Bispecific antibodies and their use

Abstract

The application provides a bispecific antibody targeting CD3 and a tumor target and an application thereof, and the bispecific antibody comprises a first antigen binding region, a second antigen binding region and an Fc part; the Fc part comprises a first Fc peptide segment and a second Fc peptide segment; the first antigen binding region comprises an scFv fragment and a binding protein of a first molecule or a fragment thereof, the binding protein of the first molecule or the fragment thereof is connected to the N end of the scFv fragment, and the C end of the scFv fragment is connected to the N end of the first Fc peptide segment; the second antigen binding region comprises a binding protein of a second molecule or a fragment thereof, the binding protein of the second molecule or the fragment thereof is connected to the N end of the second Fc peptide segment; the first molecule and the second molecule are the same, and the scFv fragment has CD3 binding activity. The bispecific antibody of the application has weak T cell binding, strong tumor cell binding, good safety, high anticancer activity, high clinical application and drug development value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, this invention relates to a bispecific antibody and its application. More specifically, this invention relates to an antibody, nucleic acid molecule, vector or transformant, cell, pharmaceutical composition and reagent kit and their application. Background Technology

[0002] Cancer is a major disease affecting human survival and development. According to the latest data, there are approximately 19 million new cancer cases and 10 million cancer deaths worldwide each year, and both incidence and mortality rates are on the rise. Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy have significant side effects and are prone to recurrence. In recent years, immunotherapy, including tumor-targeting antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a source of new hope in the fight against cancer. Immunotherapy, represented by PD-1 / L1, has shown great potential. However, even the PD-1 / L1 therapy, which currently has the broadest approved indications, has an overall response rate of only 30%, meaning many more patients cannot benefit from it. One major reason is that immune checkpoint therapy is ineffective against "cold tumors." T cells recognize neoantigens, which are antigens of tumor gene mutations, through their surface T cell receptors (TCRs). Some tumors have low gene mutation frequencies and few types of neoantigens, and are called "cold tumors." Current immune checkpoint therapies, such as PD-1 / L1 therapy, aim to fight cancer by restoring the function of T cells themselves. However, in "cold tumors," T cells cannot effectively recognize tumors, making immune checkpoint therapy ineffective against "cold tumors."

[0003] CD3-based bispecific antibodies (hereinafter referred to as "CD3 bispecific antibodies") recruit T cells to reach the tumor site, bridging T cells and the tumor, promoting T cell activation and tumor killing. These bispecific antibodies do not require neoantigens and can guide T cells to kill "cold tumors." After binding to T cells and tumor cells, CD3 bispecific antibodies trigger strong activation signals, thus to some extent "ignoring" the inhibitory signals of immune checkpoint molecules. However, CD3 bispecific antibodies also promote the production of large amounts of pro-inflammatory cytokines, such as TNFα and IL-6, triggering a strong cytokine storm and excessive immune response, causing damage to the body, and in severe cases, potentially endangering life. Therefore, CD3-based bispecific antibodies have good clinical application prospects, but their safety needs further improvement.

[0004] One approach to addressing the safety concerns of CD3 bispecific antibodies is to increase their affinity for tumor targets while simultaneously decreasing their affinity for CD3 and weakening their binding to T cells. This would allow the CD3 bispecific antibody drug to be distributed more extensively within the tumor, increasing local drug concentration, reducing peripheral drug concentration, and minimizing off-target toxicity. Therefore, there is an urgent clinical need to develop CD3 bispecific antibodies with improved safety and higher clinical application value. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, one object of this invention is to provide a bispecific antibody targeting CD3 and tumor targets. This bispecific antibody can bind to both CD3 and tumor targets, exhibiting low CD3 binding activity, weak binding to T cells, and weak ability to bridge T cells and tumor cells. Simultaneously, it effectively promotes the activity of PBMCs in killing tumor cells, enhances T cell activation performance, and promotes drug distribution to the tumor site rather than peripheral or lymphoid tissues. Therefore, the bispecific antibody of this invention has good safety, high anticancer activity, and high value for clinical application and drug development.

[0006] This invention is based on the inventor's discoveries and understanding of the following facts and problems: To improve the safety of CD3 and MICA bispecific antibodies, the inventors conducted extensive screening and experimental verification, resulting in a novel configuration of the CD3 and MICA bispecific antibody. Further experimental results demonstrated that this CD3 and MICA bispecific antibody exhibits weak binding to T cells, strong binding to tumor cells, and high pro-cytotoxic activity, effectively promoting PBMC-mediated killing of tumor cells and demonstrating good anti-cancer activity. Moreover, this bispecific antibody exhibits reduced T cell binding activity, high T cell activation performance, and enhanced safety, making it valuable for clinical applications and drug development.

[0007] Therefore, in a first aspect, the present invention provides a bispecific antibody. According to an embodiment of the present invention, the bispecific antibody comprises: a first antigen-binding region, a second antigen-binding region, and an Fc portion; The Fc portion includes a first Fc peptide segment and a second Fc peptide segment. The first antigen-binding region comprises an scFv fragment and a binding protein or a fragment thereof of a first molecule, wherein the binding protein or a fragment thereof of the first molecule is linked to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is linked to the N-terminus of the first Fc peptide. The second antigen-binding region contains a second molecule of binding protein or a fragment thereof, and the second molecule of binding protein or a fragment thereof is linked to the N-terminus of the second Fc peptide. The first molecule and the second molecule are identical, and the scFv fragment has CD3 binding activity.

[0008] The bispecific antibody according to embodiments of the present invention can bind to CD3 and tumor targets. This bispecific antibody exhibits low CD3 binding activity, weak binding to T cells, and a weak ability to bridge T cells and tumor cells. Simultaneously, it effectively promotes the activity of PBMCs in killing tumor cells, enhances T cell activation performance, and promotes drug distribution to the tumor site rather than the peripheral or lymphatic tissue. Therefore, the bispecific antibody of the present invention has good safety, high anticancer activity, and high value for clinical application and drug development.

[0009] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the aforementioned bispecific antibody. The nucleic acid according to embodiments of the invention can encode a bispecific antibody capable of simultaneously targeting CD3 and tumor targets.

[0010] In a third aspect, the present invention provides a vector or transformant. According to embodiments of the invention, the vector or transformant contains the aforementioned nucleic acid. Thus, the aforementioned bispecific antibody can be effectively expressed using the constructed vector or transformant.

[0011] In a fourth aspect, the present invention provides a cell. According to embodiments of the invention, the cell carries the aforementioned nucleic acid or the aforementioned vector or transformant, or expresses the aforementioned bispecific antibody. The cell according to embodiments of the invention is obtained by transfecting or transforming the vector or transformant, and the cell can efficiently express the aforementioned bispecific antibody under suitable conditions.

[0012] In a fifth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises: the aforementioned bispecific antibody, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned cells. According to embodiments of the invention, the bispecific antibody effectively promotes PBMC killing of tumor cells, exhibiting high anti-cancer activity; the bispecific antibody has low CD3 binding activity and good safety. The resulting drug can be further used for the prevention and / or treatment of CD3 and / or tumor target-mediated diseases.

[0013] In a sixth aspect, the present invention provides a kit. According to embodiments of the invention, the kit comprises: the aforementioned bispecific antibody, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned cells. According to embodiments of the invention, the kit can bind to CD3 protein and / or tumor targets, and can effectively identify CD3 protein and / or tumor targets.

[0014] Those skilled in the art will understand that the features and advantages described above for antibodies, nucleic acids, vectors or transformants, cells or pharmaceutical compositions also apply to this kit and will not be repeated here.

[0015] In a seventh aspect of the invention, the invention proposes the use of the aforementioned bispecific antibody, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned cells in the preparation of a kit for detecting CD3 and / or tumor targets.

[0016] Those skilled in the art will understand that the features and advantages described above for antibodies, nucleic acids, vectors or transformants, cells or pharmaceutical compositions are equally applicable to the use in this preparation kit, and will not be repeated here.

[0017] In an eighth aspect of the invention, the invention provides for the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned carrier or transformant, the aforementioned cell or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of CD3 and / or tumor target-mediated diseases.

[0018] Those skilled in the art will understand that the features and advantages described above for antibodies, nucleic acid molecules, vectors or transformants, cells and pharmaceutical compositions also apply to this use, and will not be repeated here.

[0019] 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

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram of a CD3×MICA bispecific antibody according to a specific embodiment of the present invention; Figure 2 The image shows the ELISA results of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies to MICA protein according to specific embodiments of the present invention. Figure 3 The image shows the ELISA results of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies to CD3E&D proteins according to specific embodiments of the present invention. Figure 4The image shows the ELISA results of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies to CD3E&D and MICA proteins according to specific embodiments of the present invention. Figure 5 The image shows the flow cytometry results of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies to CD8+ T cells according to specific embodiments of the present invention. Figure 6 The image shows the flow cytometry results of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies to HCT-15 colorectal cancer cells according to specific embodiments of the present invention. Figure 7 The image shows the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting PBMC killing of HCT-15 colorectal cancer cells according to specific embodiments of the present invention. Figure 8 The image shows the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting PBMC killing of NCI-H1299 lung cancer cells according to specific embodiments of the present invention. Figure 9 The image shows the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting the killing of PANC-1 pancreatic cancer cells by PBMCs according to specific embodiments of the present invention. Figure 10 The image shows the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting the killing of A498 renal cell carcinomas by PBMCs according to specific embodiments of the present invention. Figure 11The image shows the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting PBMC killing of PC-3 prostate cancer cells according to specific embodiments of the present invention. Figure 12 The figure shows the results of Cross3×h5A1002 1:1 and Cross3×h5A1002 2:1 antibodies promoting the secretion of immune-activating cytokines IL-2 and IFN-γ after co-incubation of PBMCs and HCT-15 colorectal cancer cells according to specific embodiments of the present invention. Figure 13 The image shows the results of Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1, and SP34×h5A1002 2:1 antibodies promoting PBMC killing of HCT-15 colorectal cancer cells according to a specific embodiment of the present invention. Figure 14 The image shows the results of Cross3×h5A1002 2:1, Cross3×1D5 1:1, and Cross3×1D5 2:1 antibodies promoting PBMC killing of HCT-15 colorectal cancer cells according to specific embodiments of the present invention. Figure 15 The image shows the results of Cross3×h5A1002 2:1, Cross3×13A9 1:1, and Cross3×13A9 2:1 antibodies promoting PBMC killing of HCT-15 colorectal cancer cells according to a specific embodiment of the present invention. Figure 16 The image shows the results of Cross3×h5A1002 2:1, Cross3×619 1:1, and Cross3×619 2:1 antibodies promoting PBMC killing of HCT-15 colorectal cancer cells according to specific embodiments of the present invention. Figure 17 The diagram shows the in vivo efficacy of the Cross3×h5A1002 2:1 antibody according to a specific embodiment of the present invention in the A-375 human melanoma model of immune reconstituted mice. Figure 18 The figure shows the in vivo efficacy results of the Cross3×h5A1002 2:1 antibody according to a specific embodiment of the present invention in the NCI-H1299 human lung cancer model of immune reconstituted mice; Figure 19 This figure shows the in vivo efficacy results of the Cross3×h5A1002 2:1 antibody-promoted immune reconstitution mouse PC-3 human prostate cancer model according to a specific embodiment of the present invention. Detailed Implementation

[0021] 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.

[0022] Furthermore, 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 technical features indicated. 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.

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] Terms and Definitions To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0025] In this article, the term "antibody" generally refers to an antibody that recognizes one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, bispecific antibodies, heavy-chain-only antibodies, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, provided they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854-4861). Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunologically active portion of any of these polypeptides, i.e., molecules or portions thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases.

[0026] In this paper, certain regions within the variable region exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the location where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0027] In this article, the term "anti-CD3 antibody" refers to an antibody capable of binding to CD3. This antibody is also referred to herein as a "CD3-binding antibody." The term "anti-MICA antibody" refers to an antibody capable of binding to MICA. This antibody is also referred to herein as a "MICA-binding antibody."

[0028] In this document, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment," and can include a portion of a complete antibody, generally an antigen-binding region or variable region. This includes, but is not limited to: Fv, scFv, Fab, Fab', Fab'-SH, scFab (single chain Fab fragment, scFab), F(ab')2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylation," or "PEGylation") (a PEGylation fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol).

[0029] In this article, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (such as a mouse) with the constant region of an antibody from another species (such as a human).

[0030] In this article, the term "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv backbone region (FR)) amino acid sequences in the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with the non-CDR amino acid sequences in the constant and variable regions of an antibody from another species (e.g., human). That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when all non-CDR amino acid sequences in both the constant and variable regions are humanized, it is called a humanized antibody. The humanization method can be performed using conventional antibody engineering techniques, and will not be elaborated upon here.

[0031] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0032] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0033] In this document, the term "conservatively modified amino acid sequence such as" refers to an amino acid modification that does not significantly affect or alter the binding properties of an antibody containing that amino acid sequence. Such modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine).

[0034] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0035] In this document, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining an active bispecific antibody with a liquid carrier, a finely fragmented solid carrier, or both.

[0036] In this article, the term "pharmaceuticalally acceptable ingredient" refers to a substance that is suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0037] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those incompatible with the bispecific antibody of the present invention, for example, any adverse biological effects they produce or interactions that occur in a harmful manner with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0038] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, bispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0039] In this document, the term "treatment" refers to the use of drugs to achieve desired pharmacological and / or physiological effects. These effects may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in susceptible individuals who have not yet been diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any use of medicine to treat, cure, alleviate, improve, reduce, or suppress an individual's disease, including but not limited to administering medicines containing the bispecific antibodies described herein to individuals in need.

[0040] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0041] This invention proposes a bispecific antibody, nucleic acid molecule, vector or transformant, cell, pharmaceutical composition, kit and its application targeting CD3 and tumor targets, which will be described in detail below.

[0042] Bispecific antibodies This invention provides a bispecific antibody. The bispecific antibody comprises: a first antigen-binding region, a second antigen-binding region, and an Fc portion; The Fc portion includes a first Fc peptide segment and a second Fc peptide segment. The first antigen-binding region comprises an scFv fragment and a binding protein or a fragment thereof of a first molecule, wherein the binding protein or a fragment thereof of the first molecule is linked to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is linked to the N-terminus of the first Fc peptide. The second antigen-binding region contains a second molecule of binding protein or a fragment thereof, and the second molecule of binding protein or a fragment thereof is linked to the N-terminus of the second Fc peptide. The first molecule and the second molecule are identical, and the scFv fragment has CD3 binding activity.

[0043] The bispecific antibody according to embodiments of the present invention can bind to CD3 and tumor targets. This bispecific antibody has low CD3 binding activity and a weak ability to bridge T cells and tumor cells. Simultaneously, it can effectively promote the activity of PBMCs in killing tumor cells, has enhanced T cell activation performance, and can promote drug distribution to the tumor site rather than the periphery or lymphoid tissue. Therefore, the bispecific antibody of the present invention has good safety, high anticancer activity, and high value for clinical application and drug development.

[0044] According to embodiments of the present invention, the first molecule and the second molecule are selected from MICA, PDL1, CD47, TIGIT, CD73, CD33, CEACAM1, CEACAM5, CEACAM6, STING, WNT, Beta catenin, B7H3, VISITA, CD19, BCMA, CD22, CD20, CD123, CD38, CEA, CD25, CD46, CD138, PSCA, PSMA, PSA, MUC1, MUC16, NY-ESO-1, GD2, WT1, Mesothelin, MAGE-A3, GPC3, PRAME, Globo H, AFP, Trop2, FORL1, SP, Sca-1, CD133, or EPCAM.

[0045] According to embodiments of the present invention, both the first molecule and the second molecule are MICA. The bispecific antibody of the present invention can bind to both CD3 and MICA, and exhibits low CD3 binding activity and weak bridging ability between T cells and tumor cells. Therefore, this bispecific antibody has low T cell binding activity but high tumor binding activity, effectively promoting PBMC killing of tumor cells and promoting drug distribution to the tumor site rather than the periphery or lymphatic tissue. Consequently, this bispecific antibody has good safety and high anticancer activity.

[0046] According to embodiments of the present invention, the binding protein or a fragment thereof is selected from at least one of an antibody or its antigen-binding fragment, a receptor, and a ligand.

[0047] According to embodiments of the present invention, the binding protein of the first molecule or a fragment thereof is a first Fab fragment, and / or the binding protein of the second molecule or a fragment thereof is a second Fab fragment.

[0048] It should be noted that the amino acid sequences involved in this invention are shown from the N-terminus to the C-terminus.

[0049] According to an embodiment of the present invention, the C-terminus of the CH1 fragment of the first Fab fragment is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide; and / or the C-terminus of the CH1 fragment of the second Fab fragment is connected to the N-terminus of the second Fc peptide.

[0050] In some embodiments of the present invention, the binding protein of the first molecule or a fragment thereof is a first scFab fragment, and / or the binding protein of the second molecule or a fragment thereof is a second scFab fragment.

[0051] In some embodiments of the present invention, the C-terminus of the CH1 fragment of the first scFab fragment is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide; and / or The C-terminus of the CH1 fragment of the second scFab fragment is linked to the N-terminus of the second Fc peptide.

[0052] According to embodiments of the present invention, the scFv fragment of the bispecific antibody is conformally masked by a first Fab fragment or a first scFab fragment. Consequently, the CD3 binding activity of the bispecific antibody is reduced, its ability to bridge T cells and tumor cells is weakened, and its safety is good. Unexpectedly, the tumor cell binding activity of the bispecific antibody with this conformation is significantly improved.

[0053] According to embodiments of the present invention, the first Fab fragment and the second Fab fragment possess MICA binding activity. In the bispecific antibody according to embodiments of the present invention, the CD3 antigen-binding portion has little effect on the affinity of the MICA antigen-binding portion. Therefore, to obtain a bispecific antibody with good safety and high anticancer activity, it is preferable that the MICA antigen-binding portion is selected from the antigen-binding fragment of an anti-MICA antibody with strong MICA binding ability. For example, the MICA antigen-binding region is selected from the Fab antigen-binding fragment of the h5A1002 antibody.

[0054] In some embodiments of the present invention, the first Fab fragment and the second Fab fragment are not the same.

[0055] In some embodiments of the present invention, the first Fab fragment and the second Fab fragment are the same.

[0056] According to an embodiment of the present invention, both the first Fab fragment and the second Fab fragment include: a second heavy chain variable region CDR1~3 and a second light chain variable region CDR1~3; wherein, The second heavy chain variable region CDR1 has an amino acid sequence as shown in SEQ ID NO: 7 or its conserved modified form. The second heavy chain variable region CDR2 has an amino acid sequence as shown in SEQ ID NO: 8 or its conserved modified form. The second heavy chain variable region CDR3 has an amino acid sequence as shown in SEQ ID NO: 9 or its conserved modified form. The second light chain variable region CDR1 has an amino acid sequence as shown in SEQ ID NO: 10 or its conserved modified form. The second light chain variable region CDR2 has an amino acid sequence as shown in SEQ ID NO: 11 or its conserved modified form. The second light chain variable region CDR3 has an amino acid sequence as shown in SEQ ID NO: 12 or its conserved modified form.

[0057] According to embodiments of the present invention, the bispecific antibody of the present invention not only has low CD3 binding activity and weak ability to bridge T cells and tumor cells, but also has high tumor cell binding activity, and significantly enhances the activity of PBMC in killing tumor cells.

[0058] It should be noted that one or more amino acid residues in the heavy chain variable region CDR sequence or the light chain variable region CDR sequence of the present invention may be replaced by other amino acid residues from the same side chain family, and the retention function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conserved modifications does not exceed one or two.

[0059] According to an embodiment of the present invention, the first Fab fragment and / or the second Fab fragment includes a second heavy chain framework region and / or a second light chain framework region.

[0060] According to embodiments of the present invention, at least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies.

[0061] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment each independently comprise: a second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 29 or a conserved modified form thereof, and a second light chain variable region having an amino acid sequence as shown in SEQ ID NO: 30 or a conserved modified form thereof.

[0062] According to an embodiment of the present invention, the first Fab fragment and / or the second Fab fragment further comprises a CH1 fragment and a CL fragment; The C-terminus of the second heavy chain variable region is connected to the N-terminus of the CH1 segment, the C-terminus of the second light chain variable region is connected to the N-terminus of the CL segment, and the CH1 segment and the CL segment are connected by interchain disulfide bonds.

[0063] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment each independently comprise: The second heavy chain variable region and CH1 fragment having the amino acid sequence shown in SEQ ID NO: 31, and the second light chain variable region and CL fragment having the amino acid sequence shown in SEQ ID NO: 32.

[0064] According to an embodiment of the present invention, the scFv fragment comprises: a first heavy chain variable region CDR1~3 and a first light chain variable region CDR1~3; wherein, The first heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 1. The first heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2. The first heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3. The first light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 4. The first light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 5. The first light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 6.

[0065] According to embodiments of the present invention, when the scFv fragment of the bispecific antibody of the present invention has the above-mentioned CDRs amino acid sequence, it has significantly reduced CD3 binding activity and significantly enhanced tumor cell binding ability. Moreover, the bispecific antibody further enhances the activity of PBMC in killing tumor cells, thereby further improving the anticancer efficacy and safety.

[0066] According to an embodiment of the present invention, the scFv fragment includes a first heavy chain frame region and / or a first light chain frame region.

[0067] According to embodiments of the present invention, at least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from at least one of mouse antibodies, human antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof, preferably at least one of mouse antibodies, human antibodies, and primate antibodies.

[0068] It should be noted that the immunoglobulins discussed herein can be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules, including engineered subclasses with altered Fc moieties that provide reduced or enhanced effector cell activity. Immunoglobulins can be derived from any species.

[0069] According to an embodiment of the present invention, the scFv fragment includes: A first heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 33 or a conserved modified form thereof, and a first light chain variable region having an amino acid sequence as shown in SEQ ID NO: 34 or a conserved modified form thereof.

[0070] It should be noted that one or more amino acid residues in the heavy chain variable region or light chain variable region of the present invention may be replaced by other amino acid residues from the same side chain family, and the retention function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conserved modifications does not exceed one or two.

[0071] According to an embodiment of the present invention, the scFv fragment further comprises: linker peptide 1; Wherein, the N-terminus of the linker peptide 1 is connected to the C-terminus of the first heavy chain variable region, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the first light chain variable region; or The N-terminus of the linker peptide 1 is connected to the C-terminus of the first light chain variable region, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the first heavy chain variable region.

[0072] The linker peptide 1 has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0073] According to an embodiment of the present invention, the scFv fragment has an amino acid sequence as shown in SEQ ID NO: 35.

[0074] According to an embodiment of the present invention, the first antigen-binding region further includes a linker peptide 2; In this embodiment, the C-terminus of CH1 of the first Fab fragment is connected to the N-terminus of the linker peptide 2, and the C-terminus of the linker peptide 2 is connected to the N-terminus of the scFv fragment.

[0075] According to an embodiment of the present invention, the linker peptide 2 has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0076] According to an embodiment of the present invention, the linker peptide 2 has an amino acid sequence as shown in SEQ ID NO: 36 or 37.

[0077] According to an embodiment of the present invention, the first Fc peptide and the second Fc peptide are connected by a knock-into-hole structure.

[0078] According to an embodiment of the present invention, both the first Fc peptide and the second Fc peptide are human Fc peptides.

[0079] According to an embodiment of the present invention, the human Fc peptide is a human IgG1 Fc peptide.

[0080] According to an embodiment of the present invention, the first Fc peptide has an amino acid sequence as shown in SEQ ID NO: 38, and the second Fc peptide has an amino acid sequence as shown in SEQ ID NO: 39 or 40.

[0081] According to an embodiment of the present invention, the bispecific antibody comprises: A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 13, 18, 19, 22, 25 or 28, or an amino acid sequence having at least 80% identity with it; A second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 14, 20, 23 or 26 or an amino acid sequence having at least 80% identity with it; A third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, 21, 24 or 27 or an amino acid sequence having at least 80% identity with it; A fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, 21, 24 or 27 or having an amino acid sequence that is at least 80% identical to it.

[0082] It should be noted that, without substantially affecting the CD3 and MICA binding activity of the multispecific antibody (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the amino acid sequence of the polypeptide chain contained in the multispecific antibody of the present invention to obtain variants of the amino acid sequence of the polypeptide chain of the bispecific antibody. These are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties may be substituted in the polypeptide chain. The amino acid sequence of the above-mentioned variants of the present invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with a reference amino acid sequence.

[0083] According to an embodiment of the present invention, the bispecific antibody comprises: A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 13, a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 14, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 15, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO: 15; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 18, a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 14, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 15, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO: 15; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 19, a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 14, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 15, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO: 15; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 22, a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 20, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 21, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO: 21; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 25, a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 23, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 24, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO: 24; or A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 28, a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 26, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 27, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO: 27.

[0084] Nucleic acid This invention provides a nucleic acid. The nucleic acid encodes the aforementioned antibody or its antigen-binding fragment. The nucleic acid molecule encodes the aforementioned bispecific antibody. According to embodiments of the invention, the nucleic acid molecule can encode a bispecific antibody capable of simultaneously targeting CD3 and tumor targets.

[0085] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, if the amino acid sequence of the molecules in this invention includes either DNA or RNA form, disclosing one implies that the other is also disclosed.

[0086] Vector or transformant This invention provides a vector or transformant containing the aforementioned nucleic acid. The vector or transformant according to embodiments of the invention may include optional control sequences, such as those operably linked to the nucleic acid molecule. The control sequences are one or more control sequences that direct the expression of the nucleic acid molecule in a host. The resulting vector or transformant can effectively express the aforementioned bispecific antibody.

[0087] When linking the aforementioned nucleic acid molecules to the vector or transformant, such as an expression vector, the nucleic acid molecules can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. Of course, these control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. The nucleic acid molecules and control elements simply need to be operatively linked.

[0088] According to embodiments of the present invention, the vector may refer to a cloning vector or an expression vector, and can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this invention is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0089] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector such that the control elements within the vector, such as transcriptional control amino acid sequences and translational control amino acid sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the proteins encoded by the nucleic acid molecules.

[0090] According to an embodiment of the present invention, the carrier is a eukaryotic carrier or a prokaryotic carrier.

[0091] According to embodiments of the present invention, the vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0092] cell This invention provides a cell. The cell carries the aforementioned nucleic acid, or the aforementioned vector or transformant, or expresses the aforementioned bispecific antibody. The cell according to embodiments of the invention is obtained by transfecting or transforming the aforementioned vector or transformant, and the cell can efficiently express the aforementioned bispecific antibody under suitable conditions.

[0093] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell, or a bacteriophage.

[0094] According to an embodiment of the present invention, the prokaryotic cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0095] According to an embodiment of the present invention, the eukaryotic cell is a fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0096] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomyces cerevisiae, or Trichoderma.

[0097] According to an embodiment of the present invention, the insect cell is a grass armyworm cell; according to an embodiment of the present invention, the plant cell is a tobacco plant cell; according to an embodiment of the present invention, the mammalian cell is a BHK cell, a CHO cell, a COS cell, a myeloma cell, or a human embryonic kidney 293 cell; and does not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0098] According to an embodiment of the present invention, the cell is a mammalian cell.

[0099] According to an embodiment of the present invention, the cells are BHK cells, CHO cells, COS cells, or NSO cells.

[0100] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibody or its antigen-binding fragment described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the antibody or its antigen-binding fragment 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 recombinant antibody based on the specific environment of their laboratory.

[0101] Pharmaceutical Composition This invention provides a pharmaceutical composition. The pharmaceutical composition comprises: the aforementioned bispecific antibody, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned cells. The bispecific antibody according to embodiments of the present invention can effectively promote the killing of tumor cells by PBMCs, exhibiting high anti-cancer activity; moreover, it has low CD3 binding activity and good safety. The resulting drug can be further used for the prevention and / or treatment of CD3 and / or tumor target-mediated diseases.

[0102] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0103] According to embodiments of the present invention, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0104] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0105] It should be noted that the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0106] The medicaments of this invention contain a safe and effective amount of the active ingredient of this invention and pharmaceutically acceptable excipients. Such excipients 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 forms of the medicaments of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they are prepared using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The medicaments are preferably manufactured under aseptic conditions.

[0107] The effective amount of the active ingredient described in this invention can 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.

[0108] The pharmaceutically acceptable excipients described in this invention 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.

[0109] Reagent test kit This invention provides a kit. The kit comprises the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, or the aforementioned cells. The kit according to embodiments of this invention can bind to CD3 protein and / or tumor targets, and can effectively identify CD3 protein and / or tumor targets.

[0110] Those skilled in the art will understand that the features and advantages described above for antibodies, nucleic acids, vectors or transformants, cells or pharmaceutical compositions also apply to this kit and will not be repeated here.

[0111] In some embodiments of the present invention, the tumor target is MICA. As previously described, the bispecific antibody according to embodiments of the present invention can specifically bind to CD3 and MICA. A CD3 protein and / or MICA protein-related kit developed using this property can be used for CD3 protein and / or MICA protein-related research, such as for detecting and / or enriching and / or isolating and purifying human or other mammalian CD3 protein and / or MICA protein. This kit can effectively detect, enrich, or isolate and purify CD3 protein and / or MICA protein in biological samples, and further be used for scientific research, such as qualitative or quantitative detection of CD3 protein and / or MICA protein molecules in biological samples. More specifically, it can be used in immunoblotting, immunoprecipitation, and other kits involving the detection of CD3 protein and / or MICA protein by utilizing the specific binding properties of antibodies. These kits may contain any one or more of the following: antagonist, the bispecific antibody of the present invention, or a pharmaceutical reference material; a protein purification column; an immunoglobulin affinity purification buffer; and a cell assay diluent. The bispecific antibody of the present invention can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, CD3 and / or MICA-mediated diseases can be tested by examining the subject's serum or blood.

[0112] Uses in the preparation kit The present invention provides the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant or the aforementioned cells in the preparation of a kit for detecting CD3 and / or tumor targets.

[0113] In some embodiments of the present invention, the tumor target is MICA.

[0114] Those skilled in the art will understand that the features and advantages described above for antibodies, nucleic acids, vectors or transformants, cells or pharmaceutical compositions are equally applicable to the use in this preparation kit, and will not be repeated here.

[0115] Uses in drug preparation The present invention provides the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, the aforementioned cell or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of CD3 and / or tumor target-mediated diseases.

[0116] Those skilled in the art will understand that the features and advantages described above for antibodies, nucleic acid molecules, vectors or transformants, cells and pharmaceutical compositions also apply to this use, and will not be repeated here.

[0117] According to embodiments of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0118] According to embodiments of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0119] According to an embodiment of the present invention, the tumor target is MICA. The bispecific antibody and nucleic acid, carrier, transformant, or pharmaceutical composition according to embodiments of the present invention can be further prepared into a drug that can be used clinically for the prevention or treatment of CD3 and / or MICA-mediated diseases.

[0120] According to embodiments of the present invention, the MICA-mediated related diseases are cancer, transplant rejection, autoimmune diseases, and infectious diseases.

[0121] According to embodiments of the present invention, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0122] Disease treatment methods This invention provides a method for preventing and / or treating CD3 and / or MICA-mediated diseases. According to embodiments of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, the aforementioned cells, or the aforementioned pharmaceutical composition.

[0123] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0124] The effective amount of the antibody or its antigen-binding fragment, nucleic acid, carrier, transformant, or pharmaceutical composition described in this invention can 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.

[0125] In some specific implementations, the CD3-mediated diseases include autoimmune diseases.

[0126] In some specific implementations, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, and acute idiopathic polyneuritis.

[0127] In some specific implementations, the MICA-mediated diseases are cancer, transplant rejection, autoimmune diseases, and infectious diseases.

[0128] In some specific implementations, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0129] The amino acid sequences involved in this invention are detailed in Table 1.

[0130] Table 1. Amino Acid Sequence Description

[0131] The present invention will be described in detail below through examples. In the examples or test cases, experimental methods without specific conditions are performed under conventional conditions.

[0132] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0133] Example 1: Antibody Production The specific experimental procedures for antibody production are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10⁻⁶. 6 / mL, to obtain ExpiCHO cell solution. (2) When the bispecific antibody is in configuration A ( Figure 1 ), add the pcDNA3.4 vector (synthesized by Nanjing GenScript) containing CD3 antibody, MICA antibody heavy chain, and MICA antibody light chain to 2 mL of OptiSFM medium (purchased from Thermo Fisher) in a 1:1:1 ratio to obtain solution a; or, when the bispecific antibody is in configuration B ( Figure 1 (2) Add pcDNA3.4 vector (synthesized by Nanjing GenScript) containing MICA heavy chain-CD3 antibody, MICA antibody heavy chain, and MICA antibody light chain to 2 mL OptiSFM medium (purchased from Thermo Fisher) in a 1:1:1 ratio to obtain solution a. (3) Add 160 μL ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) to 2 mL OptiSFM medium (purchased from Thermo Fisher) to obtain solution b. (4) Then mix solution a and solution b to obtain transfection mixture, and add the entire transfection mixture to 50 mL ExpiCHO cell solution within 5 minutes. (5) After culturing at 37℃ and 5% CO2 for 1 day, add 8 mL Feed and 300 μL Enhancer (purchased from Thermo Fisher), and culture at 32℃ and 5% CO2 for 9 days. Harvest the culture supernatant, adding 8 mL Feed on day 5. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from Nanomicro).

[0134] In this embodiment, a total of 11 CD3 and MICA bispecific antibodies were prepared to investigate the various properties of the bispecific antibodies of the present invention. See [link to documentation for antibody conformation and amino acid sequence descriptions]. Figure 1 Tables 1 and 2.

[0135] Table 2. Structure and corresponding amino acid sequence of the bispecific antibody from Example 1

[0136] Note: For the configurations of antibodies A and B, please refer to [link / reference]. Figure 1 .

[0137] Example 2: Antibody ELISA Binding Assay of the Present Invention ELISA assays are used to detect the binding properties of bispecific antibodies. The antigen protein is coated into a 96-well plate, and the intensity of the signal after antibody addition is used to determine the binding properties between the bispecific antibody and the antigen protein.

[0138] (1) Dilute MICA-His protein (purchased from Acro) with PBS buffer to 2 μg / ml, add 100 μl / well to a 96-well plate, and incubate overnight at 4°C. Remove the PBS buffer from the 96-well plate, wash the plate 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, add 200 μl / well of PBS / 10% BSA, and incubate at 37°C for 2 h to block. After removing the blocking buffer and washing the plate 6 times with PBST, add 100 μl / well of the following bispecific antibodies to be tested: Cross3×h5A1002 1:1 (amino acid sequence of CD3 single-chain antibody as shown in SEQ ID NO: 16, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 13, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), and UCHT1×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 15). These solutions are serially diluted with PBST / 0.05% BSA (maximum concentration 20 μg / ml, 8 gradients, 5-fold dilution). As shown in Figure 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15. The control hIgG1LALA (purchased from Baiying Biotechnology) was incubated at 37°C for 1 h. The reaction system was removed, and the plate was washed 6 times with PBST. 100 μl / well of HRP (horseradish peroxidase)-labeled anti-human IgG antibody secondary antibody (purchased from Jacksonlab) was diluted with PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing 6 times with PBST, 80 μl / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 min. The reaction was terminated by adding 80 μl / well of 4M sulfuric acid. The absorbance was read at 450 nm using a microplate reader.

[0139] The results are as follows Figure 2 As shown, the antibodies Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 can all bind to the MICA protein, and their binding abilities are comparable.

[0140] (2) Dilute CD3E&D protein (purchased from Acro) to 2 μg / ml with PBS buffer, add 100 μl / well to a 96-well plate, and incubate overnight at 4°C. Remove the PBS buffer from the 96-well plate, wash the plate 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, add 200 μl / well of PBS / 10% BSA, and incubate at 37°C for 2 h to block. After removing the blocking buffer and washing the plate 6 times with PBST, add 100 μl / well of the following bispecific antibodies to be tested: Cross3×h5A1002 1:1 (amino acid sequence of CD3 single-chain antibody as shown in SEQ ID NO: 16, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 13, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), and UCHT1×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 15). These solutions are serially diluted with PBST / 0.05% BSA (maximum concentration 20 μg / ml, 8 gradients, 5-fold dilution). As shown in Figure 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15. The control hIgG1LALA (purchased from Baiying Biotechnology) was incubated at 37°C for 1 h. The reaction system was removed, and the plate was washed 6 times with PBST. 100 μl / well of HRP (horseradish peroxidase)-labeled anti-human IgG antibody secondary antibody (purchased from Southern Biotech) was diluted with PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing 6 times with PBST, 80 μl / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 min. The reaction was terminated by adding 80 μl / well of 4M sulfuric acid. The absorbance was read at 450 nm using a microplate reader.

[0141] The results are as follows Figure 3 As shown, the antibodies Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 can all bind to CD3E&D proteins; the ability of Cross3×h5A1002 2:1 to bind to CD3E&D is weaker than that of Cross3×h5A1002 1:1; and the Cross3×h5A1002 2:1 antibody has the weakest activity in binding to CD3.

[0142] The above experimental results indicate that after adjusting the structure of the bispecific antibody ( Figure 1 (Configuration B) The antigen-binding fragment of the anti-CD3 antibody is successfully masked in configuration, thus weakening its ability to bind CD3.

[0143] Example 3: Antibody ELISA Bridging Assay ELISA assays are used to detect the bridging binding properties of bispecific antibodies. CD3E&D antigen proteins are coated into 96-well plates. After antibody addition, biotin-labeled MICA protein is used for detection. The signal strength is used to determine the binding properties of the bispecific antibody to CD3E&D and MICA proteins.

[0144] Dilute MICA-His protein (purchased from Acro) to 2 μg / ml with PBS buffer, add 100 μl / well to each well of a 96-well plate, and incubate overnight at 4°C. Remove the PBS buffer from the 96-well plate, wash 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer, add 200 μl / well of PBS / 10% BSA, and incubate at 37°C for 2 hours to block. After removing the blocking buffer and washing the plate 6 times with PBST, add 100 μl / well of the following bispecific antibodies to be tested: Cross3×h5A1002 1:1 (amino acid sequence of CD3 single-chain antibody as shown in SEQ ID NO: 16, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 13, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), and UCHT1×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 15). These solutions are serially diluted with PBST / 0.05% BSA (maximum concentration 20 μg / ml, 8 gradients, 5-fold dilution). As shown in Figure 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15. The control hIgG1LALA (purchased from Baiying Biotechnology) was incubated at 37°C for 1 hour. The reaction system was removed, and the plate was washed 6 times with PBST. Then, MICA diluted to an appropriate concentration was added. 002α3-Fc-Biotin was added and incubated at 37°C for 1 h. The reaction mixture was then removed, and the plate was washed 6 times with PBST. HRP (horseradish peroxidase)-labeled Streptavidin secondary antibody (purchased from Southern Biotech) was diluted 100 μl / well with PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing 6 times with PBST, 80 μl / well of TMB (tetramethylbenzidine) was added, and the plate was incubated at room temperature for 3 min. The reaction was terminated by adding 80 μl / well of 4M sulfuric acid. The absorbance was read at 450 nm using a microplate reader.

[0145] The results are as follows Figure 4 As shown, the Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies can bridge CD3E&D and MICA proteins; the bridging ability of the Cross3×h5A1002 2:1 antibody is weaker than that of the Cross3×h5A1002 1:1 antibody; and the Cross3×h5A1002 2:1 antibody has the weakest bridging ability.

[0146] according to Figure 2 , 3 The results analysis showed that the weak bridging ability of the Cross3×h5A1002 2:1 antibody was due to the low affinity between the Cross3×h5A1002 2:1 antibody and CD3.

[0147] Example 4: Antibody Flow Cytometry Combined with Experiment Flow cytometry is used to detect the binding properties of bispecific antibodies. Bispecific antibodies are added to cells, and the strength of the signal after antibody addition is used to determine the binding properties between the antibody and the cell.

[0148] (1) Dilute PBMC with PBS to 2×10 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted bispecific antibodies Cross3×h5A1002 1:1 (amino acid sequence of CD3 single-chain antibody as shown in SEQ ID NO: 16, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 13, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), UCHT1×h5A1002 1:1 (amino acid sequence of CD3 single-chain antibody as shown in SEQ ID NO: 17, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), UCHT1×h5A1002 A 2:1 ratio was used (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), and the control hIgG1LALA (purchased from Baiying Biotechnology) was incubated at 4℃ for 30 min. 1 ml of PBS was added to the EP tube, and the cells were centrifuged at 3500 rpm for 5 min at 4℃. The supernatant was discarded, and the cells were washed once more with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100 μl / tube of PBS. 1 μl / tube of Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson Lab) and 0.5 μl / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody were added, and the cells were incubated at 4℃ 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 / tube of PBS and analyzed by flow cytometry.

[0149] The results are as follows Figure 5 As shown, Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies can bind to CD8 T cells. The 2:1 configuration (configuration B) bispecific antibody has a weaker T cell binding ability than the 1:1 configuration (configuration A) bispecific antibody. Among them, the Cross3×h5A1002 2:1 bispecific antibody has the weakest T cell binding activity.

[0150] (2) Dilute HCT-15 tumor cells with PBS to 2×10⁻⁶. 6 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4℃ for 30min. Add serially diluted bispecific antibodies Cross3×h5A1002 1:1 (amino acid sequence of CD3 single-chain antibody as shown in SEQ ID NO: 16, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 13, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), UCHT1×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 18, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15). As shown in Figure 15), the control hIgG1LALA (purchased from Baiying Biotechnology) was incubated at 4℃ for 30 min. 1 ml of PBS was added to the EP tube, and the cells were centrifuged at 3500 rpm for 5 min at 4℃. The supernatant was discarded, and the cells were washed once more with PBS. After centrifugation, the supernatant was discarded again, and the cells were resuspended in 100 μl / tube of PBS. 1 μl / tube of Alexa-647-labeled goat anti-human IgG antibody secondary antibody (purchased from Jackson Lab) was added, and the cells were incubated at 4℃ 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 / tube of PBS and analyzed by flow cytometry.

[0151] The results are as follows Figure 6 As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies can bind to tumor cells. (2) To the inventors' surprise, the 2:1 configuration (configuration B) bispecific antibody binds to the EC of HCT-15 tumor cells. 50 Bispecific antibodies with a ratio lower than 1:1 (configuration A) have a stronger ability to bind to tumor cells. Among them, Cross3×h5A1002 2:1 and UCHT1×h5A1002 2:1 antibodies have comparable activity in binding to tumors.

[0152] Example 5: Experiment on bispecific antibody promoting PBMC killing of tumor cells To detect the ability of bispecific antibodies to promote the killing of HCT-15 colorectal cancer cells, NCI-H1299 lung cancer cells, PANC-1 pancreatic cancer cells, A-498 renal cancer cells, and PC-3 prostate cancer cells by PBMCs.

[0153] (a) Add 50 μL of complete RPMI-1640 medium to a 16-well RTCA plate and calibrate it. (b) Dilute tumor cells to 2 × 10⁻⁶ using complete RPMI-1640 medium. 5 / mL, at a volume of 50μL / well, were individually added to the RTCA plate obtained in step (1), and then the cell coefficient was detected for 24h at 37℃ and 5% CO2 using the xCELLigenceRTCA MP instrument; (c) Using complete RPMI-1640 medium, serially diluted bispecific antibodies Cross3×h5A1002 1:1 (amino acid sequence of CD3 single-chain antibody as shown in SEQ ID NO: 16, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (amino acid sequence of MICA antibody heavy chain-CD3 single-chain antibody as shown in SEQ ID NO: 13, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15), UCHT1×h5A1002 1:1 (amino acid sequence of CD3 single-chain antibody as shown in SEQ ID NO: 17, amino acid sequence of MICA antibody heavy chain as shown in SEQ ID NO: 14, and amino acid sequence of MICA antibody light chain as shown in SEQ ID NO: 15) were prepared. 15), UCHT1×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), control hIgG1LALA (purchased from Baiying Biotechnology) (the highest concentration is 0.2μg / ml, 8 gradients, 5-fold dilution), added to the RTCA plate obtained in step (2), with an addition volume of 20μl / well; (4) Dilute PBMC (purchased from Selene Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI-1640 medium. 6 Add 80 μl / well to the RTCA plate obtained in step (3); (5) The reaction system obtained in step (4) was subjected to 37°C and 5% CO2 for 24 hours, and the cell coefficient was detected using an xCELLigence RTCA MP instrument.

[0154] The results are as follows Figure 7 As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promote the killing of HCT-15 colorectal cancer cells by PBMCs. (2) To the inventors' surprise, the 2:1 configuration (configuration B) bispecific antibody has a stronger killing ability than the 1:1 configuration (configuration A) bispecific antibody, and Cross3×h5A1002 2:1 has the highest killing activity.

[0155] The results are as follows Figure 8 As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promote the killing of NCI-H1299 lung cancer cells by PBMCs. (2) To the inventors' surprise, the 2:1 configuration (configuration B) bispecific antibody has a stronger killing ability than the 1:1 configuration (configuration A) bispecific antibody, and the Cross3×h5A1002 2:1 bispecific antibody has the highest killing activity.

[0156] The results are as follows Figure 9 As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promote the killing of PANC-1 pancreatic cancer cells by PBMCs. (2) To the inventors' surprise, the 2:1 configuration (configuration B) bispecific antibody has a stronger killing ability than the 1:1 configuration (configuration A) bispecific antibody, and Cross3×h5A1002 2:1 has the highest killing activity.

[0157] The results are as follows Figure 10 As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promote the killing of A498 renal cells by PBMCs. The 2:1 configuration (configuration B) bispecific antibody has a stronger killing ability than the 1:1 configuration (configuration A) bispecific antibody, and Cross3×h5A1002 2:1 has the highest killing activity.

[0158] The results are as follows Figure 11As shown. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promote the killing of PC-3 prostate cancer cells by PBMCs. (2) To the inventors' surprise, the 2:1 configuration (configuration B) bispecific antibody has a stronger killing ability than the 1:1 configuration (configuration A) bispecific antibody, and Cross3×h5A1002 2:1 has the highest killing activity.

[0159] The above results show that the 2:1 configuration (configuration B) bispecific antibody of the present invention significantly enhances the ability of PBMCs to kill HCT-15 rectal cancer cells, NCI-H1299 lung cancer cells, PANC-1 pancreatic cancer cells, A498 renal cancer cells, and PC-3 prostate cancer cells. Specifically, when the CD3 antigen-binding region is the antigen-binding fragment of the Cross3 antibody, the 2:1 configuration (configuration B) bispecific antibody of the present invention exhibits the strongest activity in promoting PBMC tumor cell killing.

[0160] Example 6: Experiment on bispecific antibody-induced PBMC cytokine secretion Bispecific antibodies were added to a co-incubation system of PBMCs and tumor cells. After 48 hours of culture, the culture supernatant was collected, and the cytokine content in the supernatant was detected to determine the characteristics of cytokine release induced by the bispecific antibodies.

[0161] (1) The tumor was diluted to 1×10⁻⁶ using complete RPMI 1640 medium. 5 Add / ml to a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 24 hours; (2) Using complete RPMI 1640 medium, the bispecific antibody Cross3×h5A1002 1:1 (the amino acid sequence of the CD3 single chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), and control hIgG1LALA (purchased from Baiying Biotechnology) were serially diluted and added to 96-well plates, 20 μl / well; (3) PBMC (purchased from Selene Biotechnology) was diluted to 1.25 × 10⁻⁶ using complete RPMI 1640 medium. 6 Add 80 μl / well to a 96-well plate; (4) Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 48 hours; (5) Centrifuge at 300g for 10 min at room temperature and collect the cell culture supernatant; (6) The cytokine content in the supernatant was detected using the CBA kit (purchased from BD).

[0162] The results are as follows Figure 12 As shown. (1) Cross3×h5A1002 1:1 and Cross3×h5A1002 2:1 antibodies promote the secretion of immune-activating cytokines IL-2 and IFN-γ by PBMCs. (2) Cross3×h5A1002 2:1 antibody has a stronger ability to promote the secretion of immune-activating cytokines by PBMCs.

[0163] The above results show that the 2:1 configuration (configuration B) bispecific antibody of the present invention has an enhanced ability to promote the secretion of immune-activating cytokines by PBMCs. Considering that the 2:1 configuration (configuration B) bispecific antibody binds weakly to T cells and strongly to tumor cells, more drug can be distributed locally to the tumor after administration, thus enhancing the anti-cancer efficacy.

[0164] Example 7: Experiments on the effector-target ratio of bispecific antibodies promoting tumor cell killing by PBMCs The bispecific antibody of the present invention was tested under different effector-to-target ratios (PBMC: tumor) to demonstrate its ability to promote the killing of HCT-15 colorectal cancer cells by PBMC.

[0165] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate. (2) Dilute tumor cells to 2×10⁻⁶ using complete RPMI-1640 medium. 5 / mL, at a volume of 50μL / well, were individually added to the RTCA plate obtained in step (1), and then the cell coefficient was detected for 24h at 37℃ and 5% CO2 using the xCELLigenceRTCA MP instrument; (3) Using complete RPMI-1640 medium, serially diluted bispecific antibodies Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), UCHT1×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), SP34×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 19, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15), SP34×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 19, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15). NO: 15), control hIgG1LALA (purchased from Baiying Biotechnology) (highest concentration 0.2 μg / ml, 8 gradients, 5-fold dilution), added to the RTCA plate obtained in step (2), with an addition volume of 20 μl / well; (4) Dilute PBMC (purchased from Selene Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI-1640 medium. 6 0.625 × 10⁻⁶ cells / ml (target-effect ratio 10:1) 6 0.3125×10⁻⁶ cells / ml (target-effect ratio 5:1) 6 Add 80 μl / well to the RTCA plate obtained in step (3) (target-effect ratio 2.5:1); (5) The reaction system obtained in step (4) was subjected to 37°C and 5% CO2 for 24 hours, and the cell coefficient was detected using an xCELLigence RTCA MP instrument.

[0166] The results are as follows Figure 13 As shown, under different effector-target ratios, the antibodies Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1, and SP34×h5A1002 2:1 all promoted the killing of HCT-15 colorectal cancer cells by PBMCs, and Cross3×h5A1002 2:1 showed the highest killing activity.

[0167] Example 8: Experiments on how other MICA bispecific antibodies promote PBMC killing of tumor cells The ability of the bispecific antibody of the present invention in its 2:1 configuration (configuration B), formed based on other MICA antibodies, to promote the killing of HCT-15 colorectal cancer cells by PBMCs was examined to evaluate whether the ability of the 2:1 configuration (configuration B) bispecific antibody of the present invention to promote the killing of tumor cells by PBMCs is generally superior to that of the 1:1 configuration (configuration A). Other MICA antibodies were selected from: 1D5 (Genentech monoclonal antibody, US20200055939A1), 13A9 (Genentech monoclonal antibody, US20200055939A1), and 619 (Cullinan monoclonal antibody, US20210253711A1).

[0168] 1.2:1 configuration (configuration B) antibody expression: Same as in Example 1.

[0169] 2. Investigation on the ability of PBMCs to kill tumor cells: Refer to Example 7 and proceed as follows.

[0170] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate. (2) Dilute tumor cells to 2×10⁻⁶ using complete RPMI-1640 medium. 5 / mL, at a volume of 50μL / well, were individually added to the RTCA plate obtained in step (1), and then the cell coefficient was detected for 24h at 37℃ and 5% CO2 using the xCELLigenceRTCA MP instrument; (3) Using complete RPMI-1640 medium, serially diluted bispecific antibodies Cross3×h5A1002 2:1 (amino acid sequences of MICA antibody heavy chain-CD3 single chain antibody as shown in SEQ ID NO: 13, MICA antibody heavy chain amino acid sequence as shown in SEQ ID NO: 14, and MICA antibody light chain amino acid sequence as shown in SEQ ID NO: 15), Cross3×1D5 1:1 (amino acid sequences of CD3 single chain antibody as shown in SEQ ID NO: 16, MICA antibody heavy chain amino acid sequence as shown in SEQ ID NO: 20, and MICA antibody light chain amino acid sequence as shown in SEQ ID NO: 21), Cross3×1D5 2:1 (amino acid sequences of MICA antibody heavy chain-CD3 single chain antibody as shown in SEQ ID NO: 2 ... The following are examples of antibody sequences: Cross3×13A9 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 24), Cross3×13A9 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 25, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 24), Cross3×13A9 1:1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 26, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 27), and Cross3×619 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 27). As shown in SEQ ID NO:26, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:27, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:27. The control hIgG1LALA (purchased from Baiying Biotechnology) (highest concentration of 0.2 μg / ml, 8 gradients, 5-fold dilution) was added to the RTCA plate obtained in step (2) with an addition volume of 20 μl / well. (4) Dilute PBMC (purchased from Selene Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI-1640 medium. 6Add 80 μl / well to the RTCA plate obtained in step (3); (5) The reaction system obtained in step (4) was subjected to 37°C and 5% CO2 for 24 hours, and the cell coefficient was detected using an xCELLigence RTCA MP instrument.

[0171] The results are as follows Figure 14 As shown. (1) The 2:1 configuration of the 1D5-based bispecific antibody has a weaker killing effect than the 1:1 bispecific antibody. (2) Furthermore, the Cross3×h5A1002 2:1 bispecific antibody has a higher killing effect than the 1D5-based bispecific antibody.

[0172] The results are as follows Figure 15 As shown. (1) The 2:1 configuration of the 13A9-based bispecific antibody has a weaker killing effect than the 1:1 bispecific antibody. (2) Furthermore, the Cross3×h5A1002 2:1 bispecific antibody has a higher killing effect than the 13A9-based bispecific antibody.

[0173] The results are as follows Figure 16 As shown. (1) The 2:1 configuration of the 619-based bispecific antibody has a weaker killing effect than the 1:1 bispecific antibody. (2) Furthermore, the Cross3×h5A1002 2:1 bispecific antibody has a higher killing effect than the 619-based bispecific antibody.

[0174] The results show that, only when the MICA antibody is selected from the h5A1002 antibody, the 2:1 configuration (configuration B) bispecific antibody of the present invention exhibits an enhanced ability to promote PBMC killing of tumor cells.

[0175] Example 9: Effects of CD3×MICA antibody on cancer resistance in mice In vivo efficacy experiments were used to detect the anticancer function of the Cross3×h5A1002 2:1 bispecific antibody of the present invention in promoting immune reconstitution in mice.

[0176] (1) On day -14, human PBMCs (purchased from Selvi Biotech) were transfused into NSG mice (purchased from Southern Model) via the tail vein at a dose of 5 × 10⁻⁶. 6 / Only; (2) On day 10, NSG mice were implanted with tumors subcutaneously on the right ventral side, and each mouse was injected with 2×10⁻⁶ tumors. 6 One A-375 human melanoma cells, 2 × 10 6 1 NCI-H1299 human lung cancer cells, or 5 × 10 6 PC3 individuals with prostate cancer cells; (3) On day 0, the mice were weighed and the tumor volume was measured. Based on the weighing results and tumor volume, the mice were divided into 6 groups. (4) On days 0, 4, 7, and 11, mice were injected via tail vein with the Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15) (0.1 mg / kg) and the solvent control PBS, 250 μl per mouse; (4) After the above-mentioned antibodies were injected, the tumor volume was measured twice a week and the mice were weighed.

[0177] The results are as follows Figure 17 , 19 As shown in Figure 20, the Cross3×h5A1002 2:1 antibody of the present invention can effectively promote immune reconstitution in mice and is used to treat tumor-bearing mice, resulting in complete tumor regression.

[0178] The above experimental results show that the bispecific antibody of this invention can bind to CD3 and MICA, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMC killing of tumor cells, and exhibiting good anti-cancer activity. The bispecific antibody of this invention can achieve high affinity binding to MICA and low affinity binding to CD3, resulting in higher anti-cancer activity and better safety. In summary, the bispecific antibody of this invention can promote immune cell anti-cancer activity, exhibiting good anti-cancer activity and higher safety, and has high clinical application and drug development value.

[0179] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

[0180] 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.

[0181] 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, It includes: a first antigen-binding region, a second antigen-binding region, and an Fc portion; The Fc portion includes a first Fc peptide segment and a second Fc peptide segment. The first antigen-binding region comprises an scFv fragment and a binding protein or a fragment thereof of a first molecule, wherein the binding protein or a fragment thereof of the first molecule is linked to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is linked to the N-terminus of the first Fc peptide. The second antigen-binding region contains a second molecule of binding protein or a fragment thereof, and the second molecule of binding protein or a fragment thereof is linked to the N-terminus of the second Fc peptide. The first molecule and the second molecule are identical, and the scFv fragment has CD3 binding activity; Both the first molecule and the second molecule are MICA; The binding protein or a fragment thereof is selected from antibodies or antigen-binding fragments thereof; The binding protein of the first molecule or a fragment thereof is a first Fab fragment, and / or the binding protein of the second molecule or a fragment thereof is a second Fab fragment; The C-terminus of the CH1 fragment of the first Fab fragment is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide; and / or The C-terminus of the CH1 fragment of the second Fab fragment is connected to the N-terminus of the second Fc peptide; The first Fab fragment and the second Fab fragment have MICA binding activity; Both the first Fab fragment and the second Fab fragment are composed of the second heavy chain variable regions CDR1~3 and the second light chain variable regions CDR1~3; wherein, The amino acid sequence of the second heavy chain variable region CDR1 is shown in SEQ ID NO:

7. The amino acid sequence of the second heavy chain variable region CDR2 is shown in SEQ ID NO:

8. The amino acid sequence of the second heavy chain variable region CDR3 is shown in SEQ ID NO:

9. The amino acid sequence of the second light chain variable region CDR1 is shown in SEQ ID NO:

10. The amino acid sequence of the second light chain variable region CDR2 is shown in SEQ ID NO:

11. The amino acid sequence of the second light chain variable region CDR3 is shown in SEQ ID NO:

12. The scFv fragment comprises: a first heavy chain variable region CDR1~3 and a first light chain variable region CDR1~3; wherein... The amino acid sequence of the first heavy chain variable region CDR1 is shown in SEQ ID NO:

1. The amino acid sequence of the first heavy chain variable region CDR2 is shown in SEQ ID NO:

2. The amino acid sequence of the first heavy chain variable region CDR3 is shown in SEQ ID NO:

3. The amino acid sequence of the first light chain variable region CDR1 is shown in SEQ ID NO:

4. The amino acid sequence of the first light chain variable region CDR2 is the same as the amino acid sequence shown in GTN. The amino acid sequence of the first light chain variable region CDR3 is shown in SEQ ID NO:

6.

2. The bispecific antibody according to claim 1, characterized in that, The first Fab fragment and / or the second Fab fragment contain a second heavy chain framework region and / or a second light chain framework region.

3. The bispecific antibody according to claim 2, characterized in that, At least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from at least one of the following: mouse antibody, primate antibody, bovine antibody, equine antibody, porcine antibody, sheep antibody, goat antibody, canine antibody, feline antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, and turkey antibody.

4. The bispecific antibody according to claim 2, characterized in that, At least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from at least one of mouse-derived antibodies and primate-derived antibodies.

5. The bispecific antibody according to claim 1, characterized in that, The first Fab fragment and the second Fab fragment each independently comprise: a second heavy chain variable region as shown in SEQ ID NO: 29, and a second light chain variable region as shown in SEQ ID NO:

30.

6. The bispecific antibody according to claim 1, characterized in that, The first Fab fragment and / or the second Fab fragment further comprise a CH1 fragment and a CL fragment; The C-terminus of the second heavy chain variable region is connected to the N-terminus of the CH1 segment, the C-terminus of the second light chain variable region is connected to the N-terminus of the CL segment, and the CH1 segment and the CL segment are connected by interchain disulfide bonds.

7. The bispecific antibody according to claim 1, characterized in that, The first Fab fragment and the second Fab fragment each independently contain: The second heavy chain variable region and CH1 fragment with amino acid sequences as shown in SEQ ID NO: 31, and the second light chain variable region and CL fragment with amino acid sequences as shown in SEQ ID NO:

32.

8. The bispecific antibody according to claim 1, characterized in that, The scFv fragment includes a first heavy chain framework region and / or a first light chain framework region.

9. The bispecific antibody according to claim 8, characterized in that, At least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from at least one of the following: mouse antibody, primate antibody, bovine antibody, equine antibody, porcine antibody, sheep antibody, goat antibody, canine antibody, feline antibody, rabbit antibody, camel antibody, donkey antibody, deer antibody, mink antibody, chicken antibody, duck antibody, goose antibody, and turkey antibody.

10. The bispecific antibody according to claim 8, characterized in that, At least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from at least one of mouse-derived antibodies and primate-derived antibodies.

11. The bispecific antibody according to claim 1, characterized in that, The scFv fragment contains: The first heavy chain variable region as shown in SEQ ID NO: 33, and the first light chain variable region as shown in SEQ ID NO:

34.

12. The bispecific antibody according to claim 1, characterized in that, The scFv fragment further comprises: linker peptide 1; Wherein, the N-terminus of the linker peptide 1 is connected to the C-terminus of the first heavy chain variable region, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the first light chain variable region; or The N-terminus of the linker peptide 1 is connected to the C-terminus of the first light chain variable region, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the first heavy chain variable region.

13. The bispecific antibody according to claim 12, characterized in that, The amino acid sequence of the linker peptide 1 is shown as (GGGGS)n, where n is an integer greater than or equal to 1.

14. The bispecific antibody according to claim 12, characterized in that, The amino acid sequence of the linker peptide 1 is shown as (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

15. The bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the scFv fragment is shown in SEQ ID NO:

35.

16. The bispecific antibody according to claim 1, characterized in that, The first antigen-binding region further includes linker peptide 2; In this embodiment, the C-terminus of CH1 of the first Fab fragment is connected to the N-terminus of the linker peptide 2, and the C-terminus of the linker peptide 2 is connected to the N-terminus of the scFv fragment.

17. The bispecific antibody according to claim 16, characterized in that, The amino acid sequence of the linker peptide 2 is shown as (GGGGS)n, where n is an integer greater than or equal to 1.

18. The bispecific antibody according to claim 16, characterized in that, The amino acid sequence of the linker peptide 2 is shown as (GGGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

19. The bispecific antibody according to claim 16, characterized in that, The amino acid sequence of the linker peptide 2 is shown in SEQ ID NO: 36 or 37.

20. The bispecific antibody according to claim 1, characterized in that, The first Fc peptide and the second Fc peptide are connected by a knock-into-hole structure.

21. The bispecific antibody according to claim 20, characterized in that, Both the first and second Fc peptide segments are human Fc peptide segments.

22. The bispecific antibody according to claim 21, characterized in that, The human Fc peptide is the human IgG1 Fc peptide.

23. The bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the first Fc peptide is shown in SEQ ID NO: 38, and the amino acid sequence of the second Fc peptide is shown in SEQ ID NO: 39 or 40.

24. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody includes: The first polypeptide chain with the amino acid sequence shown in SEQ ID NO: 13, the second polypeptide chain with the amino acid sequence shown in SEQ ID NO: 14, the third polypeptide chain with the amino acid sequence shown in SEQ ID NO: 15, and the fourth polypeptide chain with the amino acid sequence shown in SEQ ID NO:

15.

25. A nucleic acid, characterized in that, Encoding the bispecific antibody according to any one of claims 1 to 24.

26. A vector or transformant, characterized in that, It contains the nucleic acid as described in claim 25.

27. The carrier or transformant according to claim 26, characterized in that, The vector is a eukaryotic vector or a prokaryotic vector.

28. The carrier or transformant according to claim 26, characterized in that, The vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

29. A cell characterized in that, Carrying the nucleic acid of claim 25, the vector or transformant of any one of claims 26-28; or Express the bispecific antibody according to any one of claims 1 to 24.

30. The cell according to claim 29, characterized in that, The cells are prokaryotic cells, eukaryotic cells, or bacteriophages.

31. A pharmaceutical composition, characterized in that, It comprises: the bispecific antibody according to any one of claims 1 to 24, the nucleic acid according to claim 25, or the vector or transformant according to any one of claims 26 to 28.

32. The pharmaceutical composition according to claim 31, characterized in that, This further includes pharmaceutically acceptable excipients.

33. The pharmaceutical composition according to claim 32, characterized in that, The excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

34. The pharmaceutical composition according to claim 31, characterized in that, The pharmaceutical composition is an injectable preparation.

35. A reagent kit, characterized in that, It comprises: the bispecific antibody according to any one of claims 1 to 24, the nucleic acid according to claim 25, the vector or transformant according to any one of claims 26 to 28, or the cell according to claim 29 or 30.

36. Use of the bispecific antibody of any one of claims 1 to 24, the nucleic acid of claim 25, the vector or transformant of any one of claims 26 to 28, or the cell of claim 29 or 30 in the preparation of a kit for detecting CD3 and / or MICA.

37. Use of the bispecific antibody of any one of claims 1 to 24, the nucleic acid of claim 25, the vector or transformant of any one of claims 26 to 28, the cell of claim 29 or 30, or the pharmaceutical composition of any one of claims 31 to 34 in the preparation of a medicament for the treatment of at least one of melanoma, rectal cancer, lung cancer, kidney cancer, pancreatic cancer, and prostate cancer.

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