Bispecific antibodies against TIGIT and PD-L1, pharmaceutical compositions thereof and uses thereof

Abstract

The present invention belongs to the field of biomedicine and relates to a bispecific antibody against TIGIT and PD-L1, and its pharmaceutical composition and its use. The bispecific antibody of the present invention comprises a first protein functional region targeting TIGIT and a second protein functional region targeting PD-L1, wherein the first protein functional region is an anti-TIGIT antibody or an antigen-binding fragment thereof, the heavy chain variable region of the anti-TIGIT antibody comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 10, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 11, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 12, and the light chain variable region of the anti-TIGIT antibody comprises an LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, an LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 15. The bispecific antibody of the present invention has high affinity to dual targets, i.e., TIGIT and PD-L1, and has good applicability.

Description

Detailed Description of the Invention

[0001] Technical Field The present invention relates to an anti-TIGIT / anti-PD-L1 bispecific antibody, its pharmaceutical composition, and its use.

[0002] Background technology The T cell immunoreceptor with Ig and ITIM domains (TIGIT; also known as WUCAM, Vstm3, and VSIG9) mediates the expression of activated CD8 + T cells and CD4 + It is a new type of immunosuppressive receptor expressed by T cells, natural killer (NK) cells, regulatory T cells (Treg), and follicular helper T cells.

[0003] TIGIT is involved in a complex regulatory network in tumor immunity, including multiple immunosuppressive receptors (e.g., CD96 / TACTILE, CD112R / PVRIG), competitive co-stimulatory receptors (DNAM-1 / CD226) and multiple ligands (e.g., CD155 (PVR / NECL-5), CD112 (Nectin-2 / PVRL2)). DNAM-1, TIGIT, and CD96 are expressed on T cells and NK cells and share CD155 as a ligand.

[0004] The role of TIGIT in tumor immunosuppression is similar to that of PD-1 / PD-L1. Current studies have proposed several mechanisms by which effector T cells and NK cells are inhibited by TIGIT. (1) TIGIT binding to CD155 on the surface of T / NK cells phosphorylates immunoreceptor tyrosine inhibitory motifs (ITIMs) in TIGIT cells, directly transmitting inhibitory signals. (2) TIGIT binding to CD155 on DCs promotes the production of tolerogenic DCs, reduces interleukin (IL) 12 production, and increases IL-10, thereby indirectly inhibiting T cell responses. (3) Inhibitory TIGIT competitively binds to CD155 with higher affinity than that of the costimulatory receptor CD226, thereby limiting CD226-mediated activation, and in addition, TIGIT also directly binds to CD226 in a cis-like manner on cells, thereby disrupting the ability of TIGIT to bind to CD155 homodimers. (4) TIGIT-expressing Tregs are highly inhibitory, and upon activation, TIGIT+ Tregs produce IL-10 and fibrinogen-like protein 2 (Fgl2), which mediate T cell inhibition. (5) The Fap2 protein from Fusobacterium nucleatum (an anaerobic gram-positive commensal bacterium associated with colorectal cancer) can directly bind to TIGIT, but not to CD226, thereby inhibiting tumor immunity mediated by NK cells and T cells and modulating the innate immune response.

[0005] Programmed cell death 1 ligand 1 (PD-L1), also known as CD274, is a member of the B7 family and a ligand of PD-1. PD-L1 is a type I transmembrane protein with a total of 290 amino acids, and contains one IgV-like region, one IgC-like region, one hydrophobic transmembrane region, and one intracellular region of 30 amino acids. Unlike other B7 family molecules, PD-L1 has the effect of negatively regulating immune response. Research has found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages, and dendritic cells. In addition to lymphocytes, PD-L1 is also expressed in various other tissues, such as intradermal cells in the thymus, heart, placenta, and in various types of non-lymphoid cells, such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, and head and neck cancer cells (Akintunde Akinleye & Zoaib Rasool, Journal of Hematology & Oncology Volume 12, Article number: 92 (2019)). PD-L1 has a broad range of effects in regulating autoreactive T and B cells and immune tolerance, and plays a role in T and B cell responses in peripheral tissues. High expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.

[0006] Bispecific antibodies are a direction for antibody drug development, but they face many challenges, such as large differences in preclinical evaluation models, low expression levels, low stability, complex processes, and quality control. Therefore, the research and development of bispecific antibodies has always been difficult.

[0007] Therefore, there is a need to develop a bispecific antibody targeting TIGIT and PD-L1 that has good specificity, good efficacy and is easy to prepare.

[0008] Contents of the Invention After thorough research and creative work, the inventors obtained an anti-TIGIT antibody, and constructed an anti-TIGIT / anti-PD-L1 bispecific antibody based on this antibody. The inventors surprisingly found that the anti-TIGIT antibody of the present invention (also referred to as antibody or antibody of the present invention) and the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (also referred to as bispecific antibody or bispecific antibody of the present invention), which have high binding affinity to PD-L1 and TIGIT (the bispecific antibody is even better than the positive control antibody of one or more embodiments), can block the binding of PD-L1 to its ligand PD-1 and the binding of TIGIT to its ligand CD155 / CD112, respectively, thereby reducing or eliminating the transmission of inhibitory signals to cells, and the administration of the antibody of the present invention can significantly inhibit tumor growth in animal models. Thus, the following invention is provided:

[0009] In one aspect of the present invention, A first protein functional region that targets TIGIT, and A second protein functional region that targets a different target than TIGIT (e.g., PD-L1) A bispecific antibody comprising: the first protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof; the heavy chain variable region of the anti-TIGIT immunoglobulin comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 10, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 12; A bispecific antibody is designed in which the light chain variable region of the anti-TIGIT immunoglobulin comprises LCDR1 having the amino acid sequence shown in SEQ ID NO: 13, LCDR2 having the amino acid sequence shown in SEQ ID NO: 14, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 15.

[0010] The variable regions of the light and heavy chains determine the binding to antigen, and the variable region of each chain contains three hypervariable regions called complementarity determining regions (CDRs), of which the CDRs of the heavy chain (H) contain HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) contain LCDR1, LCDR2, and LCDR3. The CDRs contained in the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment of the present invention are preferably determined according to the Kabat, Chothia, AbM HVR or IMGT numbering system. Unless otherwise stated, the CDRs contained in the antibody or antigen-binding fragment of the present invention are preferably determined according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. (1991)).

[0011] In some embodiments of the invention, in a bispecific antibody, the first protein functional region and the second protein functional region are directly linked or linked via a linker (also referred to as a linker peptide), Preferably, the linker is (GGGGS) m and m is a positive integer, for example, 1, 2, 3, 4, 5, or 6; Preferably, the linker is (GGGGS) n G, n is a positive integer, for example, 1, 2, 3, 4, 5, or 6; Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO:2, Here, the GGGGS in parentheses is represented by SEQ ID NO:22.

[0012] In some embodiments of the invention, in a bispecific antibody the number of first protein functional regions and second protein functional regions is independently one, two or more than two.

[0013] In some embodiments of the invention, the number of first protein functional regions is one and the number of second protein functional regions is two in the bispecific antibody.

[0014] In some embodiments of the invention, the number of first protein functional regions is two and the number of second protein functional regions is one in the bispecific antibody.

[0015] In some embodiments of the invention, in the bispecific antibody: the first protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof, and the second protein functional region is a single domain antibody or a single chain antibody that targets a target different from TIGIT; Preferably, the single domain antibody is an anti-PD-L1 single domain antibody, Preferably, the single chain antibody is an anti-PD-L1 single chain antibody.

[0016] In some embodiments of the invention, in the bispecific antibody: The first protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof, and the second protein functional region is an anti-PD-L1 single domain antibody or an anti-PD-L1 single chain antibody.

[0017] In some embodiments of the invention, in the bispecific antibody: the first protein functional region is an anti-TIGIT single chain antibody and the second protein functional region is an immunoglobulin or antigen-binding fragment thereof, the target of which is different from TIGIT; Preferably, the immunoglobulin targeting a target different from TIGIT is an anti-PD-L1 immunoglobulin.

[0018] In some embodiments of the invention, in the bispecific antibody: The first protein functional region is an anti-TIGIT single chain antibody and the second protein functional region is an anti-PD-L1 immunoglobulin or an antigen-binding fragment thereof.

[0019] In some embodiments of the invention, in the bispecific antibody: the anti-PD-L1 single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 16, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 18; Preferably, the amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:3.

[0020] In some embodiments of the invention, in the bispecific antibody: For the anti-TIGIT immunoglobulin, the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 19, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 20; Preferably, For the anti-TIGIT immunoglobulin, the amino acid sequence of the heavy chain is shown in SEQ ID NO:1 and the amino acid sequence of the light chain is shown in SEQ ID NO:5.

[0021] In some embodiments of the invention, in the bispecific antibody, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region fragments.

[0022] In some embodiments of the invention, in the bispecific antibody: The constant region of the anti-TIGIT immunoglobulin or of the immunoglobulin that targets a target different from TIGIT is derived from a human antibody, Preferably, the constant region is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.

[0023] In some embodiments of the invention, in the bispecific antibody: In an anti-TIGIT immunoglobulin or an immunoglobulin targeting a target different from TIGIT, the heavy chain constant region is a human Ig gamma 1 chain C region (e.g., NCBI ACCESSION: P01857) or a human Ig gamma 4 chain C region (e.g., NCBI ACCESSION: P01861.1), and the light chain constant region is a human Ig kappa chain C region (e.g., NCBI ACCESSION: P01834), and preferably the heavy chain constant region of the anti-TIGIT immunoglobulin also comprises the L234A and L235A mutations according to the EU numbering system.

[0024] In some embodiments of the invention, in a bispecific antibody, the single domain antibodies or single chain antibodies are bound to the C-terminus or N-terminus of an anti-TIGIT immunoglobulin, for example, the number of single domain antibodies or single chain antibodies is two, and one end of each single domain antibody or single chain antibody is bound to the C-terminus or N-terminus, respectively, of two heavy chains of the anti-TIGIT immunoglobulin.

[0025] In some embodiments of the invention, in the bispecific antibody, the anti-PD-L1 single domain antibodies or anti-PD-L1 single chain antibodies are linked to the C-terminus or N-terminus of the anti-TIGIT immunoglobulin, for example, the number of anti-PD-L1 single domain antibodies or anti-PD-L1 single chain antibodies is two, and one end of each anti-PD-L1 single domain antibody or anti-PD-L1 single chain antibody is linked to the C-terminus or N-terminus, respectively, of the two heavy chains of the anti-TIGIT immunoglobulin.

[0026] In some embodiments of the invention, in the bispecific antibody, the single domain antibody is an anti-PD-L1 single domain antibody, and the peptide chain obtained by binding the single domain antibody to an anti-TIGIT immunoglobulin has the amino acid sequence shown in SEQ ID NO:4.

[0027] In some embodiments of the invention, the bispecific antibody is a tetramer formed by two identical first peptide chains and two identical second peptide chains, wherein: The amino acid sequence of the first peptide chain is shown in SEQ ID NO:4, and the amino acid sequence of the second peptide chain is shown in SEQ ID NO:5.

[0028] In some embodiments of the invention, the bispecific antibody comprises: A first protein functional region that targets TIGIT, and a second protein functional region that targets PD-L1; the number of first protein functional regions is 1 and the number of second protein functional regions is 2; wherein the first protein functional region is an anti-TIGIT immunoglobulin and the second protein functional region is an anti-PD-L1 single domain antibody; For the anti-TIGIT immunoglobulin, the amino acid sequence of the heavy chain is shown in SEQ ID NO:1 and the amino acid sequence of the light chain is shown in SEQ ID NO:5; The amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:3. The two anti-PD-L1 single domain antibodies are linked to the C-terminus of each heavy chain of the anti-TIGIT immunoglobulin by the same or different linkers, Preferably, the linker is (GGGGS) m and m is a positive integer, for example, 1, 2, 3, 4, 5, or 6; Preferably, the linker is (GGGGS) n G, n is a positive integer, for example, 1, 2, 3, 4, 5, or 6; Preferably, the amino acid sequence of the linker is as shown in SEQ ID NO:2, Here, the GGGGS in parentheses is represented by SEQ ID NO:22.

[0029] In one or more embodiments of the invention, the bispecific antibody is of the IgG-VHH type.

[0030] In one or more embodiments of the invention, the bispecific antibody is of the IgG-scFv type, i.e., Morrison pattern.

[0031] In one or more embodiments of the present invention, in a bispecific antibody, a single domain antibody or a single chain antibody is attached to the C-terminus of an immunoglobulin heavy chain. Since an immunoglobulin consists of two heavy chains, one immunoglobulin molecule is attached to two single domain antibody molecules or two single chain antibody molecules. Preferably, the two single domain antibody molecules are identical. Preferably, the two single chain antibody molecules are identical. Preferably, the single domain antibody or single chain antibody is attached to the C-terminus of an immunoglobulin heavy chain via an amide bond formed by the aforementioned linker.

[0032] In one or more embodiments of the invention, for a bispecific antibody, its monovalent affinity for binding to human PD-L1 antigen, cynomolgus PD-L1 antigen, human TIGIT antigen, and / or cynomolgus TIGIT antigen is equivalent to or better than the monovalent affinity of the single-end antibody molecule for binding to anti-human PD-L1 antigen, anti-cynomolgus PD-L1 antigen, anti-human TIGIT antigen, and / or anti-cynomolgus TIGIT antigen, respectively.

[0033] Another aspect of the invention relates to an isolated nucleic acid molecule encoding a bispecific antibody according to any one of the sections of the invention.

[0034] A further aspect of the invention pertains to a vector comprising the isolated nucleic acid molecule of the invention.

[0035] A further aspect of the present invention relates to a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention.

[0036] A further aspect of the invention relates to a method for preparing a bispecific antibody according to any one of the invention articles, comprising the steps of culturing a host cell of the invention under suitable conditions and recovering the bispecific antibody from the cell culture.

[0037] Another aspect of the present invention relates to a conjugate comprising a bispecific antibody and a conjugation moiety, wherein the bispecific antibody is a bispecific antibody according to any one of the sections of the present invention, and the conjugation moiety is a detectable label, preferably the conjugation moiety is a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme.

[0038] Another aspect of the invention is a kit comprising a bispecific antibody according to any one of the items of the invention or a conjugate of the invention, Preferably, the kit further comprises a second antibody capable of specifically binding to the bispecific antibody, and optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance or an enzyme.

[0039] Another aspect of the invention relates to a pharmaceutical composition comprising a bispecific antibody according to any one of the items of the invention or a conjugate of the invention, optionally further comprising a pharma- ceutically acceptable excipient.

[0040] Another aspect of the invention relates to the use of a bispecific antibody according to any one of the sections of the invention or a conjugate of the invention in the manufacture of a medicament for the prevention and / or treatment of a malignant tumor, preferably selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer.

[0041] Yet another aspect of the present invention relates to a method for treating and / or preventing a malignant tumor comprising the step of administering an effective amount of a bispecific antibody according to any one of the items of the present invention or a conjugate of the invention to a subject in need thereof, preferably wherein the malignant tumor is selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer.

[0042] In some embodiments of the invention, in the method for treating and / or preventing malignant tumors, the step of administering an effective amount of a bispecific antibody according to any one of the sections of the invention to a subject in need thereof is performed before or after surgical treatment and / or before or after radiation therapy.

[0043] In some embodiments of the invention, in a method of treating and / or preventing a malignant tumor, the method comprises: The single dose of the bispecific antibody of the present invention is 0.1 to 100 mg, preferably 4.8 to 24 mg or 1 to 10 mg per kilogram of body weight, or the single dose of the bispecific antibody of the present invention is 10 to 1000 mg, preferably 50 to 500 mg, 100 to 400 mg, 150 to 300 mg, 150 to 250 mg, or 200 mg per kilogram of body weight, Preferably, administration is performed every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks or every 3 weeks; Preferably, administration is by intravenous drip or intravenous injection.

[0044] The bispecific antibody according to any one of the sections of the invention or the conjugate of the invention is used for the treatment and / or prevention of malignant tumors, preferably the malignant tumors are selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer.

[0045] In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the cell culture, molecular genetics, nucleic acid chemistry and immunological experimental procedures used herein are common procedures that are widely used in the corresponding fields.Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0046] "EC 50 The term "concentration" refers to the concentration that produces 50% of the maximum effect.

[0047] The term "antibody" refers to an immunoglobulin molecule, usually composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the antibody's isotype can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. In the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells), and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into highly variable regions called complementarity determining regions (CDRs) and interspersed, more conserved regions called framework regions (FRs). VH and VL each consist of three CDRs and four FRs arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy / light chain pair each form an antibody binding site.For assignment of amino acids to regions or domains, see Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or the definitions in Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al., Nature, 1989; 342: 878-883; or according to the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF [J]. Nucleic acids research, 2009; 38(suppl_1): of D301-D307.

[0048] The term "antibody" is not limited to any particular method of producing an antibody, including, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be of various isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

[0049] The terms "mcAb" and "monoclonal antibody" refer to an antibody or antibody fragment from a group of highly homologous antibody molecules, i.e., a group of antibody molecules identical except for naturally occurring natural mutations that may occur naturally. Monoclonal antibodies are highly specific to a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least two or more different antibodies, and such different antibodies usually recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. nature, 1975; Vol. 256(No. 5517): 495), but can also be obtained using recombinant DNA technology (see, for example, U.S. Patent No. 4,816,567).

[0050] The term "single chain antibody (single chain variable fragment, ScFv)" refers to antibody heavy chain variable regions (V H ) and the antibody light chain variable region (V L ), where V L Domains and V H The domains can pair and form monovalent molecules via linkers, resulting in the generation of a single polypeptide chain (see, e.g., Bird et al., Science 1988; 242: 423 426 and Huston et al., Proc. Natl. Acad. Sci. USA 1988; 85: 5879 5883). Such scFv molecules include the NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V LThe linker may have the general structure of -COOH.Suitable conventional linkers may consist of repeating GGGGS amino acid sequence or its variants.For example, the linker with amino acid sequence (GGGGS)4 (SEQ ID NO:23) may be used, but its variants may also be used (Holliger et al., Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448). Other linkers useful in the present invention are described by Alfthan et al., Protein Eng. 1995; 8: 725-731, Choi et al., Eur. J. Immunol. 2001; 31: 94-106, Hu et al., Cancer Res. 1996; 56: 3055-3061, Kipriyanov et al., J. Mol. Biol. 1999; 293: 41-56, and Roovers et al., Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59. Description.

[0051] As used herein, the term "isolated" or "isolating" refers to being obtained by artificial means from a natural state. If an "isolated" substance or component is naturally occurring, it may be due to a change in its natural environment, or the substance is separated from its natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide naturally occurs in a living animal, and the same polynucleotide or polypeptide of high purity isolated from this natural state is called an "isolated" polynucleotide or polypeptide. The term "isolated" or "isolating" does not exclude artificial or synthetic mixtures, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0052] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, thereby 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; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages, such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Vectors can include a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors can also include an origin of replication.

[0053] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells, such as Escherichia 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, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0054] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and a target antigen. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) has a specific binding affinity of about 10 -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 Affinity (K D ) refers to an antibody that binds to an antigen.

[0055] As used herein, "K D The term "antibody-antigen binding affinity" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and 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. Typically, antibodies have a dissociation equilibrium constant of about 10 -5 Less than m, e.g., about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 The dissociation equilibrium constant (K D ) binds to an antigen (e.g., TIGIT protein or PD-L1 protein). D can be determined using methods known to those of skill in the art, for example, using a Fortebio molecular interaction analyzer.

[0056] As used herein, the terms "monoclonal antibody" and "McAb" have the same meaning and are used interchangeably. The terms "polyclonal antibody" and "PcAb" also have the same meaning and are used interchangeably. In addition, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0057] As used herein, the term "pharmaceutical acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient. It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting substances, surfactants, adjuvants, ionic strength enhancing substances. For example, pH adjusting substances include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancing substances include, but are not limited to, sodium chloride.

[0058] As used herein, the term "effective amount" refers to an amount sufficient to at least partially obtain a desired effect. For example, a prophylactically effective amount is an amount sufficient to prevent, inhibit or delay the onset of a disease (e.g., tumor), and a therapeutically effective amount is an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already have the disease. The determination of such an effective amount is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the overall condition of the patient, such as age, weight, and sex, the method of administering the drug, and other treatments used in combination, etc.

[0059] As used herein, when referring to the amino acid sequence of PD-L1 protein (programmed cell death ligand 1, NCBI GenBank ID: NP_054862.1), it includes the full-length human PD-L1 protein, or the human PD-L1 extracellular fragment PD-L1 ECD (e.g., the amino acid sequence shown in SEQ ID NO: 6) or a fragment comprising PD-L1 ECD, and also includes fusion proteins of PD-L1 ECD, such as fragments fused to mouse or human IgG Fc protein fragments (mFc or hFc). However, one of skill in the art will appreciate that mutations or changes (including but not limited to substitutions, deletions, and / or additions) in the amino acid sequence of the PD-L1 protein can occur naturally or be artificially introduced without affecting biological function. Thus, in the present invention, the term "PD-L1 protein" is intended to include any such sequence, including the sequences shown and natural or artificial variants thereof. Moreover, when describing a sequence fragment of PD-L1 protein, it includes not only the sequence fragment but also the sequence fragment corresponding to this natural or artificial variant.

[0060] As used herein, when referring to the amino acid sequence of TIGIT protein (T cell immunoreceptor with Ig domain and ITIM domain, NCBI GenBank ID: NP_776160.2), it includes the full-length human TIGIT protein, or the extracellular fragment of human TIGIT, TIGIT ECD (e.g., the amino acid sequence shown in SEQ ID NO: 8), or a fragment containing TIGIT ECD, and also includes a full-length fusion protein of TIGIT protein or a fusion protein of TIGIT ECD, such as a fragment fused to Fc protein fragment (mFc or hFc) of mouse or human IgG. However, those skilled in the art understand that mutations or changes (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of TIGIT protein can occur naturally or be artificially introduced without affecting its biological function. Thus, in the present invention, the term "TIGIT protein" is intended to include any such sequence, including its natural or artificial variants. Moreover, when describing a sequence fragment of TIGIT protein, it also includes a sequence fragment corresponding to this natural or artificial variant.

[0061] As used herein, when referring to the amino acid sequence of PD-1 protein (NCBI GenBank: NP_005009.2), it includes the full-length human PD-1 protein, or the extracellular fragment of human PD-1, PD-1 ECD, or a fragment containing PD-1 ECD, and also includes a full-length fusion protein of PD-1 protein or a fusion protein of PD-1 ECD, 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 changes (including but not limited to substitutions, deletions, and / or additions) in the amino acid sequence of PD-1 protein can occur naturally or be artificially introduced without affecting its biological function. Thus, in the present invention, the term "PD-1 protein" is intended to include any such sequence, including natural or artificial variants thereof. Also, when describing a sequence fragment of PD-1 protein, it also includes the sequence fragment corresponding to the natural or artificial variant.

[0062] In the present invention, the terms "single domain antibody", "VHH" and "nanobody" have the same meaning and refer to the cloning of the variable region of an antibody heavy chain to construct a nanobody (VHH) consisting of only one heavy chain variable region, which is the smallest complete functional antigen-binding fragment. Usually, after obtaining an antibody that naturally lacks a light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0063] In the present invention, unless otherwise stated, the terms "first" (e.g., first protein functional region, first peptide chain) and "second" (e.g., second protein functional region, second peptide chain) are used to distinguish or clarify references and do not have a typical sequential meaning.

[0064] In the present invention, a single end antibody molecule, unless otherwise specified, refers to an antibody molecule that is identical or similar to the first protein functional region molecule or the second protein functional region molecule of a bispecific antibody, such as an anti-TIGIT monoclonal antibody, an anti-PD-L1 monoclonal antibody, or an anti-PD-L1 single domain antibody that is identical or similar to the first protein functional region molecule or the second protein functional region molecule of a bispecific antibody.

[0065] Advantageous Effects of the Invention The present invention achieves one or more of the following effects: (1) The anti-TIGIT antibody of the present invention has excellent affinity and specificity. (2) The bispecific antibody of the present invention can specifically bind to TIGIT with very good properties. (3) The bispecific antibody of the present invention can specifically bind to PD-L1 with very good efficacy. (4) The bispecific antibody of the present invention can simultaneously bind to human TIGIT protein and human PD-L1 protein. (5) There is synergy between the first protein functional region and the second protein functional region of the bispecific antibody of the present invention. For example, the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention can attenuate the CD155 / CD112-mediated TIGIT downstream inhibitory signaling pathway and the PD-L1-mediated PD-1 downstream inhibitory signaling pathway, respectively, and this activity is better than that of the combined use of two single-terminal antibody molecules, and / or this anti-tumor activity in vivo is better than that of the combined use of two single-terminal antibody molecules. (6) The bispecific antibodies of the present invention are easy to produce. (7) The bispecific antibody of the present invention has good stability and a long half-life. [Brief description of the drawings]

[0066] [Figure 1] FIG. 1 shows a schematic structural diagram of a bispecific antibody of the present invention. [Figure 2A] Figure 2 shows the binding curves of bispecific antibodies of the invention to human PD-L1 overexpressed on CHO cells. [Figure 2B] Figure 2 shows the binding curves of bispecific antibodies of the invention to cynomolgus PD-L1 overexpressed on CHO cells. [Figure 2C] 1 shows the curves of bispecific antibodies of the present invention blocking the binding of human PD-L1 to overexpressed human PD-1 on CHO cells. [Figure 3A] 1 shows the binding curves of the bispecific antibodies of the present invention to human TIGIT overexpressed on CHO cells. [Figure 3B] 1 shows the binding curves of the bispecific antibodies of the present invention to cynomolgus TIGIT overexpressed on CHO cells. [Figure 3C] 1 shows the binding curve of a bispecific antibody of the present invention to mouse TIGIT overexpressed on CHO cells. [Figure 3D] 1 shows a curve in which a bispecific antibody of the present invention blocks the binding of human CD155 to human TIGIT overexpressed on CHO cells. [Figure 3E]1 shows a curve in which a bispecific antibody of the present invention blocks binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells. [Figure 4] 1 shows the simultaneous binding curves of the bispecific antibody of the present invention to human PD-L1 protein and human TIGIT protein. [Diagram 5] 1 shows the curves of bispecific antibodies of the present invention blocking the PD-1 / PD-L1 signaling pathway and the TIGIT / CD155 / CD112 signaling pathway. [Figure 6A] 6A and 6B show statistical graphs of cytokine release in a mixed lymphocyte assay using bispecific antibodies of the invention, where the PBMC cell samples used in FIG. 6A and FIG. 6B were obtained from different donors. [Figure 6B] 6A and 6B show statistical graphs of cytokine release in a mixed lymphocyte assay using bispecific antibodies of the invention, where the PBMC cell samples used in FIG. 6A and FIG. 6B were obtained from different donors. [Figure 7] FIG. 1 shows pharmacodynamic curves of bispecific antibodies of the invention in the A375 and B-NDG mouse model co-inoculated with human PBMCs. [Figure 8] FIG. 1 shows dose-dependent pharmacodynamic curves of bispecific antibodies of the invention in a B-NDG mouse model co-inoculated with A375 and human PBMCs. [Figure 9] 1 shows pharmacodynamic curves of bispecific antibodies of the invention in a human PD-L1 / PD-1 / TIGIT transgenic mouse CT26 tumor model. [Figure 10] FIG. 1 shows half-life curves of bispecific antibodies of the invention in mice. [Figure 11] 1 shows the binding curve of the anti-TIGIT antibody of the present invention to human TIGIT overexpressed on CHO cells. [Figure 12] 1 shows the binding curve of the anti-TIGIT antibody of the present invention to cynomolgus monkey TIGIT overexpressed on CHO cells. [Figure 13] 1 shows the binding curve of the anti-TIGIT antibody of the present invention to mouse TIGIT overexpressed on CHO cells. [Figure 14] 1 shows a curve in which the anti-TIGIT antibody of the present invention blocks the binding of human CD155 to human TIGIT overexpressed on CHO cells. [Figure 15] 1 shows a curve in which the anti-TIGIT antibody of the present invention blocks the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells. [Figure 16] 1 shows the binding curve of the anti-TIGIT antibody of the present invention to TIGIT on activated human primary T cells.

[0067] The sequences encompassed by the invention are set forth below in Table A, with the CDRs each determined according to the Kabat numbering system. [Table A-1] [Table A-2] [Table A-3] EXAMPLES

[0068] Specific Models for Carrying Out the Invention The present invention will be further described below in combination with specific examples. It should be understood that such examples are used merely to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, the experimental methods that do not specify specific conditions are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by the manufacturer. Unless otherwise stated, percentages and fractions are by weight.

[0069] Control antibody Atezolizumab: anti-PD-L1 monoclonal antibody, trademark: Tecentriq, Roche.

[0070] Control antibody Tiragolumab: anti-TIGIT monoclonal antibody, Roche.

[0071] Example 1: Expression and purification of anti-TIGIT / anti-PD-L1 bispecific antibodies In this example, an anti-TIGIT antibody heavy chain variable region (VH) sequence (SEQ ID NO: 19) was synthesized using full gene synthesis and recombined with a wild-type hIgG1 antibody heavy chain to form a complete "VH-CH1-CH2-CH3" antibody heavy chain (SEQ ID NO: 1), and an anti-PD-L1 VHH (SEQ ID NO: 3) was attached at the heavy chain C-terminus by two linkers G4S (SEQ ID NO: 2) to obtain the full-length amino acid sequence of peptide chain #1 shown in SEQ ID NO: 4. In addition, an anti-TIGIT light chain variable region (VL) sequence (SEQ ID NO: 20) was synthesized using full gene synthesis and recombined with a kappa light chain to form a complete "VL-CL" antibody light chain structure. The resulting full-length amino acid sequence of peptide chain #2 is shown in SEQ ID NO: 5.

[0072] The heavy and light chain sequences were constructed in the pcDNA3.1 expression frame using molecular cloning technology, respectively, and conventional expression was performed by the Expi-293 expression system. The transfection method was performed according to the product instructions, where the supernatant was collected after 5 days from cell culture, and the target protein was purified using Protein A magnetic beads (purchased from GenScript). The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of magnetic beads), then added to the sample to be purified and incubated at room temperature for 1 hour with gentle shaking during that period. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. Elution buffer (0.1 M sodium citrate, pH 3.2) was added at 3-5 volumes relative to the magnetic beads, shaken at room temperature for 5-10 minutes, placed back on the magnetic stand, and the elution buffer was collected and transferred to a collection tube containing neutralization buffer (1 M Tris, pH 8.54) and mixed thoroughly.

[0073] An anti-TIGIT / anti-PD-L1 bispecific antibody (also referred to as anti-TIGIT / PD-L1 bispecific antibody in the present invention) was obtained, the schematic structure of which is shown in Figure 1.

[0074] Example 2: Detection of antibody affinity Using biofilm layer optical interference technology (ForteBio), the binding dissociation constants (K D) was determined. Fortebio affinity measurements were performed according to existing methods (Este, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013.5(2):270-8), where the amino acid sequences of the human PD-L1 extracellular fragment, the cynomolgus monkey PD-L1 extracellular fragment, the human TIGIT extracellular fragment, and the cynomolgus monkey TIGIT extracellular fragment are shown in SEQ ID NOs: 6 to 9, respectively.

[0075] The detailed procedure was as follows: the sensor was equilibrated offline with analysis buffer for 30 min, then detected online for 60 s to establish a baseline, and purified intact antibody was loaded onto the AHQ sensor at a thickness of 1 nm for affinity detection. The antibody-loaded sensor was exposed to 100 nM human or cynomolgus PD-L1, TIGIT-his antigen until the plateau phase, and then the sensor was transferred to analysis buffer for at least 2 min to measure the dissociation rate. Kinetic analysis was performed using a 1:1 binding model.

[0076] K of bispecific antibodies and their corresponding single-terminal antibody molecules in binding to human and cynomolgus monkey PD-L1 and TIGIT D The values ​​are shown in Table 1 below. [Table 1]

[0077] The results showed that the monovalent affinity values ​​of the anti-TIGIT / PD-L1 bispecific antibodies to human PD-L1 antigen, cynomolgus PD-L1 antigen, human TIGIT antigen, and cynomolgus TIGIT antigen were comparable to or even better than the monovalent affinity values ​​of the single-terminal antibody molecules to human PD-L1 antigen, cynomolgus PD-L1 antigen, human TIGIT antigen, and cynomolgus TIGIT antigen, respectively.

[0078] Example 3: Binding and blocking activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on CHO cells overexpressing human / cynomolgus PD-L1 3.1 Detection of the binding activity of anti-TIGIT / anti-PD-L1 bispecific antibodies to human / cynomolgus PD-L1 overexpressed on CHO cells based on flow cytometry detection method In detail, CHO-S cells overexpressing human PD-L1 (CHO-huPD-L1 cells) and CHO-S cells overexpressing cynomolgus PD-L1 (CHO-cynoPD-L1 cells) were generated by transfecting human PD-L1 and cynomolgus PD-L1 cloned into MCS pCHO1.0 vector (purchased from Invitrogen) through pressure screening. The overexpressed cells after growth culture were adjusted to an appropriate cell density and added to a 96-well flow cytometry plate, then centrifuged, serially diluted test samples were added, and incubated at 4°C for 30 minutes. Then, washed twice with PBS, fluorescent secondary antibodies similarly diluted to appropriate concentrations were added, and incubated at 4°C for 30 minutes. Then, washed twice with PBS, cells resuspended in PBS were added, detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated. Graphic analysis was performed using Graphpad software, and EC 50 Got the value.

[0079] The results are shown in Table 2, Figure 2A, and Figure 2B. [Table 2]

[0080] The results showed that the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention had binding activity to human / cynomolgus PD-L1 overexpressed on CHO cells, and this binding activity was comparable to that of the PD-L1 terminal monoclonal antibody molecule (anti-PD-L1 VHH).

[0081] 3.2 Detection of the blocking activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on the binding of human PD-L1 to human PD-1 overexpressed on CHO cells based on flow cytometry detection In detail, CHO-S cells overexpressing human PD-1 (CHO-huPD-1 cells) were generated by transfecting with pCHO1.0 vector (purchased from Invitrogen) in which human PD-1 was cloned into MCS by pressure screening. Purified test antibodies were diluted with PBS, and the diluted samples were added to a 96-well flow cytometry plate at 60 μL / well. Biotinylated human PD-L1 protein was then added at 60 μL / well to reach a final concentration of 0.5 μg / mL, mixed, and incubated at 4°C for 30 min. CHO-huPD-1 cells after expansion culture were grown at a cell density of 2 × 10 6 The cells were adjusted to reach 100 cells / mL and added to a 96-well flow cytometry plate at 100 μL / well, centrifuged, and the supernatant was discarded. The above co-incubated antibody-antigen mixture was added at 100 μL / well and incubated at 4 °C for 30 min. After washing twice with PBS, streptomycin avidin-R-phycoerythrin conjugate (SAPE) diluted 100-fold in PBS was added at 100 μL / well and incubated at 4 °C for 30 min. After washing twice with PBS, cells resuspended in PBS were added at 100 μL / well and detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated. Graphical analysis was performed using Graphpad software to determine IC 50 Got the value.

[0082] The results are shown in Table 2 and Figure 2C. The results showed that the blocking activity of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention on the binding of human PD-L1 to human PD-1 overexpressed on CHO cells was comparable to or even better than that of this PD-L1 terminal monoclonal antibody molecule (anti-PD-L1 VHH).

[0083] Example 4: Binding and blocking activities of anti-TIGIT / anti-PD-L1 bispecific antibodies on CHO cells overexpressing human / cynomolgus / mouse TIGIT 4.1 Detection of the binding activity of anti-TIGIT / anti-PD-L1 bispecific antibodies to human / cynomolgus / mouse TIGIT overexpressed on CHO cells based on flow cytometry detection method In detail, CHO-S cells overexpressing human TIGIT (CHO-huTIGIT cells), CHO-S cells overexpressing cynomolgus monkey TIGIT (CHO-cynoTIGIT cells), and CHO-S cells overexpressing mouse TIGIT (CHO-muTIGIT cells) were generated by transfecting pCHO1.0 vectors (purchased from Invitrogen) in which human TIGIT cDNA, cynomolgus monkey TIGIT cDNA, and mouse TIGIT cDNA were cloned into MCS by pressure screening. The overexpressing cells after growth culture were adjusted to a suitable cell density, added to a 96-well flow cytometry plate, centrifuged, and then serially diluted test samples were added and incubated at 4°C for 30 minutes. After washing twice with PBS, fluorescent secondary antibodies similarly diluted to appropriate concentrations were added and incubated at 4°C for 30 minutes. After washing twice with PBS, cells resuspended in PBS were added and detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated. Graphical analysis was performed using Graphpad software to calculate the EC 50 Got the value.

[0084] The results are shown in Table 3 and Figures 3A to 3C. [Table 3]

[0085] The results showed that the binding activity of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention to human / cynomolgus / mouse TIGIT overexpressed on CHO cells was comparable to or even better than that of the TIGIT terminal monoclonal antibody molecule (anti-TIGITmAb).

[0086] 4.2 Detection of the blocking activity of anti-TIGIT / anti-PD-L1 bispecific antibodies based on flow cytometry of the binding of human CD155 to human TIGIT overexpressed on CHO cells and of mouse CD155 to mouse TIGIT overexpressed on CHO cells In detail, CHO-huTIGIT cells after expansion culture were cultured at a cell density of 2 × 10 6 The monoclonal antibodies were adjusted to cells / mL and added to a 96-well flow cytometry plate at 100 μL / well and centrifuged for further use. The purified monoclonal antibodies were diluted in PBS for a total of 12 points by 3-fold dilution starting from 400 nM. The diluted samples were added to the above 96-well flow cytometry plate with cells at 60 μL / well and incubated at 4°C for 30 minutes. Human CD155 protein with mouse IgG2aFc tag was then added at 60 μL / well to reach a final concentration of 2 μg / mL and incubated at 4°C for 30 minutes. After washing twice with PBS, APC goat anti-mouse IgG antibody diluted 100-fold in PBS was added at 100 μL / well and incubated at 4°C for 30 minutes. After washing twice with PBS, cells resuspended in PBS were added at 100 μL / well and detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated.

[0087] After expansion culture, CHO-muTIGIT cells were grown at a cell density of 2 × 10 6The monoclonal antibodies were adjusted to reach 100 cells / mL, added to a 96-well flow cytometry plate at 100 μL / well, and centrifuged for later use. The purified monoclonal antibodies were diluted in PBS for a total of 12 points by 3-fold dilution starting from 400 nM. The diluted samples were added to the above 96-well flow cytometry plate with cells at 60 μL / well and incubated at 4°C for 30 minutes. Then, mouse CD155 protein with mouse IgG2aFc tag was added at 60 μL / well to reach a final concentration of 2 μg / mL and incubated at 4°C for 30 minutes. After washing twice with PBS, APC goat anti-mouse IgG antibody diluted 100-fold in PBS was added at 100 μL / well and incubated at 4°C for 30 minutes. After washing twice with PBS, the cells resuspended in PBS were added at 100 μL / well and detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated. Graphical analysis was performed using Graphpad software, and IC 50 Got the value.

[0088] The results are shown in Table 3 and Figures 3D-3E. The results showed that the blocking activity of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention for the binding of human CD155 to human TIGIT overexpressed on CHO cells and mouse CD155 to mouse TIGIT overexpressed on CHO cells was comparable to that of the TIGIT-terminal monoclonal antibody molecule (anti-TIGITmAb).

[0089] Example 5: Simultaneous binding activity of anti-TIGIT / anti-PD-L1 bispecific antibodies to human TIGIT and human PD-L1 The simultaneous binding activity of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention to human TIGIT protein and human PD-L1 protein was detected based on enzyme-linked immunosorbent assay (ELISA).

[0090] In detail, human TIGIT protein was dissolved according to the instructions, diluted to 1μg / mL in 1x ELISA coating solution, coated onto 96-well ELISA plates at 100μL / well, covered with film, and incubated at 4℃ overnight. The coating solution was discarded, washed 3 times with 1x PBST, and blocked at room temperature for 2 hours with 5% BSA / PBS at 200μL / well. The blocking solution was discarded, and the test antibodies serially diluted in 1% BSA / PBS were added at 100μL / well and incubated at room temperature for 2 hours. The antibody diluent was discarded, washed 3 times with 1x PBST, and biotin-labeled PD-L1 protein diluted in 1% BSA / PBS was added at 100μL / well to reach a final concentration of 1μg / mL and incubated at room temperature for 1 hour. The antigen dilution solution was discarded, the plate was washed three times with 1x PBST, and SA-HRP diluted with 1% BSA / PBS was added at 100μL / well and incubated at room temperature for 1 hour. The SA-HRP dilution solution was discarded, the plate was washed three times with 1x PBST, and ELISA color development solution was added at 100μL / well and incubated at room temperature for 1-3 minutes. Then, ELISA stop solution was added at 50μL / well, and absorbance values ​​were read at 450nm. Concentration-absorbance value binding curves were plotted using Graphpad software.

[0091] The results are shown in Figure 4. The anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention could simultaneously bind to human TIGIT protein and human PD-L1 protein.

[0092] Example 6: Blocking activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on PD-1 / PD-L1 signaling pathway and TIGIT / CD155 / CD112 signaling pathway in luciferase reporter gene system To further detect the activity of anti-TIGIT / anti-PD-L1 bispecific antibody on simultaneously blocking PD-1 / PD-L1 signaling pathway and TIGIT / CD155 / CD112 signaling pathway at the cellular level, in this example, the following luciferase reporter gene system was constructed. Briefly, lentivirus was used to transfect cells to construct a CHO-K1 cell line (CHO-K1-CD155-CD112-PD-L1) overexpressing human CD155, human CD112, human PD-L1, and OKT-3scFv, and a Jurkat cell line (Jurkat-TIGIT-PD-1-luc) overexpressing human TIGIT, human PD-1, and NF-AT luciferase reporter gene (purchased from Promega), and then the reporter gene system was used to carry out related experiments.

[0093] In detail, CHO-K1-CD155-CD112-PD-L1 functional cells were obtained by dissociation, adjusted to reach the desired cell density, and added to a 96-well white-bottom plate at 100μL / well and cultured overnight to allow adhesion. The next day, a suspension of Jurkat-TIGIT-PD-1-luc effector cells was prepared, and the test samples were serially diluted with reaction medium. The white-bottom plate was removed, the culture supernatant was removed by pipetting, the diluted samples above were added to the white-bottom plate at 40μL / well, and the suspension of Jurkat-TIGIT-PD-1-luc effector cells was added at 40μL / well, and incubation was performed for 6 hours at 37℃ in a 5% CO2 incubator. During this time, the Bio-Glo™ reagent was allowed to return to room temperature. After the culture was completed, the cells were removed and equilibrated at room temperature for 5 minutes. Bio-Glo™ reagent was added at 80 μL / well and the fluorescent signal was read using a multifunctional microplate reader.

[0094] The results are shown in Figure 5. The results show that the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention can release the CD155 / CD112-mediated TIGIT downstream inhibitory signaling pathway and the PD-L1-mediated PD-1 downstream inhibitory signaling pathway, respectively, which can up-regulate the reporter gene luciferase expression, and this activity is better than that of the combined use of two single-terminal antibody molecules.

[0095] Example 7: Activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on T cell activation in a mixed lymphocyte assay In this example, a mixed lymphocyte reaction (MLR) assay was used to detect the activity of anti-TIGIT / anti-PD-L1 bispecific antibodies in T cell activation. The detailed experimental method was as follows:

[0096] PBMC cells (purchased from SAILYBIO, XFB-HP100B) were resuscitated and centrifuged. The PBMC cells were resuspended in 10 mL of X-VIVO-15 medium (purchased from LONZA) and cultured for 2 hours at 37°C in a cell culture incubator to remove non-adherent cells. 10 mL of DC medium (X-VIVO-15 medium supplemented with 10 ng / mL GM-CSF (purchased from R&D) and 20 ng / mL IL-4 (purchased from R&D)) was added and cultured for 3 days, then 5 mL of DC medium was supplemented and culture was continued until the 6th day. Then, DC maturation medium (X-VIVO-15 medium supplemented with 1000 U / mL TNF-α (purchased from R&D), 10 ng / mL IL-6 (purchased from R&D), 5 ng / mL IL-1β (purchased from R&D), and 1 μM PGE2 (purchased from Tocris)) was added and cultured for 2 days, and then matured DC cells were harvested and the cell density was adjusted to 2 × 10 using X-VIVO-15 medium. 5 Adjusted to cells / mL.

[0097] PBMC cells from another donor (purchased from SAILY BIO, XFB-HP100B) were resuscitated and centrifuged. The PBMC cells were resuspended in 10 mL of X-VIVO-15 medium. T cells were enriched using a T cell isolation kit (purchased from Stemcell) and resuspended using X-VIVO-15 to a cell density of 2 × 10 6 The T cell suspension was mixed with the mature DC cells collected above at a volume ratio of 1:1 and added to a 96-well U-bottom plate at 100 μL / well.

[0098] The antibody samples to be tested were diluted with X-VIVO-15 culture medium for a total of 5 points by 10-fold dilution starting from 200nM, and added to the above mixed cell wells at 100μL / well and cultured for 5 days.Then, the supernatant was collected and the IFN-γ expression level was detected using ELISA (purchased from eBioscience) method.

[0099] The results are shown in Figures 6A and 6B. The results showed that the anti-TIGIT / anti-PD-L1 bispecific antibody exhibited good biological activity in MLR experiments, and the T cell activation level was comparable to that of the combined activity of two single-terminal antibody molecules.

[0100] Example 8: In vivo pharmacodynamic testing of anti-TIGIT / anti-PD-L1 bispecific antibodies in B-NDG mice inoculated with a mixture of A375 and human PBMCs In this experiment, the anti-tumor effect of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention was determined in a model of B-NDG mice inoculated with a mixture of A375 (purchased from Addexbio, C0020004, human malignant melanoma cells) and human PBMC cells (Shanghai Miles-bio, A10S033014 / PB100C) (A375 huPBMC model), where a humanized tumor mouse model with locally reconstituted human immune system was generated by inoculating immunodeficient mice with human immune cells (PBMCs).

[0101] In detail, A375 cells and human PBMCs were first mixed in an equal volume ratio of 1:1 to obtain 0.1 mL of cell suspension, which was then subcutaneously injected into the right groin area of ​​the mouse abdomen to establish the A375 huPBMC model. The average tumor volume was approximately 200 mm 3 When the tumor size reached 100 mg / kg, the mice were divided into groups. Six mice in each group were treated by intraperitoneal administration of PBS or antibody at various doses and the same administration volume. The changes in tumor volume and body weight of the mice in each group were monitored at a monitoring frequency of once every 2 to 3 days, and the monitoring was continued for 2 to 3 weeks. The doses and administration methods are shown in Table 4.

[0102] [Table 4]

[0103] The results are shown in Figure 7. The results show that the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention has a significant anti-tumor effect, which is better than that of the combination of two single-terminal antibody molecules.

[0104] Example 9: In vivo dose-dependent pharmacodynamic study of anti-TIGIT / anti-PD-L1 bispecific antibodies in B-NDG mice inoculated with a mixture of A375 and human PBMCs In this experiment, the A375 huPBMC model was established by subcutaneous mixed inoculation (the steps for establishing the model were the same as in Example 8). The average tumor volume was approximately 300 mm 3 When the tumor size reached 100 mg / kg, the mice were divided into groups. Six mice in each group were treated with various doses and the same volume of PBS or bispecific antibody by intraperitoneal administration. The changes in tumor volume and body weight of the mice in each group were monitored once every 2-3 days, and the monitoring was continued for 2-3 weeks. The doses and administration methods are shown in Table 5.

[0105] [Table 5]

[0106] The results are shown in Figure 8. The anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention could significantly inhibit the growth of tumors in mice in a dose-dependent manner.

[0107] Example 10: In vivo pharmacodynamic testing of anti-TIGIT / anti-PD-L1 bispecific antibodies in huPD-L1 / PD-1 / TIGIT KI mice In this experiment, the anti-tumor effect of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention was determined by implanting CT-26-huPD-L1 tumor cells (human PD-L1 knocked-into CT26 mouse colon carcinoma cells, purchased from Jiangsu Gempharmatech) into human PD-L1 / PD-1 / TIGIT transgenic mice (huPD-L1 / PD-1 / TIGIT KI mice).

[0108] In detail, a CT-26-huPD-L1 cell suspension was first prepared, containing approximately 5 × 10 5 0.1 mL of cells was injected subcutaneously into the right groin of the mouse abdomen to establish the CT-26-huPD-L1 tumor-bearing mouse model. The mean tumor volume was 80–120 mm 3 When the tumor size reached 100 mg / kg, the mice were divided into groups. Six mice in each group were treated with various doses and the same volume of PBS or antibody by intraperitoneal injection. The changes in tumor volume and body weight of the mice in each group were monitored once every 2-3 days, and the monitoring was continued for 2-3 weeks. The doses and administration methods are shown in Table 6.

[0109] [Table 6]

[0110] The results are shown in Figure 9. The efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention was comparable to that of the clinical drug combination (atezolizumab + tiragolumab), and the anti-tumor activity was superior to that of the two monoclonal antibody groups.

[0111] Example 11: In vivo half-life study of anti-TIGIT / anti-PD-L1 bispecific antibodies in mice A single tail vein injection method was used to detect the half-life of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention in mice.

[0112] In detail, three male and three female Balb / c laboratory mice were housed in a 12 / 12-h light / dark controlled environment at a temperature of 24°C ± 2°C and humidity of 40%-70%, with free access to water and food. On the day of the experiment, Balb / c mice were administered a single tail vein injection of monoclonal antibody molecules at a dose of 10 mg / kg. Blood collection time points: blood was collected from the mouse orbit at 5 min, 0.5 h, 2 h, 6 h, 24 h, 48 h, 96 h, 168 h, 336 h, and 504 h after administration. Whole blood samples were left to stand at 2°C-8°C for 30 min and centrifuged at 12,000 rpm for 5 min to collect serum. The resulting serum was centrifuged at 2°C-8°C, 12,000 rpm for 5 min, and stored at -80°C. The amount of bispecific antibody molecules in serum was detected by ELISA.

[0113] The results are shown in Figure 10. The results showed that the half-life of the anti-TIGIT / anti-PD-L1 bispecific antibody of the invention after a single injection in mice was 189 hours.

[0114] Preparation Example 1: Preparation of anti-TIGIT monoclonal antibody The heavy chain variable region of anti-TIGIT monoclonal antibody (amino acid sequence shown in SEQ ID NO: 19) was recombined with human IgG1 heavy chain constant region and human IgG1 heavy chain constant region with L234A modification and L235A modification. In addition, the light chain variable region (amino acid sequence shown in SEQ ID NO: 20) was recombined with human kappa light chain constant region, and the anti-TIGIT monoclonal antibody was named 55796-G1 and 55796-G1LALA, respectively. Transient expression and purification were carried out via HEK293 expression system. The specific operations were as follows: pcDNA3.1 vectors carrying antibody heavy chain and antibody light chain were introduced into HEK293 cells using chemical transfection method and cultured at 37°C and 8% CO2 for 7 days. The cell clearing was collected and centrifuged at 13,000 rpm for 20 minutes. The supernatant was taken out and purified with protein A, and the antibody purity was detected using SEC, and the endotoxin content was controlled simultaneously.

[0115] The prepared anti-TIGIT monoclonal antibodies 55796-G1 and 55796-G1LALA were used in the following test examples 1 to 3. Test Example 1: Affinity detection of anti-TIGIT monoclonal antibodies Using biofilm layer optical interference technology (ForteBio), the binding dissociation constants (K D ) was determined. Fortebio affinity measurements were performed according to existing methods (Este, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. Mabs, 2013.5(2):270-8). The extracellular amino acid sequences of human TIGIT, cynomolgus monkey TIGIT, and mouse TIGIT are shown in SEQ ID NOs: 8, 9, and 21, respectively.

[0116] Monovalent affinities of intact antibodies (i.e., full-length IgGs originally obtained from Adimab) against human, cynomolgus, and mouse TIGIT-his proteins were measured. Sensors were equilibrated offline with assay buffer for 20 min, then online detection was performed for 120 s to establish a baseline, and affinity detection was performed by loading intact TIGIT antibodies to reach a thickness of 1 nm on the AHQ sensor. Sensors loaded with antibody were incubated with 100 nM TIGIT-his antigen until the plateau phase, and then the sensors were transferred to assay buffer for at least 2 min to measure dissociation rates. Kinetic analysis was performed using a 1:1 binding model.

[0117] In the above determination method, the measured K D The values ​​are shown in Table 7 below. [Table 7]

[0118] The results in Table 7 showed that: (1) the monovalent affinity of the anti-TIGIT monoclonal antibody to human TIGIT-his protein was higher than that of the control molecule tiragolumab; (2) the monovalent affinity of the anti-TIGIT monoclonal antibody to cynomolgus monkey TIGIT-his protein was comparable to that of the control molecule tiragolumab; and (3) the anti-TIGIT monoclonal antibody had cross-binding activity with mouse TIGIT.

[0119] Test Example 2: Binding and blocking activities of anti-TIGIT monoclonal antibodies against CHO cells overexpressing human / cynomolgus / mouse TIGIT 2.1 Flow cytometry-based detection of binding activity of anti-TIGIT monoclonal antibodies to human / cynomolgus / mouse TIGIT overexpressed on CHO cells In detail, CHO-S cells overexpressing human TIGIT (CHO-huTIGIT cells), CHO-S cells overexpressing cynomolgus monkey TIGIT (CHO-cynoTIGIT cells), and CHO-S cells overexpressing mouse TIGIT (CHO-muTIGIT cells) were generated by transfecting pCHO1.0 vectors (purchased from Invitrogen) in which human TIGIT cDNA, cynomolgus monkey TIGIT cDNA, and mouse TIGIT cDNA were cloned into MCS by pressure screening. After growth culture, the overexpressing cells were adjusted to an appropriate cell density and added to a 96-well flow cytometry plate. After centrifugation, serially diluted test samples were added and incubated at 4°C for 30 min. After washing twice with PBS, fluorescent secondary antibodies similarly diluted to the appropriate concentration were added, incubated at 4°C for 30 min, and washed twice with PBS. Cells resuspended in PBS were added and detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated. Graphical analysis was performed using Graphpad software, and EC 50 The results are shown in Table 8 and Figures 11 to 13.

[0120] 2.2 Detection of the blocking activity of anti-TIGIT monoclonal antibodies based on flow cytometry in blocking the binding of human CD155 to human TIGIT overexpressed on CHO cells and in blocking the binding of mouse CD155 to mouse TIGIT overexpressed on CHO cells In detail, CHO-huTIGIT cells after expansion culture were cultured at a cell density of 2 × 10 6The monoclonal antibodies were adjusted to cells / mL and added to a 96-well flow cytometry plate at 100 μL / well and centrifuged for later use. The purified monoclonal antibodies were diluted in PBS for a total of 12 points by 3-fold dilution starting from 400 nM. The diluted samples were added to the above 96-well flow cytometry plate with cells at 60 μL / well and incubated at 4°C for 30 minutes. Human CD155 protein with mouse IgG2aFc tag was then added at 60 μL / well to reach a final concentration of 2 μg / mL, incubated at 4°C for 30 minutes, and washed twice with PBS. APC goat anti-mouse IgG antibody diluted 100-fold in PBS was added at 100 μL / well, incubated at 4°C for 30 minutes, and washed twice with PBS. The cells resuspended in PBS were added at 100 μL / well and detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated.

[0121] After expansion culture, CHO-muTIGIT cells were grown at a cell density of 2 × 10 6 The monoclonal antibodies were adjusted to cells / mL and added to a 96-well flow cytometry plate at 100 μL / well and centrifuged for later use. The purified monoclonal antibodies were diluted in PBS for a total of 12 points by 3-fold dilution starting from 400 nM. The diluted samples were added to the above 96-well flow cytometry plate with cells at 60 μL / well and incubated at 4°C for 30 minutes. Then, mouse CD155 protein with mouse IgG2aFc tag was added at 60 μL / well to reach a final concentration of 2 μg / mL, incubated at 4°C for 30 minutes, and washed twice with PBS. APC goat anti-mouse IgG antibody diluted 100-fold in PBS was added at 100 μL / well, incubated at 4°C for 30 minutes, and washed twice with PBS. The cells resuspended in PBS were added at 100 μL / well and detected on a CytoFlex flow cytometer and the corresponding MFI was calculated. Graphical analysis was performed using Graphpad software, and IC 50 The results are shown in Table 8 and Figures 14 to 15.

[0122] [Table 8]

[0123] The anti-TIGIT monoclonal antibody of the present invention showed the following results from Table 8 and Figures 11 to 13: (1) its binding activity to human TIGIT protein overexpressed on the surface of CHO cells was better than that of the control molecule tiragolumab; (2) its binding activity to cynomolgus monkey TIGIT protein overexpressed on the surface of CHO cells was better than that of the control molecule tiragolumab; (3) it showed significant binding to mouse TIGIT protein overexpressed on the surface of CHO cells.

[0124] The following was observed for the anti-TIGIT monoclonal antibody of the present invention from Table 8 and Figures 14-15: (1) its ability to block the binding of human CD155 to human TIGIT protein overexpressed on the surface of CHO cells was better than that of the control molecule tiragolumab; (2) the anti-TIGIT antibody molecule binding to mouse TIGIT protein overexpressed on the cell surface of CHO cells could significantly block the binding of mouse CD155 to mouse TIGIT protein overexpressed on the surface of CHO cells.

[0125] Test Example 3: Binding of anti-TIGIT monoclonal antibodies to TIGIT on the surface of primary T cells The binding activity of the anti-TIGIT antibody of the present invention to TIGIT on the surface of activated T cells was detected based on a flow cytometry detection method.

[0126] In detail, human PBMCs were selected according to the experimental protocol provided by STEMCELL (stemcell, Cat. No.: #17951C) to obtain human total T cells. The concentration of T cells was adjusted to 1.0 × 10 using X-VIVO15 medium (purchased from lonza, Cat. No. 04-418Q). 6The activated T cells were adjusted to 1000 cells / mL, and then 1 μL of IL-2 stock solution (1 million IU) was added, while CD3 / CD28 Dynabeads (purchased from Gibco, No.: 11132D) were added 1:1 (beads to cells), and cultured at 37°C in a 5% CO2 incubator for 48 hours. The activated T cells were adjusted to the appropriate cell density and added to a 96-well flow cytometry plate. After centrifugation, serially diluted test samples were added and incubated at 4°C for 30 minutes. After washing twice with PBS, fluorescent secondary antibodies similarly diluted to the appropriate concentration were added, incubated at 4°C for 30 minutes, and washed twice with PBS. Cells resuspended in PBS were added and detected on a CytoFlex flow cytometer, and the corresponding MFI was calculated.

[0127] The results are shown in Figure 16. The results showed that the anti-TIGIT antibodies 55796-G1 and 55796-G1LALA of the present invention can bind to TIGIT molecules on the surface of T cells, and their binding activity is better than that of the control molecule tiragolumab.

[0128] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that, according to all the teachings disclosed, various modifications and substitutions can be made to such details, and such modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and all equivalents thereof.

Claims

1. a first protein functional region that targets TIGIT; and A second protein functional region that targets a target different from TIGIT. A bispecific antibody comprising: the first protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof, the anti-TIGIT immunoglobulin comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region of the anti-TIGIT immunoglobulin comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 10, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 12; A bispecific antibody, wherein the light chain variable region of the anti-TIGIT immunoglobulin comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 13, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:

15.

2. The bispecific antibody described in claim 1, wherein the target different from TIGIT is PD-L1.

3. the first protein functional region and the second protein functional region are directly linked or linked via a linker; Preferably, the linker is (GGGGS) ｍ and m is a positive integer, for example, 1, 2, 3, 4, 5, or 6; Preferably, the linker is (GGGGS) ｎ G, n is a positive integer, for example, 1, 2, 3, 4, 5, or 6; 2. The bispecific antibody of claim 1, wherein the amino acid sequence of the linker is as set forth in SEQ ID NO:

2.

4. 2. The bispecific antibody of claim 1 , wherein the number of said first protein functional regions and said second protein functional regions is independently 1, 2 or more than 2.

5. the first protein functional region is an anti-TIGIT immunoglobulin or an antigen-binding fragment thereof, and the second protein functional region is a single domain antibody or a single chain antibody that targets a target different from TIGIT; Preferably, the single domain antibody is an anti-PD-L1 single domain antibody; The bispecific antibody of claim 1, wherein the single chain antibody is an anti-PD-L1 single chain antibody.

6. the first protein functional region is an anti-TIGIT single chain antibody, and the second protein functional region is an immunoglobulin or antigen-binding fragment thereof that targets a target different from TIGIT; The bispecific antibody according to claim 1, wherein the immunoglobulin targeting a target different from TIGIT is an anti-PD-L1 immunoglobulin.

7. the anti-PD-L1 single domain antibody comprises one heavy chain variable region, the heavy chain variable region comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 16, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 18; The bispecific antibody of claim 5, wherein the amino acid sequence of the anti-PD-L1 single domain antibody is set forth in SEQ ID NO:

3.

8. the heavy chain variable region of the anti-TIGIT immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 19, and the light chain variable region of the anti-TIGIT immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 20; Preferably, the anti-TIGIT immunoglobulin comprises a heavy chain and a light chain, 2. The bispecific antibody of claim 1 , wherein the heavy chain of the anti-TIGIT immunoglobulin has the amino acid sequence set forth in SEQ ID NO: 1 and the light chain of the anti-TIGIT immunoglobulin has the amino acid sequence set forth in SEQ ID NO:

5.

9. The anti-TIGIT immunoglobulin comprises a constant region, the constant region of the anti-TIGIT immunoglobulin or the constant region of the immunoglobulin targeting a target different from TIGIT is derived from a human antibody; 2. The bispecific antibody of claim 1, wherein the constant region is selected from the group consisting of human IgG1, IgG2, IgG3 or IgG4 constant regions.

10. The anti-TIGIT immunoglobulin comprises a heavy chain constant region and a light chain constant region, The bispecific antibody of claim 1, wherein the heavy chain constant region of the anti-TIGIT immunoglobulin or the heavy chain constant region of the immunoglobulin targeting a target different from TIGIT is a human Ig gamma 1 chain C region or a human Ig gamma 4 chain C region, and the light chain constant region is a human Ig kappa chain C region, and preferably the heavy chain constant region of the anti-TIGIT immunoglobulin further comprises the L234A and L235A mutations according to the EU numbering system.

11. 6. The bispecific antibody of claim 5 , wherein the single domain antibodies or single chain antibodies are bound to the C-terminus or N-terminus of the anti-TIGIT immunoglobulin, and preferably the number of the single domain antibodies or single chain antibodies is two, with one end of each single domain antibody or single chain antibody being bound to the C-terminus or N-terminus of two heavy chains of the anti-TIGIT immunoglobulin, respectively.

12. The bispecific antibody of claim 5, wherein the single domain antibody is an anti-PD-L1 single domain antibody, and the peptide chain obtained by binding the single domain antibody to the anti-TIGIT immunoglobulin has the amino acid sequence shown in SEQ ID NO:

4.

13. a tetramer formed by two identical first peptide chains and two identical second peptide chains; 6. The bispecific antibody of claim 5, wherein the amino acid sequence of the first peptide chain is set forth in SEQ ID NO: 4 and the amino acid sequence of the second peptide chain is set forth in SEQ ID NO:

5.

14. An isolated nucleic acid molecule encoding a bispecific antibody according to any one of claims 1 to 13.

15. A vector comprising the isolated nucleic acid molecule described in claim 14.

16. A host cell comprising the isolated nucleic acid molecule of claim 14, or a vector comprising the isolated nucleic acid molecule of claim 14.

17. 14. A conjugate comprising a bispecific antibody and a coupling moiety, wherein the bispecific antibody is the bispecific antibody of any one of claims 1 to 13, and the coupling moiety is a detectable label, preferably a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance, or an enzyme.

18. A kit comprising the bispecific antibody of any one of claims 1 to 13, Preferably, the kit further comprises a second antibody capable of specifically binding to the bispecific antibody, and optionally, the second antibody further comprises a detectable label, for example, a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance, or an enzyme.

19. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 13, optionally further comprising a pharmaceutically acceptable excipient.

20. 14. Use of a bispecific antibody according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment or prevention of a malignant tumor, preferably said malignant tumor being selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, and head and neck cancer.

21. 14. The bispecific antibody of any one of claims 1 to 13 for use in the treatment or prevention of a malignant tumor, preferably wherein the malignant tumor is selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, and head and neck cancer.

22. A pharmaceutical composition for treating or preventing a malignant tumor, comprising the bispecific antibody of any one of claims 1 to 13, wherein the malignant tumor is preferably selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, and head and neck cancer.

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