen-binding fragment according to any one of claims 1 to 7, a complex according to claim 12 or a pharmaceutical composition according to any one of claims 17 to 19, and the second product comprises at least one anti-PD-1 antibody or at least one anti-PD-L1 antibody. ​ ​ ​ The first product and the second product further independently comprise one or more pharmaceutically acceptable adjuvants. Combination products.

21. the mass ratio of the anti-TIGIT antibody or antigen-binding fragment thereof to the anti-PD-1 antibody or anti-PD-L1 antibody is between 1:5 and 5:1, calculated based on the mass of the antibodies; 21. The combination product of claim 20.

22. In the preparation of a medicament for treating and / or preventing a tumor, Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the conjugate according to claim 12, the bispecific antibody according to any one of claims 15 to 16, or the pharmaceutical composition according to any one of claims 17 to 19.

23. The use according to claim 22, wherein the tumor is selected from one or more of liver cancer, kidney cancer, brain tumor, urothelial carcinoma, bone tumor, bile duct cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, malignant melanoma, pancreatic cancer, cervical tumor, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, plasmacytoma, endometrial cancer, prostate cancer and testicular cancer.

24. Used to treat and / or prevent tumors, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or the bispecific antibody according to any one of claims 15 to 16.

25. The antibody or antigen-binding fragment thereof, or bispecific antibody described in claim 24, wherein the tumor is selected from one or more of liver cancer, kidney cancer, brain tumor, urothelial carcinoma, bone tumor, bile duct cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, ovarian cancer, colorectal cancer, malignant melanoma, pancreatic cancer, cervical tumor, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, plasmacytoma, endometrial cancer, prostate cancer, and testicular cancer.

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