Bispecific antibody targeting PVRIG and TIGIT and application thereof

Abstract

The invention provides a bispecific antibody targeting PVRIG and TIGIT and an application of the bispecific antibody. The bispecific antibody comprises a first antigen binding region and a second antigen binding region, wherein the first antigen binding region has PVRIG molecular binding activity; and a second antigen binding region, wherein the second antigen binding region has TIGIT molecule binding activity; wherein the first antigen binding region is connected with the second antigen binding region. The bispecific antibody prepared by the invention can be combined with human PVRIG and TIGIT proteins with high affinity, and can effectively block the combination of PVRIG and CD112 and the combination of TIGIT and CD155, thereby promoting the killing of human PBMC on tumor cells and enhancing the cytokine secretion function of CD8T cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to bispecific antibodies targeting PVRIG and TIGIT and their applications, and more specifically, to bispecific antibodies and their preparation methods, nucleic acid molecules, expression vectors, recombinant cells, compositions, pharmaceutical uses, drugs, and reagent kits. Background Technology

[0002] In the field of immunotherapy, treatment strategies for tumors are constantly advancing. Among them, immune checkpoint inhibitors have received widespread attention as an important therapeutic approach. Poliovirus receptor-associated immunoglobulin domain protein (PVRIG, also known as CD112R) and T-cell immunoglobulin and ITIM domain protein (TIGIT), as members of the PVR family, play a crucial role in tumor immunotherapy by regulating the activation of NK cells and T cells. PVRIG and TIGIT negatively regulate the immune response by inhibiting the CD226-mediated signaling pathway, leading to tumor immune escape. Previous studies have shown that therapeutic antibodies targeting PVRIG and TIGIT can enhance the killing function of immune cells, thereby improving their effectiveness against tumor cells.

[0003] Currently, bispecific antibodies have attracted much attention due to their simultaneous targeting of two antigen sites, demonstrating greater potential in immunotherapy. However, the development and preparation of bispecific antibodies still face some challenges.

[0004] Therefore, the present invention aims to develop a bispecific antibody that simultaneously targets PVRIG and TIGIT. Summary of the Invention

[0005] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a bispecific antibody targeting PVRIG and TIGIT.

[0006] Specifically, this application provides the following technical solution:

[0007] In a first aspect, this application proposes a bispecific antibody. According to embodiments of this application, the bispecific antibody comprises: a first antigen-binding region having PVRIG molecule binding activity; and a second antigen-binding region having TIGIT molecule binding activity; wherein the first antigen-binding region and the second antigen-binding region are linked. In some examples of this application, the bispecific antibody can bind human PVRIG and TIGIT proteins with high affinity, while effectively blocking the binding of PVRIG to CD112 and the binding of TIGIT to CD155, thereby promoting the killing of tumor cells by human PBMCs and enhancing the cytokine secretion function of CD8 T cells.

[0008] In a second aspect of this application, a nucleic acid molecule is proposed. According to embodiments of this application, the nucleic acid molecule encodes the bispecific antibody described in the first aspect of this application. In some examples of this application, the bispecific antibody encoded by the nucleic acid molecule can bind to human PVRIG and TIGIT proteins with high affinity, while effectively blocking the binding of PVRIG to CD112 and TIGIT to CD155, thereby promoting the killing of tumor cells by human PBMCs and enhancing the cytokine secretion function of CD8 T cells.

[0009] In a third aspect of this application, an expression vector is provided. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the second aspect of this application. In some examples of this application, the expression vector can efficiently express the bispecific antibody in suitable host cells. This bispecific antibody can bind to human PVRIG and TIGIT proteins with high affinity, while effectively blocking the binding of PVRIG to CD112 and TIGIT to CD155, thereby promoting the killing of tumor cells by human PBMCs and enhancing the cytokine secretion function of CD8 T cells.

[0010] In a fourth aspect of this application, a method for preparing the bispecific antibody described in the first aspect is provided. According to embodiments of this application, the method includes: introducing the expression vector described in the third aspect of this application into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the bispecific antibody. In some examples of this application, the bispecific antibody prepared by this method can bind to human PVRIG and TIGIT proteins with high affinity, while effectively blocking the binding of PVRIG to CD112 and TIGIT to CD155, thereby promoting the killing of tumor cells by human PBMCs and enhancing the cytokine secretion function of CD8 T cells.

[0011] In a fifth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. In some examples of this application, the bispecific antibody can bind to human PVRIG and TIGIT proteins with high affinity, while effectively blocking the binding of PVRIG to CD112 and TIGIT to CD155, thereby promoting the killing of tumor cells by human PBMCs and enhancing the cytokine secretion function of CD8 T cells.

[0012] In a sixth aspect of this application, a composition is proposed. According to embodiments of this application, the composition comprises: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cells described in the fifth aspect. As previously stated, the bispecific antibody can simultaneously block the PVRIG and TIGIT signaling pathways, thereby maximally restoring CD226-mediated activation of immune cells and enhancing their anti-tumor function. In some specific examples of this application, compositions containing the bispecific antibody, such as food compositions and pharmaceutical compositions, also have significant therapeutic or preventative effects against tumors.

[0013] In a seventh aspect of this application, the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fifth aspect, or the composition described in the sixth aspect in the preparation of a medicament for the treatment or prevention of cancer. As previously stated, the bispecific antibody can simultaneously block the PVRIG and TIGIT signaling pathways, thereby maximally restoring the activation of immune cells mediated by CD226 and enhancing the anti-tumor function of immune cells. Medicaments containing the bispecific antibody and a series of other substances also have significant therapeutic or preventative effects against cancer.

[0014] In an eighth aspect of this application, a medicament is proposed. According to embodiments of this application, the medicament comprises: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fifth aspect, or the composition described in the sixth aspect, and the medicament is used to treat or prevent cancer. As previously stated, the bispecific antibody can simultaneously block the PVRIG and TIGIT signaling pathways, thereby maximally restoring the activation of CD226-mediated immune cells and enhancing the anti-tumor function of immune cells. Medicaments containing the bispecific antibody and a series of other substances also have significant therapeutic or preventative effects against cancer.

[0015] In a ninth aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises the bispecific antibody described in the first aspect. In some examples of this application, the bispecific antibody can bind to human PVRIG and TIGIT proteins with high affinity; therefore, the kit comprising the bispecific antibody can be used to detect PVRIG and TIGIT proteins. The kit can be used in scientific research, such as for qualitative or quantitative detection of PVRIG and TIGIT proteins in biological samples, and can also be used to assess an individual's condition, such as determining whether the individual's PVRIG and TIGIT protein levels are higher or lower than normal levels after obtaining the individual's PVRIG and TIGIT protein levels.

[0016] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0017] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0018] Figure 1 This is a schematic diagram of the bispecific antibody structure according to an embodiment of this application;

[0019] Figure 2 This is a schematic diagram showing the detection results of the binding ability of the bispecific antibody to PVRIG and TIGIT proteins according to the embodiments of this application;

[0020] Figure 3 This is a schematic diagram showing the detection results of the binding ability of the bispecific antibody to 293T-human PVRIG cells according to the embodiments of this application;

[0021] Figure 4 This is a schematic diagram showing the detection results of the binding ability of the bispecific antibody to 293T-human TIGIT cells according to the embodiments of this application;

[0022] Figure 5 This is a schematic diagram of the detection results of bispecific antibody blocking the binding of human PVRIG to human CD112 according to an embodiment of this application;

[0023] Figure 6 This is a schematic diagram of the detection results of bispecific antibody blocking the binding of human TIGIT to human CD155 according to an embodiment of this application;

[0024] Figure 7 This is a schematic diagram of the detection results of bispecific antibody blocking the binding of 293T-human CD112 cells to human PVRIG protein according to an embodiment of this application;

[0025] Figure 8 This is a schematic diagram of the detection results of bispecific antibody blocking the binding of 293T-human TIGIT cells to human CD155 protein according to an embodiment of this application;

[0026] Figure 9 This is a schematic diagram of the detection results of bispecific antibody promoting the killing of A375 melanoma cells by human PBMCs according to the embodiments of this application;

[0027] Figure 10 This is a schematic diagram of the detection results of bispecific antibodies promoting the secretion of cytokines by human CD8 T cells according to embodiments of this application. Detailed Implementation

[0028] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0029] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0030] In this application, the amino acid sequences of the CDRs listed above are all as shown in the IMGT definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the Kabat rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes above. Although the scope of protection claimed in this application is based on the sequences shown in the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of this application.

[0031] In this application, the term "bispecific antibody" refers to an antibody that can recognize two antigenic epitopes, such as TIGIT and PVRIG antigenic epitopes.

[0032] In this application, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecule & Larva Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see AltschμL et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (AltschμL et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST AltschμL et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0033] In this application, the term "at least 80% identity" means at least 80% identity with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0034] The amino acid sequences involved in this application are shown in Table 1.

[0035] In a first aspect, this application provides a bispecific antibody. According to an embodiment of this application, the bispecific antibody includes: a first antigen-binding region having PVRIG molecule binding activity; and a second antigen-binding region having TIGIT molecule binding activity; wherein the first antigen-binding region and the second antigen-binding region are connected.

[0036] In some examples of this application, the above-mentioned bispecific antibodies may also include at least one of the following technical features:

[0037] In some examples of this application, the first antigen-binding region includes an anti-PVRIG antibody, which includes a heavy chain variable region and a light chain variable region.

[0038] In some examples of this application, the heavy chain variable region of the anti-PVRIG antibody includes a CDR1 sequence as shown in the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% identity with SEQ ID NO:1. In some specific examples of this application, the CDR1 sequence of the bispecific antibody heavy chain variable region is as shown in SEQ ID NO:1. In one specific example of this application, using the CDR1 sequence of the heavy chain variable region as shown in SEQ ID NO:1 results in high antibody affinity and specificity.

[0039] In some examples of this application, the heavy chain variable region of the anti-PVRIG antibody includes a CDR2 sequence as shown in the amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 80% identity with SEQ ID NO:2. In a specific example of this application, the CDR2 sequence of the bispecific antibody heavy chain variable region is as shown in SEQ ID NO:2. In some examples of this application, using the CDR2 sequence of the heavy chain variable region as shown in SEQ ID NO:2 results in higher antibody affinity and specificity.

[0040] In some examples of this application, the heavy chain variable region of the anti-PVRIG antibody includes a CDR3 sequence as shown in the amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 80% identity with SEQ ID NO:3. In a specific example of this application, the CDR3 sequence of the heavy chain variable region of the bispecific antibody is as shown in SEQ ID NO:3. In some examples of this application, using the CDR3 sequence of the heavy chain variable region as shown in SEQ ID NO:3 results in higher antibody affinity and specificity.

[0041] In a specific example of this application, the amino acid sequence of the variable region of the anti-PVRIG antibody heavy chain is shown in SEQ ID NO:4.

[0042] In some examples of this application, the light chain variable region of the anti-PVRIG antibody includes: a light chain variable region CDR1 sequence as shown in the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 80% identity with SEQ ID NO:5.

[0043] In some examples of this application, the light chain variable region of the anti-PVRIG antibody includes: a light chain variable region CDR2 sequence as shown in the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 80% identity with SEQ ID NO:6.

[0044] In some examples of this application, the light chain variable region of the anti-PVRIG antibody includes: a light chain variable region CDR3 sequence as shown in the amino acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 80% identity with SEQ ID NO:7.

[0045] In a specific example of this application, the amino acid sequence of the variable region of the light chain of the anti-PVRIG antibody is shown in SEQ ID NO:8.

[0046] In some examples of this application, the second antigen-binding region includes an anti-TIGIT antibody, which includes a heavy chain variable region and a light chain variable region.

[0047] In some examples of this application, the heavy chain variable region of the anti-TIGIT antibody includes a CDR1 sequence as shown in the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80% identity with SEQ ID NO:9. In some specific examples of this application, the CDR1 sequence of the heavy chain variable region of the bispecific antibody is as shown in SEQ ID NO:9. In one specific example of this application, using the CDR1 sequence of the heavy chain variable region as shown in SEQ ID NO:9 results in high antibody affinity and specificity.

[0048] In some examples of this application, the heavy chain variable region of the anti-TIGIT antibody includes a CDR2 sequence as shown in the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 80% identity with SEQ ID NO:10. In some specific examples of this application, the CDR2 sequence of the heavy chain variable region of the bispecific antibody is as shown in SEQ ID NO:10. In one specific example of this application, using the CDR2 sequence of the heavy chain variable region as shown in SEQ ID NO:10 results in higher antibody affinity and specificity.

[0049] In some examples of this application, the heavy chain variable region of the anti-TIGIT antibody includes a CDR3 sequence as shown in the amino acid sequence of SEQ ID NO:11 or an amino acid sequence having at least 80% identity with SEQ ID NO:11. In some specific examples of this application, the CDR3 sequence of the heavy chain variable region of the bispecific antibody is as shown in SEQ ID NO:11. In one specific example of this application, using the CDR3 sequence of the heavy chain variable region as shown in SEQ ID NO:11 results in high antibody affinity and specificity.

[0050] In a specific example of this application, the amino acid sequence of the variable region of the anti-TIGIT antibody heavy chain is shown in SEQ ID NO:12.

[0051] In some examples of this application, the light chain variable region of the anti-TIGIT antibody includes: a light chain variable region CDR1 sequence as shown in the amino acid sequence of SEQ ID NO:13 or an amino acid sequence having at least 80% identity with SEQ ID NO:13.

[0052] In some examples of this application, the light chain variable region CDR2 sequence is shown as the amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 80% identity with SEQ ID NO:14.

[0053] In some examples of this application, the light chain variable region CDR3 sequence is shown as the amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 80% identity with SEQ ID NO:15.

[0054] In a specific example of this application, the amino acid sequence of the variable region of the light chain of the anti-TIGIT antibody is shown in SEQ ID NO:16.

[0055] In some examples of this application, the heavy chain variable region and the light chain variable region are linked by a linker peptide. In one specific example of this application, the linker peptide has the amino acid sequence shown in SEQ ID NO:17.

[0056] In some examples of this application, the heavy chain variable region and the light chain variable region are connected by intermolecular forces.

[0057] In some examples of this application, the anti-PVRIG antibody further comprises a heavy chain constant region, wherein the heavy chain constant region is selected from at least one of murine antibodies, human antibodies, primate antibodies, or mutants thereof. In some preferred examples of this application, the heavy chain constant region is selected from human antibodies. In some more preferred examples of this application, the heavy chain constant region is selected from the human IgG1 constant region.

[0058] In some examples of this application, the anti-PVRIG antibody further comprises a light chain constant region, wherein the light chain constant region is selected from at least one of murine antibodies, human antibodies, primate antibodies, or mutants thereof. In some preferred examples of this application, the light chain constant region is selected from human antibodies. In a more preferred example of this application, the light chain constant region is selected from the human Ig kappa constant region.

[0059] In some examples of this application, the anti-TIGIT antibody further comprises a heavy chain constant region, wherein the heavy chain constant region is selected from at least one of murine antibodies, human antibodies, primate antibodies, or mutants thereof. In some preferred examples of this application, the heavy chain constant region is selected from human antibodies. In some more preferred examples of this application, the heavy chain constant region is selected from the human IgG1 constant region.

[0060] In some examples of this application, the anti-TIGIT antibody further comprises a light chain constant region selected from at least one of murine antibodies, human antibodies, primate antibodies, or mutants thereof. In some preferred examples of this application, the light chain constant region is selected from human antibodies. In a more preferred example of this application, the light chain constant region is selected from the human Ig kappa constant region.

[0061] In some examples of this application, the anti-TIGIT antibody includes a heavy chain and a light chain, the heavy chain including a heavy chain variable region and a heavy chain constant region, the light chain including a light chain variable region and a light chain constant region, and the heavy chain and the light chain linked by interchain disulfide bonds; the anti-PVRIG antibody includes a heavy chain variable region and a light chain variable region, the heavy chain variable region of the anti-PVRIG antibody is linked to the light chain variable region of the anti-PVRIG antibody via a linker peptide, the C-terminus of the heavy chain variable region of the anti-PVRIG antibody is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the light chain variable region of the anti-PVRIG antibody; the C-terminus of the heavy chain of the anti-TIGIT antibody is linked to the N-terminus of the heavy chain variable region of the anti-PVRIG antibody. In one example of this application, the linking includes direct linking (e.g., Figure 1 The structure shown in TP7 is an indirect link. The indirect link involves connecting the Fc region of the anti-TIGIT antibody to the C-terminus of the anti-PVRIG antibody heavy chain via a linker peptide.

[0062] In some examples of this application, the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:18 and an amino acid sequence as shown in SEQ ID NO:19.

[0063] In some examples of this application, the anti-PVRIG antibody includes a heavy chain and a light chain, the heavy chain including a heavy chain variable region and a heavy chain constant region, the light chain including a light chain variable region and a light chain constant region, and the heavy chain and the light chain linked by interchain disulfide bonds; the anti-TIGIT antibody includes a heavy chain variable region and a light chain variable region, the heavy chain variable region of the anti-TIGIT antibody is linked to the light chain variable region of the anti-TIGIT antibody via a linker peptide, the C-terminus of the heavy chain variable region of the anti-TIGIT antibody is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the light chain variable region of the anti-TIGIT antibody; the C-terminus of the heavy chain of the anti-PVRIG antibody is linked to the N-terminus of the heavy chain variable region of the anti-TIGIT antibody. In one example of this application, the linking includes direct linking (e.g., Figure 1 The structure shown in TP9 is an indirect link. The indirect link involves connecting the Fc region of the anti-PVRIG antibody to the C-terminus of the anti-TIGIT antibody heavy chain via a linker peptide.

[0064] In some examples of this application, the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:20 and an amino acid sequence as shown in SEQ ID NO:21.

[0065] Those skilled in the art will understand that the criteria for evaluating antibodies include affinity, specificity, selectivity, stability, purity, yield, and potency in specific cell or animal models. For bispecific antibodies, these criteria are typically determined by both the antibody sequence and conformation. The same antibody sequence in different conformations can significantly impact antibody quality and function. The inventors of this application have discovered that asymmetric antibody structures, such as the knot-into-hole conformation, result in a significant reduction in the yield and purity of the anti-PVRIG-TIGIT bispecific antibody in this application, hindering subsequent large-scale production and affecting its functional effect. Based on practical production applications, the inventors ultimately found that combining the disclosed anti-PVRIG and TIGIT antibody sequences with a symmetric conformation yields superior results in both yield and purity, and simplifies purification, which is beneficial for practical industrial applications.

[0066] In a second aspect of this application, a nucleic acid molecule is provided that encodes the bispecific antibody described in the first aspect of this application.

[0067] In some examples of this application, the aforementioned nucleic acid molecule may also include at least one of the following technical features:

[0068] In some examples of this application, the nucleic acid molecule is DNA.

[0069] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0070] In a third aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the second aspect of this application.

[0071] It should be noted that when ligating the aforementioned nucleic acid molecules to a vector, the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecules and control elements only need to be operatively linked. In this article, "operatively linked" means ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids and bacteriophages.

[0072] In a fourth aspect of this application, a method for preparing the bispecific antibody described in the first aspect is provided, comprising: introducing the expression vector described in the third aspect of this application into cells; and culturing the cells under conditions suitable for protein expression and secretion in order to obtain the bispecific antibody.

[0073] In some examples of this application, the above method may also include at least one of the following technical features:

[0074] In some examples of this application, the cells are eukaryotic cells.

[0075] In some specific examples of this application, the eukaryotic cells are mammalian cells. When the cells are mammalian cells, the expression efficiency of the antibody or its antigen-binding fragment is higher.

[0076] It should be noted that the eukaryotic cells mentioned do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0077] In a fifth aspect of this application, a recombinant cell is provided, carrying the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect. In some examples of this application, the recombinant cell is obtained by transfecting or transforming the expression vector.

[0078] It should be noted that the recombinant cells described in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this application are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0079] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the bispecific antibody described in this application. Those skilled in the art will readily understand that suitable conditions for bispecific antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the bispecific antibody based on the specific environment of their laboratory.

[0080] In a sixth aspect of this application, a composition is provided comprising: the bispecific antibody described in the first aspect of this application, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect.

[0081] It should be noted that the composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the invention. For example, the components contained in the composition may be applied to the subject as a whole or separately. When the components contained in the composition are applied to the subject separately, the individual components may be applied to the subject simultaneously or sequentially.

[0082] In a seventh aspect of this application, the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect in the preparation of a medicament for the treatment or prevention of cancer is provided.

[0083] In some examples of this application, the above-described uses may also include at least one of the following technical features:

[0084] In some examples of this application, at least one of the following is included: head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, bile duct cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

[0085] In an eighth aspect of this application, a medicament is provided, comprising: the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect, said medicament for treating or preventing cancer.

[0086] In some examples of this application, the aforementioned drug may also include at least one of the following technical features:

[0087] In some examples of this application, the cancer includes at least one of the following: head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, gastric cancer, liver cancer, bile duct cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

[0088] In some examples of this application, the drug comprises a pharmaceutically acceptable carrier and an effective amount of the bispecific antibody active ingredient.

[0089] It should be noted that the terms "effective amount" or "effective dose" refer to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0090] It should be noted that "pharmaceutically acceptable" ingredients are substances suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0091] The drug of this application contains a safe and effective amount of the active ingredient of this application and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage form of the drug of this application is an injection, an oral formulation (tablet, capsule, oral liquid), a transdermal formulation, or a sustained-release formulation. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The drug is preferably manufactured under aseptic conditions.

[0092] The effective amount of the active ingredient described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0093] Pharmaceutically acceptable carriers described in this application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0094] In a ninth aspect of this application, a kit is provided comprising the antibody or antigen-binding fragment thereof described in the first aspect.

[0095] In some examples of this application, the above-described reagent kit may also include at least one of the following technical features:

[0096] In some examples of this application, the kit is used to detect at least one of the PVRIG and TIGIT molecules.

[0097] Table 1

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] Note: The first peptide chain represents the anti-PVRIG-TIGIT bispecific antibody TP7 polypeptide chain 1; the second peptide chain represents the anti-PVRIG-TIGIT bispecific antibody TP7 polypeptide chain 2; the third peptide chain represents the anti-PVRIG-TIGIT bispecific antibody TP9 polypeptide chain 1; and the fourth peptide chain represents the anti-PVRIG-TIGIT bispecific antibody TP9 polypeptide chain 2.

[0106] The embodiments will be described in detail below. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0107] Example 1: Design and Construction of PVRIG-TIGIT Bispecific Antibody

[0108] In this embodiment, the scFv of the anti-PVRIG antibody is linked to the C-terminus of the anti-TIGIT antibody heavy chain via gene synthesis. Figure 1 A); Attach the scFv of the anti-TIGIT antibody to the C-terminus of the anti-PVRIG antibody heavy chain ( Figure 1B). The scFv of both the anti-PVRIG antibody and the anti-TIGIT antibody are linked using a "VH-linker peptide-VL" configuration. Both of the aforementioned bispecific antibodies use the human IgG1 subtype and are named TP7 and TP9, respectively. The sequences involved in this embodiment are shown in Table 1.

[0109] The aforementioned peptide sequences were constructed into PTT5 vectors (synthesized by Suzhou Genewiz) using molecular cloning technology. TP7 antibodies were prepared by transfecting the PTT5 vectors containing the encoding genes for TP7 polypeptide chains 1 and 2 into ExpiCHO-S cells (Gibco) using the ExpiFectamine CHO transfection kit (Gibco); TP9 antibodies were prepared by transfecting the PTT5 vectors containing the encoding genes for TP9 polypeptide chains 1 and 2 into ExpiCHO-S cells. 18-22 hours post-transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed and immediately added to the transfected cells. The cells were then incubated at 32°C with 5% CO2 and shaking at 120 rpm. On day 5 post-transfection, 8 ml of ExpiCHO Feed was added to the cells, mixed, and the cells were cultured further. The cell culture supernatant was harvested by centrifugation after 10-14 days and purified using a Protein A affinity chromatography column (NanoTech) to obtain the bispecific antibodies.

[0110] Example 2: Affinity determination of bispecific antibodies

[0111] The affinities of bispecific antibodies TP7 and TP9 with PVRIG protein (ACRO) and TIGIT protein (ACRO), respectively, were determined using surface plasmon resonance (SPR) technology. The specific procedures are as follows:

[0112] Turn on the Biacore 1K instrument (Cytiva), place the Protein A chip (Cytiva) inside, and then equilibrate it in HBS-EP+ buffer. Next, flow the target antibody at a concentration of 0.5 μg / mL through the chip surface at a rate of 30 μL / min to capture the antibody. Then, flow different concentrations of PVRIG or TIGIT protein through the chip surface at a rate of 30 μL / min, setting the binding time to 90 s, followed by dissociation for 900 s. Perform kinetic analysis using a 1:1 binding model.

[0113] The affinity test results of the bispecific antibodies for human PVRIG protein and human TIGIT protein are shown in Table 2.

[0114] Table 2 Results of Bispecific Antibody Affinity Detection

[0115]

[0116]

[0117] Example 3: Identification of the binding ability of bispecific antibodies to PVRIG and TIGIT proteins

[0118] Human PVRIG protein (ACRO) was diluted to 2 μg / mL with PBS buffer and added to each well in a 96-well plate at a volume of 100 μL. The plate was incubated overnight at 4°C. The PBS buffer was discarded, and the plate was washed three times with PBST (pH 7.2 PBS containing 0.1% Tween 20). Then, 300 μL / well of 5% BSA was added for blocking. The blocking solution was discarded, and the antibody was diluted to the appropriate concentration with 100 μL / well of 0.05% BSA. The plate was then incubated at room temperature for 1 hour. After washing three times with PBST, biotinylated human TIGIT protein (ACRO) was diluted to 25 ng / mL with 0.05% BSA and added to each well in a 100 μL solution. The plate was incubated at room temperature for 1 hour. After washing the plate three times with PBST, add 100 μL of horseradish peroxidase (HRP)-labeled streptavidin secondary antibody (Southern Biotech) diluted with 0.5% BSA and incubate at room temperature for 1 h. After washing the plate three times with PBST, add 80 μL / well TMB and incubate at room temperature for 3–5 min. Stop the reaction by adding 80 μL / well stop solution. Read the absorbance at 450 nm using a microplate reader.

[0119] Test results as follows Figure 2 As shown, the bispecific antibodies TP7 and TP9 can effectively bind to both PVRIG and TIGIT proteins.

[0120] Example 4: Identification of the binding ability of bispecific antibodies to 293T-human PVRIG cells

[0121] The pLVX-EF1α-IRES-zsGreen vector containing the human PVRIG encoding gene (SEQ ID NO: 22), along with the pMD2G and psPAX2 vectors, were transfected into 293T cells using polyetherimide (PEI, Polysciences). Forty-eight hours after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the lentiviral supernatant. All of the lentiviral supernatant was then added to a container containing 1×10⁻⁶ cells / mL. 4 Polyglobulin (Sigma) was added to 6-well plates containing 293T cells to a final concentration of 4 μg / mL, and the cells were cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were 293T-human PVRIG cells.

[0122] 293T-human PVRIG cells were diluted to 1×10⁻⁶ with PBS. 6 Cells were added at a rate of 90 μL / well to a 96-well plate, followed by 10 μL / well of rat serum, and blocked at 4°C for 30 min. A series of serially diluted test antibodies were then added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS, followed by 1 μL / well of Alexa-647-labeled rat anti-human Fc antibody (Biolegend), and incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry.

[0123] Test results as follows Figure 3 As shown, the activity of the anti-PVRIG-TIGIT bispecific antibody TP9 in binding to 293T-human PVRIG cells is similar to that of the anti-PVRIG antibody.

[0124] Example 5: Identification of the binding ability of bispecific antibodies to 293T-human TIGIT cells

[0125] Using the chemical reagent PEI (Polysciences), the pLVX-EF1α-IRES-zsGreen vector containing the full-length human TIGIT gene (SEQ ID NO:23), along with the pMD2G and psPAX2 vectors, were transfected into 293T cells. Forty-eight hours after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the lentiviral supernatant. All of the lentiviral supernatant was then added to a container containing 1×10⁻⁶ cells / mL. 4 Polyglobulin (Sigma) was added to 6-well plates containing 293T cells to a final concentration of 4 μg / mL, and the cells were cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were 293T-human TIGIT cells.

[0126] 293T-human TIGIT cells were diluted to 2 × 10⁻⁶ with PBS. 6 Cells were added at a rate of 90 μL / well to a 96-well plate, followed by 10 μL / well of goat serum, and blocked at 4°C for 30 min. A series of serially diluted test antibodies were then added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS, followed by 1 μL / well of Alexa-647-labeled rat anti-human Fc antibody (Biolegend), and incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry.

[0127] Test results as follows Figure 4As shown, the activity of the anti-PVRIG-TIGIT bispecific antibody TP7 in binding to 293T-human TIGIT cells is similar to that of the anti-TIGIT antibody.

[0128] Example 6: Identification of the ability of bispecific antibodies to block the binding of PVRIG to CD112

[0129] Human PVRIG protein (ACRO) was diluted to 1 μg / mL with PBS buffer and added to 100 μL / well of a 96-well plate. The plate was incubated overnight at 4°C. The PBS buffer was discarded, and the plate was washed three times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 300 μL / well of 5% BSA was added for blocking, and the plate was incubated at room temperature for 2 hours. The blocking solution was discarded, and biotinylated human CD112 protein (ACRO) was diluted to 20 ng / mL with 0.05% BSA. 50 μL of the antibody diluted with 0.05% BSA was added to each well, and the plate was incubated at room temperature for 1 hour. After washing three times with PBST, 100 μL of HRP-labeled streptavidin secondary antibody (Southern Biotech) diluted with 0.5% BSA was added, and the plate was incubated at room temperature for 1 hour. After washing the plate three times with PBST, add 80 μL of TMB per well and incubate at room temperature for 3-5 min. Then, add 80 μL of stop solution per well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader.

[0130] Test results as follows Figure 5 As shown, the activity of the bispecific antibody TP9 in blocking the binding of PVRIG to CD112 is comparable to that of the anti-PVRIG antibody.

[0131] Example 7: Identification of the ability of bispecific antibodies to block the binding of TIGIT to CD155

[0132] Human TIGIT protein (ACRO) was diluted to 2 μg / mL with PBS buffer and added to 100 μL / well of a 96-well plate. The plate was incubated overnight at 4°C. The PBS buffer was discarded, and the plate was washed three times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 300 μL / well of 5% BSA was added for blocking, and the plate was incubated at room temperature for 2 h. The blocking solution was discarded, and biotinylated human CD155 protein (ACRO) was diluted to 250 ng / mL with 0.05% BSA. 50 μL of the antibody diluted with 0.05% BSA was added to each well, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, 100 μL of HRP-labeled streptavidin secondary antibody (Southern Biotech) diluted with 0.5% BSA was added, and the plate was incubated at room temperature for 1 h. After washing the plate three times with PBST, add 80 μL of TMB per well and incubate at room temperature for 3-5 min. Then, add 80 μL of stop solution per well to terminate the reaction. Read the absorbance at 450 nm using a microplate reader.

[0133] Test results as follows Figure 6 As shown, the bispecific antibodies TP7 and TP9 have comparable activity in blocking the binding of TIGIT to CD155 to the anti-TIGIT antibody.

[0134] Example 8: Identification of the ability of bispecific antibodies to block the binding of 293T-human CD112 cells to PVRIG protein.

[0135] Using the chemical reagent PEI (Polysciences), the pLVX-EF1α-IRES-zsGreen vector containing the full-length coding gene of human CD112 (SEQ ID NO:24), along with the pMD2G and psPAX2 vectors, were transfected into 293T cells. Forty-eight hours after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the lentiviral supernatant. The entire lentiviral supernatant was then added to a container containing 1×10⁻⁶ cells / mL. 4 Polybrene (Sigma) was added to 6-well plates containing 293T cells at a final concentration of 4 μg / mL and cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were 293T-human CD112 cells.

[0136] 293T-human CD112 cells were diluted to 2 × 10⁻⁶ with PBS. 6Cells were added at a rate of 90 μL / well to a 96-well plate, followed by 10 μL / well of rat serum, and blocked at 4°C for 30 min. A series of serially diluted test antibodies and a final concentration of 0.5 μg / mL of human PVRIG protein (ACRO) tagged with mouse IgG2a Fc were added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS, followed by 1 μL / well of Alexa-647-labeled rat anti-mouse IgG2a antibody (Biolegend), and incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry.

[0137] Test results as follows Figure 7 As shown, the activity of the anti-PVRIG-TIGIT bispecific antibody TP9 in blocking the binding of 293T-human CD112 cells to the PVRIG protein is similar to that of the anti-PVRIG antibody.

[0138] Example 9: Identification of the ability of bispecific antibodies to block the binding of 293T-human TIGIT cells to CD155 protein.

[0139] 293T-human TIGIT cells were diluted to 2 × 10⁻⁶ with PBS. 6 Cells were added at a rate of 90 μL / well to a 96-well plate, followed by 10 μL / well of rat serum, and blocked at 4°C for 30 min. A series of serially diluted test antibodies and a final concentration of 1 μg / mL of human CD155 protein tagged with mouse IgG2a Fc (ACRO) were added, and the plates were incubated at 4°C for 30 min. After incubation, the cells were washed twice with PBS, followed by 1 μL / well of Alexa-647-labeled rat anti-mouse IgG2a antibody (Biolegend), and incubated at 4°C in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / well of PBS and analyzed by flow cytometry.

[0140] Test results as follows Figure 8 As shown, the activity of the anti-PVRIG-TIGIT bispecific antibody TP7 in blocking the binding of 293T-human TIGIT cells to CD155 protein is similar to that of the anti-TIGIT antibody.

[0141] Example 10: Identification of the in vitro PBMC-promoting killing ability of bispecific antibodies

[0142] A375 melanoma cells (ATCC ID CRL-1619) were collected by digestion with trypsin (Gibco), centrifuged at 200g for 5 min, and the cell pellet was resuspended in 1640 medium. Cell counts were then performed, and the cell density was adjusted to approximately 1.5 × 10⁻⁶ cells / cm². 6Cells / mL. Take 1 mL of cell suspension into a 1.5 mL EP tube, add 1 μL of Cell Trace Violet (Invitrogen), mix well, and label at 37℃ for 20 min. Wash the labeled cells three times with 1640 medium and adjust the cell density to 1×10⁻⁶ cells / mL. 5 Human peripheral blood mononuclear cells (PBMCs, seldomochondria) were collected by centrifugation at 500g, resuspended in 1640 medium, counted, and the PBMC concentration was adjusted to 1×10⁻⁶ cells / mL. 6 Cells / mL. Add 100 μL / well of diluted A375 cells and 100 μL / well of PBMCs to a 96-well round-bottom plate, and add 10 μL / well of each test antibody to a final concentration of 66 nM. Centrifuge at 250 g for 4 min at room temperature, and incubate at 37°C in a 5% CO2 incubator for 4–6 h. After incubation, add 1 μL of 7AAD (BDBioscience) to each well and mix well. Analyze using flow cytometry.

[0143] Test results as follows Figure 9 As shown, the anti-PVRIG-TIGIT bispecific antibodies TP7 and TP9 can effectively promote the killing of A375 melanoma cells by human PBMCs.

[0144] Example 11 Identification of the ability of bispecific antibodies to promote T cell cytokine secretion

[0145] PBMCs (Miaoshun) were cultured in T150 cell culture flasks using 1640 complete culture medium, with final concentrations of 1 μg / mL pp65 antigen peptide (MBL), 2 ng / mL IL2 (ACRO), and 10 ng / mL IL7 (ACRO). The flasks were incubated at 37°C in a 5% CO2 incubator, with the complete culture medium changed every 3 days. On day 12, induced PBMCs were collected, and CD8 T cells were isolated using a CD8 T cell isolation kit (Mitteni) according to the manufacturer's instructions. The cell density was adjusted to 1 × 10⁶ cells / mL. 6 100 μL / well CD8 T cells were transferred to a 96-well plate and incubated for 30 minutes. Cultured A375 melanoma cells (Shanghai Cell Bank, Chinese Academy of Sciences) were collected by trypsin digestion (Gibco) and the cell density was adjusted to 1 × 10⁶ cells / mL. 5 100 μL / well A375 cells were transferred to a 96-well plate, along with pp65 antigen peptide and a series of serially diluted test antibodies. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 18 h. After incubation, the cell culture supernatant was collected by centrifugation, and the IFN-γ level in the supernatant was detected using a pre-coated human IFN-γ ELISA kit (Dayou) according to the manufacturer's instructions.

[0146] Test results as follows Figure 10 As shown, both the anti-PVRIG-TIGIT bispecific antibodies TP7 and TP9 can effectively promote the secretion of IFN-γ by human CD8 T cells.

[0147] In summary, the anti-PVRIG-TIGIT bispecific antibody prepared in this application can bind to human PVRIG and TIGIT proteins with high affinity, and can effectively block the binding of PVRIG to CD112 and TIGIT to CD155, thereby promoting the killing of tumor cells by human PBMCs and enhancing the cytokine secretion function of CD8 T cells.

[0148] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0149] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bispecific antibody, characterized in that, include: The first antigen-binding region has PVRIG molecule binding activity; and The second antigen-binding region has TIGIT molecule binding activity; The first antigen-binding region is connected to the second antigen-binding region.

2. The bispecific antibody according to claim 1, characterized in that, The first antigen-binding region includes an anti-PVRIG antibody, which includes a heavy chain variable region and a light chain variable region; Optionally, the heavy chain variable region of the anti-PVRIG antibody includes: a heavy chain variable region CDR1 sequence as shown in the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% identity with SEQ ID NO:1; The heavy chain variable region CDR2 sequence is shown as the amino acid sequence of SEQ ID NO:2 or as an amino acid sequence having at least 80% identity with SEQ ID NO:2; The heavy chain variable region CDR3 sequence is shown as the amino acid sequence of SEQ ID NO:3 or as an amino acid sequence having at least 80% identity with SEQ ID NO:3; Optionally, the amino acid sequence of the variable region of the heavy chain of the anti-PVRIG antibody is as shown in SEQ ID NO:4; Optionally, the light chain variable region of the anti-PVRIG antibody includes: a light chain variable region CDR1 sequence as shown in the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 80% identity with SEQ ID NO:5; The light chain variable region CDR2 sequence is shown as the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 80% identity with SEQ ID NO:

6. The light chain variable region CDR3 sequence is shown as the amino acid sequence of SEQ ID NO:7 or as an amino acid sequence having at least 80% identity with SEQ ID NO:

7. The amino acid sequence of the variable region of the light chain of the anti-PVRIG antibody is shown in SEQ ID NO:

8.

3. The bispecific antibody according to claim 1, characterized in that, The second antigen-binding region includes an anti-TIGIT antibody, which includes a heavy chain variable region and a light chain variable region; Optionally, the anti-TIGIT antibody heavy chain variable region includes: a heavy chain variable region CDR1 sequence as shown in the amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 80% identity with SEQ ID NO:9; The heavy chain variable region CDR2 sequence is shown as the amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 80% identity with SEQ ID NO:

10. The heavy chain variable region CDR3 sequence is shown as the amino acid sequence of SEQ ID NO:11 or as an amino acid sequence having at least 80% identity with SEQ ID NO:11; Optionally, the amino acid sequence of the variable region of the anti-TIGIT antibody heavy chain is as shown in SEQ ID NO:12; Optionally, the light chain variable region of the anti-TIGIT antibody includes: a light chain variable region CDR1 sequence as shown in the amino acid sequence of SEQ ID NO:13 or an amino acid sequence having at least 80% identity with SEQ ID NO:13; The light chain variable region CDR2 sequence is shown as the amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 80% identity with SEQ ID NO:

14. The light chain variable region CDR3 sequence is shown as the amino acid sequence of SEQ ID NO:15 or as the amino acid sequence having at least 80% identity with SEQ ID NO:

15. Optionally, the amino acid sequence of the variable region of the light chain of the anti-TIGIT antibody is as shown in SEQ ID NO:

16.

4. The bispecific antibody according to claim 2 or 3, characterized in that, The heavy chain variable region and the light chain variable region are linked by a linker peptide; Optionally, the linker peptide has an amino acid sequence as shown in SEQ ID NO:17; Optionally, the heavy chain variable region and the light chain variable region are connected by intermolecular forces.

5. The bispecific antibody according to claim 2, characterized in that, The anti-PVRIG antibody further includes: a heavy chain constant region; Optionally, the heavy chain constant region is selected from at least one of mouse antibodies, human antibodies, primate antibodies, or mutants thereof; Optionally, the heavy chain constant region is selected from human antibodies, preferably the human IgG1 constant region; Optionally, the anti-PVRIG antibody further comprises: a light chain constant region; Optionally, the light chain constant region is selected from at least one of mouse antibodies, human antibodies, primate antibodies, or mutants thereof; Optionally, the light chain constant region is selected from human antibodies, preferably the human Ig kappa constant region.

6. The bispecific antibody according to claim 3, characterized in that, The anti-TIGIT antibody further includes: a heavy chain constant region; Optionally, the heavy chain constant region is selected from at least one of mouse antibodies, human antibodies, primate antibodies, or mutants thereof; Optionally, the heavy chain constant region is selected from human antibodies, preferably the human IgG1 constant region; Optionally, the anti-TIGIT antibody further comprises: a light chain constant region; Optionally, the light chain constant region is selected from at least one of mouse antibodies, human antibodies, primate antibodies, or mutants thereof; Optionally, the light chain constant region is selected from human antibodies, preferably the human Ig kappa constant region.

7. The bispecific antibody according to any one of claims 2 to 6, characterized in that, The anti-TIGIT antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, the light chain comprising a light chain variable region and a light chain constant region, and the heavy chain and the light chain being linked by interchain disulfide bonds. The anti-PVRIG antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region of the anti-PVRIG antibody is linked to the light chain variable region of the anti-PVRIG antibody via a linker peptide. The C-terminus of the heavy chain variable region of the anti-PVRIG antibody is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the light chain variable region of the anti-PVRIG antibody. The C-terminus of the heavy chain of the anti-TIGIT antibody is connected to the N-terminus of the variable region of the heavy chain of the anti-PVRIG antibody.

8. The bispecific antibody according to any one of claims 2 to 6, characterized in that, The anti-PVRIG antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, the light chain comprising a light chain variable region and a light chain constant region, and the heavy chain and the light chain being linked by interchain disulfide bonds. The anti-TIGIT antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region of the anti-TIGIT antibody is linked to the light chain variable region of the anti-TIGIT antibody via a linker peptide. The C-terminus of the heavy chain variable region of the anti-TIGIT antibody is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the light chain variable region of the anti-TIGIT antibody. The C-terminus of the heavy chain of the anti-PVRIG antibody is connected to the N-terminus of the variable region of the heavy chain of the anti-TIGIT antibody.

9. The bispecific antibody according to claim 7, characterized in that, The bispecific antibody has an amino acid sequence as shown in SEQ ID NO:18 and an amino acid sequence as shown in SEQ ID NO:

19.

10. The bispecific antibody according to claim 8, characterized in that, The bispecific antibody has an amino acid sequence as shown in SEQ ID NO:20 and an amino acid sequence as shown in SEQ ID NO:

21.

11. A nucleic acid molecule, characterized in that, The nucleic acid encodes the bispecific antibody according to any one of claims 1 to 10; Optionally, the nucleic acid molecule is selected from DNA.

12. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 11.

13. A method for preparing the bispecific antibody according to any one of claims 1 to 10, characterized in that, include: The expression vector according to claim 12 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the bispecific antibody; Optionally, the cells are eukaryotic cells.

14. A recombinant cell, characterized in that, The recombinant cells carry the bispecific antibody as described in any one of claims 1 to 10, the nucleic acid molecule as described in claim 11, or the expression vector as described in claim 12.

15. A composition, characterized in that, include: The bispecific antibody according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the expression vector according to claim 12, or the recombinant cell according to claim 14.

16. Use of the bispecific antibody of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the expression vector of claim 12, the recombinant cell of claim 14, or the composition of claim 15 in the preparation of a medicament for the treatment or prevention of cancer.

17. The use according to claim 16, characterized in that, The cancers include at least one of the following: head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, stomach cancer, liver cancer, bile duct cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

18. A drug, characterized in that, include: The bispecific antibody according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the expression vector according to claim 12, the recombinant cell according to claim 14, or the composition according to claim 15, wherein the drug is used to treat or prevent cancer.

19. The medicament according to claim 18, characterized in that, The cancers include at least one of the following: head and neck cancer, glioma, thyroid cancer, lung cancer, colorectal cancer, stomach cancer, liver cancer, bile duct cancer, breast cancer, ovarian cancer, endometrial cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, melanoma, multiple myeloma, and acute myeloid leukemia.

20. A reagent kit, characterized in that, The kit contains the bispecific antibody as described in any one of claims 1 to 10.

21. The reagent kit according to claim 20, characterized in that, The kit is used to detect at least one of the PVRIG and TIGIT molecules.

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