Bispecific antibodies and their applications

Abstract

The present invention proposes a bispecific antibody targeting CD3 and MICA and its application. It comprises: a first antigen binding region, the first antigen binding region comprises an anti-CD3 antibody or an antigen binding fragment thereof; a second antigen binding region, the second antigen binding region has MICA binding activity, wherein the anti-CD3 antibody or its antigen binding fragment comprises a first heavy chain variable region and a first light chain variable region having a specific CDRs amino acid sequence. The bispecific antibody of the present invention can bind to CD3 and MICA, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMC to kill tumor cells, and has good anti-cancer activity; moreover, the bispecific antibody has reduced pro-inflammatory cytokine secretion and T cell activation performance, is safer, and has good clinical application value and drug development value.

Description

Technical Field

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

[0002] Cancer is a major disease that affects human survival and development. According to the latest data, approximately 19 million new cancer cases and 10 million cancer deaths occur worldwide each year, and both incidence and mortality rates are on the rise. Besides surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy are associated with significant side effects and a high risk of recurrence. In recent years, immunotherapy, including tumor-targeted antibodies, immune checkpoint antibodies, and bispecific antibodies, has become a new hotspot and a new hope in the fight against cancer. Immunotherapy, exemplified by PD-1 / L1, has demonstrated tremendous potential. However, even the PD-1 / L1 therapy, currently the most widely approved, has an overall response rate of only 30%, leaving many patients unable to benefit. One major reason for this is that immune checkpoint therapy is ineffective against "cold tumors." T cells recognize neoantigens (neoantigens)—antigens expressed by tumor gene mutations—through the T cell receptors (TCRs) on their surface. However, some tumors have a low frequency of genetic mutations and a limited number of neoantigens, making them "cold tumors." Current immune checkpoint therapies, such as PD-1 / L1 therapy, achieve anti-cancer goals by restoring the function of T cells themselves. However, in "cold tumors", T cells cannot effectively recognize tumors, resulting in the ineffectiveness of immune checkpoint therapy for "cold tumors".

[0003] CD3-based bispecific antibodies (hereinafter referred to as "CD3 bispecific antibodies") recruit T cells to reach local tumors, bridge T cells and tumors, promote T cell activation, and kill tumors. This type of bispecific antibody does 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, so they can also "ignore" the inhibitory signals of immune checkpoint molecules to a certain extent; however, CD3 bispecific antibodies also promote the production of a large number of pro-inflammatory cytokines, such as TNFα, IL-6, etc., triggering a strong cytokine storm and excessive immune response, causing damage to the body, and severe cases may be life-threatening. Therefore, CD3-based bispecific antibodies have good application prospects in clinical practice, but their safety needs to be further improved.

[0004] One approach to addressing the safety concerns of CD3 bispecific antibodies is to increase their affinity for tumor targets while simultaneously reducing their affinity for CD3. This allows for greater local distribution of CD3 bispecific antibodies to the tumor, increasing local tumor drug concentrations while reducing peripheral drug concentrations, reducing off-target toxicity, lowering the production of pro-inflammatory cytokines, and reducing on-target toxicity. Therefore, clinically, there is an urgent need to develop CD3 bispecific antibodies with improved safety and greater clinical application value. Summary of the Invention

[0005] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to provide a bispecific antibody targeting CD3 and MICA. This bispecific antibody can bind to CD3 and MICA, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMCs to kill tumor cells, and exhibiting good anti-cancer activity. Furthermore, this bispecific antibody has reduced pro-inflammatory cytokine secretion and T cell activation, is safer, and has good clinical application value and drug development value.

[0006] The present invention is based on the inventor's discovery and understanding of the following facts and problems:

[0007] To improve the safety of CD3 and MICA bispecific antibodies, the inventors, through extensive screening and experimental verification, obtained two anti-CD3 antibodies, Cross313 and Cross325, and constructed a CD3 and MICA bispecific antibody based on them. Further experimental results demonstrated that the CD3 and MICA bispecific antibody of the present invention can bind to CD3 and MICA, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMCs to kill tumor cells, and has excellent anti-cancer activity. Furthermore, this bispecific antibody has reduced pro-inflammatory cytokine secretion and T cell activation, resulting in higher safety and promising clinical application and drug development value.

[0008] Therefore, in the first aspect of the present invention, the present invention provides a bispecific antibody. According to an embodiment of the present invention, the bispecific antibody comprises: a first antigen-binding region, the first antigen-binding region comprises an anti-CD3 antibody or an antigen-binding fragment thereof; a second antigen-binding region, the second antigen-binding region has MICA binding activity,

[0009] Wherein, the anti-CD3 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region;

[0010] The first heavy chain variable region has a CDR sequence of an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3 and SEQ ID NO: 7 or a conservative modification thereof; and / or,

[0011] The first light chain variable region has a CDR sequence of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6 and SEQ ID NO: 8 or a conservative modification thereof.

[0012] According to an embodiment of the present invention, the bispecific antibody can bind to CD3 and MICA, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMC to kill tumor cells, and has good anti-cancer activity; moreover, the bispecific antibody has reduced performance in promoting the secretion of pro-inflammatory cytokines and T cell activation performance, is safer, and has good clinical application and drug development value.

[0013] In a second aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the aforementioned bispecific antibody. According to an embodiment of the present invention, the nucleic acid molecule can encode a bispecific antibody that can simultaneously target CD3 and MICA.

[0014] In a third aspect, the present invention provides a vector or transformant. According to an embodiment of the present invention, the vector or transformant contains the aforementioned nucleic acid. Thus, the constructed vector or transformant can effectively express the aforementioned bispecific antibody.

[0015] In a fourth aspect, the present invention provides a cell. According to embodiments of the present invention, the cell carries the aforementioned nucleic acid, the aforementioned vector, or the aforementioned transformant, or expresses the aforementioned bispecific antibody. According to embodiments of the present invention, the cell is obtained by transfecting or transforming the aforementioned vector or transformant, and the cell can efficiently express the aforementioned bispecific antibody under appropriate conditions.

[0016] In a fifth aspect, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned bispecific antibody, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned cell. According to an embodiment of the present invention, the bispecific antibody can effectively promote PBMC to kill tumor cells and has anti-cancer activity; it can produce fewer pro-inflammatory cytokines, has higher safety, and has good clinical application value and drug development value. The resulting drug can be further used to prevent and / or treat related diseases mediated by CD3 and / or MICA.

[0017] In a sixth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, or the aforementioned cell. According to embodiments of the present invention, the kit can bind to CD3 protein and / or MICA protein, and can effectively identify CD3 protein and / or MICA protein.

[0018] In a seventh aspect, the present invention provides use of the aforementioned bispecific antibody, nucleic acid molecule, vector or transformant, or cell in preparing a kit for detecting CD3 and / or MICA. Those skilled in the art will appreciate that the aforementioned features and advantages of bispecific antibodies also apply to this use and are not further elaborated here.

[0019] In an eighth aspect, the present invention provides the use of the aforementioned bispecific antibodies, nucleic acid molecules, vectors or transformants, cells, or pharmaceutical compositions in the preparation of medicaments for preventing and / or treating diseases mediated by CD3 and / or MICA. According to embodiments of the present invention, the bispecific antibodies and corresponding nucleic acids, vectors or transformants, or pharmaceutical compositions of the present invention can be further prepared into medicaments that can be clinically used to prevent or treat diseases mediated by CD3 and / or MICA.

[0020] Those skilled in the art will appreciate that the features and advantages described above for antibodies or antigen-binding fragments thereof, conjugates, nucleic acid molecules, vectors or transformants, and pharmaceutical compositions are also applicable to this use and will not be elaborated here.

[0021] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0023] Figure 1 A schematic diagram of a CD3×MICA bispecific antibody according to a specific embodiment of the present invention;

[0024] Figure 2 This is a diagram showing the ELISA results of the binding of Cross3×h5A1002 antibody and Cross3×h5A1 antibody to MICA protein according to a specific embodiment of the present invention;

[0025] Figure 3This is a diagram showing the ELISA results of the binding of Cross3×h5A1002 antibody and Cross3×h5A1 antibody to CD3E&D protein according to a specific embodiment of the present invention;

[0026] Figure 4 This is a diagram showing the ELISA results of the binding of Cross3×h5A1002 antibody and Cross3×h5A1 antibody to bridge CD3E&D and MICA protein according to a specific embodiment of the present invention;

[0027] Figure 5 This is a flow cytometry result diagram of the binding of Cross3×h5A1002 antibody and Cross3×h5A1 antibody to A-375 melanoma cells according to a specific embodiment of the present invention;

[0028] Figure 6 This is a flow cytometry result diagram of the binding of Cross3×h5A1002 antibody and Cross3×h5A1 antibody to HCT-116 colorectal cancer cells according to a specific embodiment of the present invention;

[0029] Figure 7 Flow cytometry results of the binding of Cross3×h5A1002 antibody and Cross3×h5A1 antibody to NCI-H358 lung cancer cells according to a specific embodiment of the present invention;

[0030] Figure 8 Flow cytometry results of the binding of Cross3×h5A1002 antibody and Cross3×h5A1 antibody to NCI-1975 lung cancer cells according to a specific embodiment of the present invention;

[0031] Figure 9 This is a graph showing the results of Cross3×h5A1002 antibody and Cross3×h5A1 antibody promoting PBMC to kill HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0032] Figure 10 This is a flow cytometry result diagram of the binding of Cross3×h5A1002, TR66×hA1002, and UCHT1×h5A1002 antibodies to A-375 melanoma cells according to a specific embodiment of the present invention;

[0033] Figure 11 Flow cytometry results of the binding of Cross3×h5A1002, TR66×hA1002, and UCHT1×h5A1002 antibodies to HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0034] Figure 12Flow cytometry results of the binding of Cross3×h5A1002, TR66×hA1002, and UCHT1×h5A1002 antibodies to HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0035] Figure 13 This figure shows the results of Cross3×h5A1002, TR66×h5A1002, and UCHT1×h5A1002 antibodies promoting PBMC to kill A-375 melanoma cells according to a specific embodiment of the present invention;

[0036] Figure 14 ELISA results of binding of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies to CD3E&D proteins according to a specific embodiment of the present invention;

[0037] Figure 15 ELISA results of the binding of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies to MICA protein according to a specific embodiment of the present invention;

[0038] Figure 16 This is a graph showing the ELISA results of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies bridging CD3E&D and MICA protein according to a specific embodiment of the present invention;

[0039] Figure 17 Flow cytometry results of the binding of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies to A-375 melanoma cells according to a specific embodiment of the present invention;

[0040] Figure 18 This is a flow cytometry result diagram of the binding of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies to HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0041] Figure 19 Flow cytometry results of the binding of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies to CD8 T cells according to a specific embodiment of the present invention;

[0042] Figure 20This figure shows the results of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies according to a specific embodiment of the present invention promoting PBMC to kill A-375 melanoma and HCT-15 colorectal cancer cells;

[0043] Figure 21 This figure shows the results of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies according to a specific embodiment of the present invention promoting the secretion of cytokine TNFα by PBMC after co-incubation with A-375 melanoma and HCT-15 colorectal cancer cells;

[0044] Figure 22 This figure shows the results of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies according to a specific embodiment of the present invention promoting the secretion of cytokine IL-6 by PBMC after co-incubation with A-375 melanoma and HCT-15 colorectal cancer cells;

[0045] Figure 23 This figure shows the results of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies according to a specific embodiment of the present invention promoting the expression of CD25 in PBMCs after co-culture with A-375 melanoma and HCT-15 colorectal cancer cells;

[0046] Figure 24 This figure shows the results of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 antibodies according to a specific embodiment of the present invention promoting the expression of CD69 in PBMCs after co-culture with A-375 melanoma and HCT-15 colorectal cancer cells;

[0047] Figure 25 This is a flow cytometry result diagram of the binding of Cross313×h5A1002 to A-498 renal cancer cells according to a specific embodiment of the present invention;

[0048] Figure 26 This is a flow cytometry result diagram of the binding of Cross313×h5A1002 to HCC827 non-small cell lung cancer cells according to a specific embodiment of the present invention;

[0049] Figure 27 This is a flow cytometry result diagram of the binding of Cross313×h5A1002 to PC-3 prostate cancer cells according to a specific embodiment of the present invention;

[0050] Figure 28 This is a flow cytometry result diagram of the binding of Cross313×h5A1002 to NCI-N87 gastric cancer cells according to a specific embodiment of the present invention;

[0051] Figure 29 This is a flow cytometry result diagram of the binding of Cross313×h5A1002 to NCI-H1299 non-small cell lung cancer cells according to a specific embodiment of the present invention;

[0052] Figure 30 This figure shows the results of Cross313×h5A1002 promoting PBMC to kill NCI-H1299 non-small cell lung cancer cells according to a specific embodiment of the present invention;

[0053] Figure 31 This figure shows the results of Cross313×h5A1002 promoting PBMC to kill HCC827 non-small cell lung cancer cells according to a specific embodiment of the present invention;

[0054] Figure 32 This figure shows the results of Cross313×h5A1002 promoting PBMC to kill A-498 renal cancer cells according to a specific embodiment of the present invention;

[0055] Figure 33 This figure shows the results of Cross313×h5A1002 promoting PBMC to kill PANC-1 pancreatic cancer cells according to a specific embodiment of the present invention;

[0056] Figure 34 This figure shows the results of Cross313×h5A1002 promoting PBMC to kill PC-3 prostate cancer cells according to a specific embodiment of the present invention;

[0057] Figure 35 This is a graph showing the in vivo efficacy of Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 in promoting anti-cancer in immune reconstitution mice according to a specific embodiment of the present invention;

[0058] Figure 36 This is a graph showing the tumor volume of a single mouse showing that Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 according to a specific embodiment of the present invention promote anti-cancer effects in immune reconstructed mice. DETAILED DESCRIPTION

[0059] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0060] 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 the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0061] The endpoints of the ranges and any values ​​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 endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0062] Terms and Definitions

[0063] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly 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 belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0064] As used herein, the term "antibody" generally refers to an antibody that can recognize one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies only, triabodies, single chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, as long as they exhibit the desired biological activity (Miller et al. (2003) Jour. of Immunology 170: 4854 4861). Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy chains, full-length light chains, complete immunoglobulin molecules; or immunologically active portions of any of these polypeptides, i.e., molecules or portions thereof that contain an antigen binding site that immunospecifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases.

[0065] Herein, certain regions within the variable region have a higher degree of variability in amino acid composition and sequence, referred to as "hypervariable regions (HVRs)." Hypervariable regions are where antigen and antibody bind, and are therefore also referred to as complementarity-determining regions (CDRs). Both the heavy and light chain variable regions have three CDRs. For example, amino acid residues around 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and around 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 from a "hypervariable loop" (e.g., amino acid residues around 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and around 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)).

[0066] As used herein, the term "anti-CD3 antibody" refers to an antibody that can bind to CD3. Such an antibody is also referred to herein as an "antibody that binds to CD3." The term "anti-MICA antibody" refers to an antibody that can bind to MICA. Such an antibody is also referred to herein as an "antibody that binds to MICA."

[0067] As used herein, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment" and may include a portion of an intact antibody, generally the antigen-binding region or variable region. This includes, but is not limited to, Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFv-Fc fragments or bispecific antibodies (BsAbs), linear antibodies, or any fragment that can increase half-life by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" is polyethylene glycol) or by incorporation into liposomes.

[0068] As used herein, 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 amino acids of a monoclonal antibody from one species (such as mouse) with the constant region of an antibody from another species (such as human).

[0069] As used herein, the term "humanized antibody" refers to a recombinant antibody obtained by replacing the amino acid sequence of the non-CDR (Fv framework region (FR)) amino acids of the constant region and variable region of a monoclonal antibody from one species (such as a mouse) with the amino acid sequence of the non-CDR amino acids of the constant region and variable region of an antibody from another species (such as a human) using recombinant DNA technology. That is, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when the amino acid sequence of the non-CDR amino acids of the constant region and variable region is fully humanized, it is called a humanized antibody. The method of humanization can be carried out with reference to conventional antibody engineering technology and will not be described in detail here.

[0070] With respect to 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 sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions in the second sequence] minus [the total number of nucleotides in the first sequence] and then multiplying by [100%], where each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide 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 known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and setups for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2 357 768-A.

[0071] With respect to polypeptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more polypeptides or designated sequences thereof when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). The percent homology between two sequences varies with the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions x 100), where optimal alignment is determined by taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. Sequence comparison and percent identity determination between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting Examples below.

[0072] As used herein, the term "conservatively modified amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody containing the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced 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, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0073] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0074] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0075] As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic response), ie, substances with a reasonable benefit / risk ratio.

[0076] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0077] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient by a suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate, or pharmaceutical composition of the present invention can be administered by any common route as long as it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0078] As used herein, the term "treatment" refers to any process used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0079] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0080] The present invention provides a bispecific antibody, nucleic acid molecule, vector or transformant, cell, pharmaceutical composition, kit and application thereof targeting CD3 and MICA, which are described in detail below.

[0081] Bispecific antibodies

[0082] The present invention provides a bispecific antibody. The bispecific antibody comprises: a first antigen-binding region, wherein the first antigen-binding region comprises an anti-CD3 antibody or an antigen-binding fragment thereof;

[0083] a second antigen-binding region, wherein the second antigen-binding region has MICA binding activity,

[0084] Wherein, the anti-CD3 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region;

[0085] The first heavy chain variable region has a CDR sequence of an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 3 and SEQ ID NO: 7 or a conservative modification thereof; and / or,

[0086] The first light chain variable region has a CDR sequence of the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6 and SEQ ID NO: 8 or a conservative modification thereof.

[0087] It should be noted that one or more amino acid residues in the CDR region of the antibodies or antigen-binding fragments thereof of the present invention may be replaced by other amino acid residues from the same side chain family, and the retained function of the altered antibodies can be tested using the functional assays described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0088] The bispecific antibody of the present invention can bind to CD3 and MICA, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMC to kill tumor cells, and has good anti-cancer activity; moreover, the bispecific antibody has reduced performance in promoting the secretion of pro-inflammatory cytokines and T cell activation, is safer, and has good clinical application and drug development value.

[0089] According to an embodiment of the present invention, the first heavy chain variable region has the heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1, 2 and 3, or the first heavy chain variable region has the heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1, 2 and 7.

[0090] According to an embodiment of the present invention, the first light chain variable region has the heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4, 5 and 6, or the first light chain variable region has the heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 4, 5 and 8.

[0091] According to an embodiment of the present invention, the first heavy chain variable region has the heavy chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 1, 2 and 3, and the first light chain variable region has the heavy chain variable region CDR1, CDR2 and CDR3 shown in SEQ ID NOs: 4, 5 and 6; or

[0092] The first heavy chain variable region has heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 7, and the first light chain variable region has heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 8.

[0093] It should be noted that the antigen-binding fragment will be composed of a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, or contain a partial sequence of the light or heavy chain variable region, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived.

[0094] According to an embodiment of the present invention, the first heavy chain variable region further includes a heavy chain framework region, and / or the second heavy chain variable region further includes a light chain framework region.

[0095] According to an embodiment of the present invention, at least a portion of the heavy chain framework region and / or the light chain framework region is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy bovine antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0096] Preferably, it is at least one of a murine antibody, a human antibody, and a primate antibody.

[0097] It should be noted that the immunoglobulins described 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 portions that provide reduced or enhanced effector cell activity. The immunoglobulins can be derived from any species.

[0098] According to an embodiment of the present invention, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and / or, the light chain constant region includes a light chain constant region selected from κ type or λ type.

[0099] In some specific implementations, the heavy chain constant region comprises a heavy chain constant region selected from human IgG, IgA, IgM, IgE or IgD, such as human IgG1, human IgG2, human IgG3, human IgG4, human IgA, human IgM, human IgE or human IgD.

[0100] In some specific embodiments, the heavy chain constant region comprises a heavy chain constant region selected from murine IgG, IgA, IgM, IgE or IgD, such as murine IgG1, murine IgG2a, murine IgG2b, murine IgG2c, murine IgG3, murine IgA, murine IgM, murine IgE or murine IgD.

[0101] It should be noted that, in order to further improve the bioacceptability of the antibody, the antibody may be humanized, that is, the antibody is a chimeric antibody or a humanized antibody.

[0102] As previously described, one or more amino acid residues in the heavy or light chain variable region of the antibody or antigen-binding fragment thereof can be replaced by other amino acid residues from the same side chain family, and the retained function of the altered antibody can be tested using the functional assays described herein.

[0103] According to an embodiment of the present invention, the first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 31 or 34 or a conservative modification thereof, and / or the first light chain variable region has an amino acid sequence as shown in SEQ ID NO: 32 or 35 or a conservative modification thereof.

[0104] According to an embodiment of the present invention, the first heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 31, and the first light chain variable region has the amino acid sequence shown in SEQ ID NO: 32; or,

[0105] The first heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 34, and the first light chain variable region has the amino acid sequence shown in SEQ ID NO: 35.

[0106] According to an embodiment of the present invention, the N-terminus of the first light chain variable region is connected to the C-terminus of the first heavy chain variable region, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region.

[0107] According to an embodiment of the present invention, the first antigen binding region further includes a first connecting peptide, the N-terminus of the first light chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first heavy chain variable region, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first light chain variable region.

[0108] According to an embodiment of the present invention, the first antigen binding region further comprises a first anti-MICA antibody or an antigen binding fragment thereof, and the anti-MICA antibody or the antigen binding fragment thereof is a first Fab fragment, a first scFab fragment or a first scFv fragment of anti-MICA.

[0109] It should be noted that the amino acid sequences described in the present invention are all shown from N-terminus to C-terminus.

[0110] According to an embodiment of the present invention, the N-terminus of the first light chain variable region is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first light chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or the N-terminus of the first light chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first light chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment.

[0111] According to an embodiment of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof further includes a CL fragment and a CH1 fragment, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment, and the C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment.

[0112] According to an embodiment of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof further includes a second connecting peptide, the C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment, the C-terminus of the CL fragment is connected to the N-terminus of the second connecting peptide, the C-terminus of the second connecting peptide is connected to the N-terminus of the first heavy chain variable region, and the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment.

[0113] According to an embodiment of the present invention, the first antigen binding region further comprises a first Fc fragment;

[0114] wherein the N-terminus of the first light chain variable region is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first Fc fragment is connected to the C-terminus of the first light chain variable region, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first Fc fragment is connected to the C-terminus of the first heavy chain variable region; or

[0115] The N-terminus of the first Fc fragment is connected to the C-terminus of the first light chain variable region, the N-terminus of the first light chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first heavy chain variable region; or, the N-terminus of the first Fc fragment is connected to the C-terminus of the first heavy chain variable region, the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first light chain variable region; or

[0116] The N-terminus of the first Fc fragment is connected to the C-terminus of the first light chain variable region, the N-terminus of the first light chain variable region is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or, the N-terminus of the first Fc fragment is connected to the C-terminus of the first heavy chain variable region, the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first light chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or

[0117] The N-terminus of the first Fc fragment is connected to the C-terminus of the first light chain variable region, the N-terminus of the first light chain variable region is connected to the C-terminus of the first connecting peptide, the N-terminus of the first connecting peptide is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or, the N-terminus of the first Fc fragment is connected to the C-terminus of the first heavy chain variable region, the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first connecting peptide, the N-terminus of the first connecting peptide is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first light chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or

[0118] The N-terminus of the first Fc fragment is connected to the C-terminus of the CH1 fragment, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment, and the C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment; or

[0119] The C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment, the C-terminus of the CL fragment is connected to the N-terminus of the second connecting peptide, the C-terminus of the second connecting peptide is connected to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment, and the N-terminus of the first Fc fragment is connected to the C-terminus of the CH1 fragment.

[0120] According to an embodiment of the present invention, the first Fc peptide segment is selected from human Fc peptide segments, preferably human IgG1 Fc peptide segments.

[0121] According to an embodiment of the present invention, the first Fc peptide segment has an amino acid sequence as shown in SEQ ID NO:51.

[0122] According to an embodiment of the present invention, the first connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0123] According to an embodiment of the present invention, the second connecting peptide has an amino acid sequence as shown in SEQ ID NO: 50 or 53.

[0124] According to an embodiment of the present invention, the first anti-MICA antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region.

[0125] According to an embodiment of the present invention, the second heavy chain variable region has a heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 9, 10 and 11, and the second light chain variable region has a heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 12, 13 and 14.

[0126] According to an embodiment of the present invention, the second heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 37 or an amino acid sequence with at least 90% identity thereto, and the second light chain variable region has an amino acid sequence as shown in SEQ ID NO: 38 or an amino acid sequence with at least 90% identity thereto.

[0127] According to an embodiment of the present invention, the first scFab fragment has an amino acid sequence as shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% identity thereto; or

[0128] The first Fab fragment has the amino acid sequence shown in SEQ ID NO: 42 and 43, or an amino acid sequence at least 80% identical thereto;

[0129] According to an embodiment of the present invention, the first antigen binding region has an amino acid sequence as shown in SEQ ID NO: 15 or 16 or an amino acid sequence having at least 80% identity thereto; or

[0130] The first antigen binding region has an amino acid sequence as shown in SEQ ID NO:46 and SEQ ID NO:53, or an amino acid sequence with at least 80% identity thereto, or has an amino acid sequence as shown in SEQ ID NO:46 and SEQ ID NO:54, or an amino acid sequence with at least 80% identity thereto.

[0131] According to an embodiment of the present invention, the second antigen binding region comprises a second anti-MICA antibody or an antigen binding fragment thereof, and the second anti-MICA antibody or an antigen binding fragment thereof is a second scFab fragment or a second Fab fragment of anti-MICA.

[0132] It should be noted that the amino acid sequence of the second scFab fragment or the second Fab fragment of the present invention may be the same as or different from that of the first scFab fragment or the first Fab fragment.

[0133] According to an embodiment of the present invention, the second anti-MICA antibody or antigen-binding fragment thereof comprises a third heavy chain variable region and a third light chain variable region.

[0134] According to an embodiment of the present invention, the third heavy chain variable region has heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NO:9, 10 and 11, and the third light chain variable region has heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NO:12, 13 and 14.

[0135] It should be noted that, without substantially affecting the MICA binding activity of the antibody or its antigen-binding fragment (retaining at least 95% activity), those skilled in the art may replace, 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 heavy chain variable region, light chain variable region, scFab fragment, Fab fragment or Fc fragment of the antibody or its antigen-binding fragment of the present invention to obtain variants of the amino acid sequence of the heavy chain variable region, light chain variable region, scFab fragment, Fab fragment or Fc fragment of the antibody or its antigen-binding fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are substituted in the heavy chain variable region or light chain variable region, scFab fragment, Fab fragment or Fc fragment. The amino acid sequence of the above-mentioned variant of the present invention may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference amino acid sequence. The amino acid sequence identity of the present invention can be measured using amino acid sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters.

[0136] According to an embodiment of the present invention, the third heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 37 or an amino acid sequence that is at least 90% identical thereto, and the third light chain variable region has an amino acid sequence as shown in SEQ ID NO: 38 or an amino acid sequence that is at least 90% identical thereto.

[0137] According to an embodiment of the present invention, the second scFab fragment has an amino acid sequence as shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% identity thereto; or

[0138] The second Fab fragment has the amino acid sequence shown in SEQ ID NO: 42 and 43, or an amino acid sequence at least 80% identical thereto.

[0139] According to an embodiment of the present invention, the second antigen binding region further comprises a second Fc fragment; wherein the N-terminus of the second Fc fragment is connected to the C-terminus of CH1 of the second scFab fragment or the second Fab fragment.

[0140] According to an embodiment of the present invention, the second Fc peptide segment is selected from a human Fc peptide segment, preferably a human IgG1 Fc peptide segment.

[0141] According to an embodiment of the present invention, the second Fc peptide segment has an amino acid sequence as shown in SEQ ID NO: 52 or 54, or an amino acid sequence having at least 80% identity thereto.

[0142] According to an embodiment of the present invention, the first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure.

[0143] According to an embodiment of the present invention, the second scFab fragment has an amino acid sequence as shown in SEQ ID NO: 44 or an amino acid sequence having at least 80% identity thereto; or

[0144] The second Fab fragment has the amino acid sequence shown in SEQ ID NO: 42 and 43, or an amino acid sequence at least 80% identical thereto.

[0145] According to an embodiment of the present invention, the second antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence with at least 80% identity thereto; or the second antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 17 and SEQ ID NO: 18 or an amino acid sequence with at least 80% identity thereto.

[0146] As mentioned above, under the premise of not substantially affecting the CD3 and MICA binding activity of the bispecific antibody (retaining at least 95% activity), those skilled in the art can replace, 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 bispecific antibody of the present invention to obtain variants of the amino acid sequence of the polypeptide chain of the bispecific antibody. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the polypeptide chain. The amino acid sequence of the above-mentioned variant of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference amino acid sequence.

[0147] According to an embodiment of the present invention, the bispecific antibody comprises:

[0148] A first polypeptide chain having an amino acid sequence as set forth in one of SEQ ID NOs: 15, 16, 39, or 40, or an amino acid sequence at least 80% identical thereto, and a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 44, or an amino acid sequence at least 80% identical thereto; or a first polypeptide chain having an amino acid sequence as set forth in one of SEQ ID NOs: 15, 16, 39, or 40, or an amino acid sequence at least 80% identical thereto, a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence at least 80% identical thereto, and a third polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence at least 80% identical thereto; or a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence at least 80% identical thereto, a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 47 or SEQ ID NO: 48, or an amino acid sequence at least 80% identical thereto, and a third polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence at least 80% identical thereto. A fourth polypeptide chain having the amino acid sequence shown in NO: 17 or an amino acid sequence having at least 80% identity thereto.

[0149] Nucleic Acids

[0150] The present invention provides a nucleic acid. The nucleic acid encodes the aforementioned antibody or antigen-binding fragment thereof. The nucleic acid molecule encodes the aforementioned bispecific antibody. The nucleic acid molecule according to an embodiment of the present invention can encode a bispecific antibody that can simultaneously target CD3 and MICA.

[0151] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include either or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, if the amino acid sequence of a molecule in the present invention includes either a DNA form or an RNA form, disclosure of one form implies disclosure of the other.

[0152] Vector or transformant

[0153] The present invention provides a vector or transformant. The vector or transformant contains the aforementioned nucleic acid. The vector or transformant may include an optional control amino acid sequence, such as , operably linked to the nucleic acid molecule. The control amino acid sequence, such as , is one or more control amino acid sequences, such as , that can direct expression of the nucleic acid molecule in a host. The vector or transformant thus constructed can effectively express the aforementioned bispecific antibody.

[0154] When the nucleic acid molecule is linked to a vector or transformant, such as an expression vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or exogenous, i.e., not derived from the vector itself. The nucleic acid molecule and the control elements can be operably linked.

[0155] According to an embodiment of the present invention, the vector may refer to a cloning vector or an expression vector, which can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0156] As used herein, the term "operably linked" refers to the attachment of an exogenous gene to a vector so that control elements within the vector, such as transcriptional control amino acid sequences such as and translational control amino acid sequences such as , can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include viral vectors, plasmids, and bacteriophages. Expression vectors according to certain embodiments of the present invention, when introduced into appropriate recipient cells, can effectively express the aforementioned nucleic acid molecules under the mediation of a regulatory system, thereby enabling the in vitro production of large quantities of the protein encoded by the nucleic acid molecules.

[0157] According to an embodiment of the present invention, the vector is a eukaryotic vector or a prokaryotic vector.

[0158] According to an embodiment of the present invention, the vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

[0159] cell

[0160] The present invention provides a cell. The cell carries the aforementioned nucleic acid, or the aforementioned vector, or transformant, or expresses the aforementioned bispecific antibody. According to an embodiment of the present invention, the cell is obtained by transfecting or transforming the vector or transformant, and the cell can efficiently express the aforementioned bispecific antibody under appropriate conditions.

[0161] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

[0162] According to an embodiment of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0163] According to an embodiment of the present invention, the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0164] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0165] According to an embodiment of the present invention, the insect cell is a fall 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.

[0166] According to an embodiment of the present invention, the cell is a mammalian cell.

[0167] According to an embodiment of the present invention, the cell is a BHK cell, a CHO cell, a COS cell or a NSO cell.

[0168] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the antibodies or antigen-binding fragments thereof described in this application. It will be readily understood by those skilled in the art that conditions suitable for the expression of antibodies or antigen-binding fragments thereof include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the recombinant antibodies according to the specific environment of the laboratory.

[0169] Pharmaceutical composition

[0170] The present 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 cell. According to embodiments of the present invention, the bispecific antibody can effectively promote PBMCs to kill tumor cells, exhibiting anti-cancer activity; it can produce less pro-inflammatory cytokines, has higher safety, and has excellent clinical application value and drug development value. The resulting drug can be further used to prevent and / or treat diseases mediated by CD3 and / or MICA.

[0171] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0172] According to an embodiment of the present invention, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0173] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0174] It should be noted that the pharmaceutical composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the composition can 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 can be administered to the subject simultaneously or sequentially.

[0175] The medicament of the present invention contains a safe and effective amount of the active ingredient of the present invention and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be compatible with the mode of administration. The dosage form of the medicament of the present invention is an injection, oral preparation (tablets, capsules, oral liquid), transdermal preparation, or sustained-release preparation. For example, it can be prepared using conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. The medicament is preferably manufactured under sterile conditions.

[0176] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, the 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, the route of administration, and the like. For example, depending on the exigencies of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0177] The pharmaceutically acceptable excipients of the present 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 compatible with the mode of administration, as is well known to those skilled in the art.

[0178] Reagent test kit

[0179] The present invention provides a kit. The kit comprises the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, or the aforementioned cell. According to an embodiment of the present invention, the kit can bind to CD3 protein and / or MICA protein and can effectively identify CD3 protein and / or MICA protein.

[0180] As described above, the bispecific antibodies of the embodiments of the present invention can specifically bind to CD3 and MICA. The CD3 protein and / or MICA protein-related kits 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 CD3 protein and / or MICA protein from humans or other mammals.

[0181] The kit can effectively detect, enrich, or separate and purify CD3 protein and / or MICA protein in biological samples, and further be used in scientific research, such as qualitatively or quantitatively detecting CD3 protein and / or MICA protein molecules in biological samples. More specifically, the kit can be used for immunoblotting, immunoprecipitation, and other methods involving detection using the specific binding properties of CD3 protein and / or MICA protein and antibodies. These kits may contain any one or more of the following: an antagonist, a bispecific antibody of the present invention, or a drug reference material; a protein purification column; an immunoglobulin affinity purification buffer; and a cell assay diluent. The bispecific antibodies of the present invention can be used in various diagnostic tests, for example, to detect various diseases or the presence of drugs, toxins, or other proteins in vitro or in vivo. For example, the test can be performed on the serum or blood of a subject to test for diseases mediated by CD3 and / or MICA.

[0182] Use in the preparation of kits

[0183] The present invention provides use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, or the aforementioned cell in preparing a kit for detecting CD3 and / or MICA.

[0184] As previously described, the bispecific antibodies of the embodiments of the present invention are capable of specifically binding to CD3 and MICA. Therefore, these bispecific antibodies can be used to detect CD3 and / or MICA. Furthermore, they can be used to prepare CD3 and / or MICA-related kits for scientific research, such as for the qualitative or quantitative detection of CD3 and / or MICA protein molecules in biological samples. More specifically, they can be used in kits for immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of CD3 and / or MICA with antibodies. These kits may contain any one or more of the following: an antagonist, the bispecific antibody of the present invention, or a reference drug material; a protein purification column; an immunoglobulin affinity purification buffer; and a cell assay diluent. The bispecific antibodies of the present invention can be used in various diagnostic tests, for example, to detect various diseases or the presence of drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for diseases mediated by CD3 and / or MICA by testing serum or blood from a subject.

[0185] Use in preparing medicines

[0186] The present invention provides the use of the aforementioned bispecific antibodies, nucleic acid molecules, vectors or transformants, cells, or pharmaceutical compositions in the preparation of medicaments for preventing and / or treating diseases mediated by CD3 and / or MICA. According to embodiments of the present invention, the bispecific antibodies and corresponding nucleic acids, vectors or transformants, or pharmaceutical compositions of the present invention can be further prepared into medicaments that can be clinically used to prevent or treat diseases mediated by CD3 and / or MICA.

[0187] According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0188] According to an embodiment 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, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.

[0189] According to an embodiment of the present invention, the MICA-mediated related diseases are cancer, diseases caused by transplant rejection, autoimmune diseases, and infectious diseases.

[0190] According to an embodiment 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, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0191] Disease treatment methods

[0192] The present invention provides a method for preventing and / or treating diseases mediated by CD3 and / or MICA. According to an embodiment of the present invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned bispecific antibody, nucleic acid molecule, vector or transformant, cell, or pharmaceutical composition.

[0193] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, 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 infection, and the like. The subject can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, and the like.

[0194] The effective amount of the antibody or antigen-binding fragment thereof, conjugate, nucleic acid, vector or transformant or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0195] In some embodiments, the CD3-mediated related disease comprises an autoimmune disease.

[0196] In some embodiments, the autoimmune disease comprises at least one of 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, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.

[0197] In some embodiments, the MICA-mediated disease is cancer, disease caused by transplant rejection, autoimmune disease, or infectious disease.

[0198] In some embodiments, 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.

[0199] The sequences involved in the present invention are detailed in Table 1.

[0200] Table 1 Amino acid sequence description

[0201]

[0202]

[0203]

[0204]

[0205]

[0206] The present invention will be described in detail below through examples. In the examples or test examples, if no specific conditions are specified, the experimental methods were carried out under conventional conditions.

[0207] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not specified, they are carried out according to the technology or conditions described in the literature in this area or according to the product instructions.The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased commercially.

[0208] Example 1: Production of antibodies

[0209] 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 / mL to obtain ExpiCHO cell solution. (2) When the dual antibody is A and C configuration ( Figure 1 ), pcDNA3.4 vector containing CD3 antibody, MICA antibody heavy chain, and MICA antibody light chain (commissioned to Nanjing GenScript for synthesis) was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a; or, when the dual antibody is in the B and D configuration ( Figure 1 ), add the pcDNA3.4 vector containing CD3 antibody and MICA antibody (commissioned to Nanjing GenScript for synthesis) into 2 mL OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1 to obtain solution a; or, when the double antibody is of E configuration ( Figure 1), the pcDNA3.4 vector containing MICA heavy chain-CD3 antibody, MICA antibody heavy chain, and MICA antibody light chain (commissioned to Nanjing GenScript for synthesis) was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a. (3) 160 μL ExpressiFectamine CHO transfection reagent (purchased from Thermo Fisher) was added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution b. (4) Solution a and solution b were then mixed to obtain a transfection mixture, and the entire transfection mixture was added to 50 mL of ExpiCHO cell solution within 5 minutes. (5) After culturing at 37°C and 5% CO2 for 1 day, 8 mL of Feed and 300 μL of Enhancer (purchased from Thermo Fisher) were added, and the cells were transferred to 32°C and 5% CO2 for 9 days. The culture supernatant was harvested, and 8 mL of Feed was added on the 5th day. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from NanoMicro).

[0210] In this example, 11 CD3 and MICA bispecific antibodies were prepared to investigate the various properties of the bispecific antibodies of the present invention. Figure 1 , Table 1 and Table 2.

[0211] Table 2 Structure of bispecific antibody and corresponding amino acid sequence in Example 1

[0212]

[0213] Note: For the configurations of class A, B, C, D, and E antibodies, see Figure 1 .

[0214] Example 2: ELISA binding experiment of the antibody of the present invention

[0215] ELISA is used to test the binding properties of bispecific antibodies. The antigen protein is coated onto a 96-well plate, and the strength of the signal after the antibody is added is used to determine the binding properties of the bispecific antibody and the antigen protein.

[0216] (1) Dilute MICA-His protein (purchased from Acro) to 2 μg / ml in PBS buffer and add 100 μl / well to a 96-well plate. Incubate at 4°C overnight. Aspirate the PBS buffer from the 96-well plate, wash the plate six times with PBST (pH 7.2 PBS containing 0.1% Tween 20), add 200 μl / well PBS / 10% BSA, and incubate at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μl / well of the bispecific antibodies to be tested, Cross3×h5A1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 20), Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), and Cross3×h5A1002scFab (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18) were added. The cells were incubated at 37°C for 1 h. The reaction mixture was removed, the plate was washed six times with PBST, and 100 μl / well of an HRP (horseradish peroxidase)-labeled anti-human IgG secondary antibody (purchased from Jackson Labs) was added with PBST / 0.05% BSA at 37°C for 1 h. After washing the plate six times with PBST, add 80 μl / well of TMB (tetramethylbenzidine) and incubate at room temperature for 3 min. Then, add 80 μl / well of 4 M sulfuric acid to terminate the reaction. The absorbance is read at 450 nm using a microplate reader.

[0217] The results are as follows Figure 2As shown, both Cross3×h5A1 and Cross3×h5A1002 antibodies can bind to MICA protein, and the binding ability of Cross3×h5A1002 antibody is stronger than that of Cross3×h5A1.

[0218] The results are as follows Figure 15 As shown, Cross3×h5A1002 and the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention can bind to MICA protein, and the three bispecific antibodies Cross3×h5A1002, Cross313×h5A1002, and Cross325×h5A1002 have similar binding abilities to MICA protein.

[0219] (2) Dilute CD3 E&D protein (purchased from Acro) to 2 μg / ml in PBS buffer and add 100 μl / well to a 96-well plate. Incubate at 4°C overnight. Aspirate the PBS buffer from the 96-well plate, wash the plate six times with PBST (pH 7.2 PBS containing 0.1% Tween 20), add 200 μl / well PBS / 10% BSA, and incubate at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μl / well of the bispecific antibodies to be tested, Cross3×h5A1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 20), Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), and Cross3×h5A1002scFab (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18) were added. The plates were incubated at 37°C for 1 h. The reaction mixture was removed, the plate was washed six times with PBST, and 100 μl / well of an HRP (horseradish peroxidase)-labeled anti-human IgG secondary antibody (purchased from Southern Biotech) diluted in PBST / 0.05% BSA was added and incubated at 37°C for 1 h. After washing the plate six times with PBST, add 80 μl / well of TMB (tetramethylbenzidine) and incubate at room temperature for 3 min. Then, add 80 μl / well of 4 M sulfuric acid to terminate the reaction. The absorbance is read at 450 nm using a microplate reader.

[0220] The results are as follows Figure 3As shown, the Cross3×h5A1 and Cross3×h5A1002 antibodies of the present invention can bind to CD3E&D protein, and the two antibodies have similar binding abilities to CD3E&D protein.

[0221] The above test results suggest that the MICA antigen-binding portion has little effect on the affinity of the CD3 antigen-binding portion. Therefore, the MICA antigen-binding region of the bispecific antibody of the present invention is preferably selected from an anti-MICA antibody or antigen-binding fragment with stronger MICA binding ability. Exemplarily, the MICA antigen-binding region is selected from the h5A1002 antibody or its binding fragment, h5A1002 scFab or h5A1002 Fab.

[0222] The results are as follows Figure 14 As shown, Cross3×h5A1002 and the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention can bind to CD3E&D protein, and compared with Cross3×h5A1002, the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention have weaker binding ability to CD3E&D protein.

[0223] Example 3: Antibody ELISA bridging experiment

[0224] ELISA assays were used to test the bridging binding properties of bispecific antibodies. CD3E&D antigen proteins were coated onto 96-well plates. After the addition of the antibodies, the biotinylated MICA protein was used for detection. The strength of the signal was used to determine the binding properties of the bispecific antibody bridging CD3E&D and MICA proteins.

[0225] (1) Dilute MICA-His protein (purchased from Acro) to 2 μg / ml in PBS buffer and add 100 μl / well to a 96-well plate. Incubate at 4°C overnight. Aspirate the PBS buffer from the 96-well plate, wash the plate six times with PBST (pH 7.2 PBS containing 0.1% Tween 20), add 200 μl / well PBS / 10% BSA, and incubate at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. Then, 100 μl / well of the bispecific antibodies to be tested, Cross3×h5A1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 20), Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), and Cross3×h5A1002scFab (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18) were added. The cells were incubated at 37°C for 1 h. The reaction mixture was removed, the plate was washed six times with PBST, and MICA*002α3-Fc-Biotin diluted to an appropriate concentration was added and the cells were incubated at 37°C for 1 h. Remove the reaction mixture, wash the plate six times with PBST, and then add 100 μl / well of HRP (horseradish peroxidase)-conjugated streptavidin secondary antibody (purchased from Southern Biotech) diluted in PBST / 0.05% BSA. Incubate at 37°C for 1 hour. Wash the plate six times with PBST, then add 80 μl / well of TMB (tetramethylbenzidine). Incubate at room temperature for 3 minutes. Terminate the reaction by adding 80 μl / well of 4 M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0226] The results are as follows Figure 4 As shown, Cross3×h5A1 and Cross3×h5A1002 antibodies can bridge CD3E&D and MICA proteins, and the bridging ability of Cross3×h5A1002 antibody is stronger than that of Cross3×h5A1.

[0227] The results are as follows Figure 16 As shown, Cross3×h5A1002 and the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention can bridge CD3E&D and MICA proteins, and the bridging ability of the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention is weaker than that of Cross3×h5A1002.

[0228] Example 4: Antibody Flow Cytometry Binding Experiment

[0229] Flow cytometry experiments are used to detect the binding properties of bispecific antibodies. Bispecific antibodies are added to cells, and the strength of the signal after the addition of the antibody is used to determine the binding properties of the antibody and cells.

[0230] (1) Dilute the tumor cells to 2×10 6 / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, 10μl / tube of goat serum was added thereto, and the tube was blocked at 4℃ for 30min. Serial dilutions of the bispecific antibodies Cross3×h5A1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 20), Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross3×h5A1002scFab (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA single-chain antibody is shown in SEQ ID NO: 23), TR66×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 24, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: NO: 18), UCHT1×h5A1002 (the amino acid sequence of the 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: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross313×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 15, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross325×h5A1002 (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: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), and control hIgG1LALA (purchased from Bio-Tech), and incubated at 4°C for 30 min. Add 1 ml of PBS to the EP tube and centrifuge at 3500 rpm for 5 minutes at 4°C. Discard the supernatant and wash once more with PBS. Discard the supernatant after centrifugation. Resuspend the cells in 100 μl / tube of PBS and add 1 μl / tube of Alexa-647-labeled goat anti-human IgG secondary antibody (Jackson Labs). Incubate at 4°C in the dark for 30 minutes. Wash twice with PBS and centrifuge. Discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze by flow cytometry.

[0231] The results are as follows Figure 5 、 6 As shown in Figures 7 and 8, Cross3×h5A1 and Cross3×h5A1002 antibodies can bind to A-375 melanoma cells, HCT-116 colorectal cancer cells, NCI-H358 lung cancer cells, and NCI-H1975 lung cancer cells, and the ability of Cross3×h5A1002 antibody to bind to tumor cells is stronger than that of Cross3×h5A1.

[0232] The results are as follows Figure 10 、 19 As shown, Cross3×h5A1002, TR66×h5A1002, and UCHT1×h5A1002 antibodies were able to bind to A-375 melanoma cells and HCT-15 colorectal cancer cells.

[0233] The results are as follows Figure 17 、 26 As shown, Cross3×h5A1002 and the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention can bind to A-375 melanoma cells and HCT-15 colorectal cancer cells, and the binding ability of the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention to tumor cells is comparable to that of the Cross3×h5A1002 antibody.

[0234] (2) Dilute PBMC to 2×10 6 / ml, added to a 1.5ml EP tube at a volume of 100μl / tube, 10μl / tube of goat serum was added thereto, and the tube was blocked at 4℃ for 30min. Serially diluted bispecific antibodies Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), TR66×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 24, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), UCHT1×h5A1002 (the amino acid sequence of the 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: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross313×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 15, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 1 The cells were incubated at 4°C 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°C. The supernatant was discarded and the tube was washed once 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 secondary antibody (purchased from Jackson Lab) and 0.5 μl / tube of PerCP-Cy5.5-labeled anti-human CD8 antibody were added and incubated at 4°C for 30 min in the dark. The cells were washed twice with PBS and centrifuged and the supernatant was discarded. Resuspend the cells with 200 μl / tube of PBS and analyze them using flow cytometry.

[0235] The results are as follows Figure 12 As shown, Cross3×h5A1002, TR66×h5A1002, and UCHT1×h5A1002 antibodies can bind to CD8 T cells, and the T cell binding abilities of Cross3×h5A1002 and TR66×h5A1002 are similar, but weaker than that of UCHT1×h5A1002.

[0236] The results are as follows Figure 19 As shown, Cross3×h5A1002 and the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention can bind to CD8 T cells, and the T cell binding ability of the Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention is weaker than that of Cross3×h5A1002.

[0237] Example 5: Experiment on Bispecific Antibodies Promoting PBMC to Kill Tumor Cells

[0238] The ability of bispecific antibodies to promote PBMC to kill A-375 melanoma cells, HCT-15 colorectal cancer cells, NCI-H1299 lung cancer cells, HCC827 lung cancer cells, A-498 renal cancer cells, PANC-1 pancreatic cancer cells, and PC-3 prostate cancer cells was tested.

[0239] (a) Complete RPMI-1640 medium was added to a 16-well RTCA plate at a volume of 50 μL / well and calibrated on the instrument.

[0240] (b) Tumor cells were diluted to 2×10 5 / mL, and added separately to the RTCA plate obtained in step (1) at a volume of 50 μL / well, and then the cell coefficient was detected using the xCELLigence RTCA MP device at 37°C and 5% CO2 for 24 hours;

[0241] (c) Serial dilutions of the bispecific antibodies Cross3×h5A1 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 19, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 20), Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross3×h5A1002scFab (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA single-chain antibody is shown in SEQ ID NO: 23), TR66×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 24, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), UCHT1×h5A1002 (the amino acid sequence of the 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: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross313×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 15, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross325×h5A1002 (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: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), control hIgG1LALA (purchased from Baiying Bio) Figure 9 、 17 , 21 with the highest concentration of 5 μg / ml, 8 gradients, 5-fold dilution; Figure 20 The highest concentration was 5 μg / ml, 8 gradients, 10-fold dilutions), added to the RTCA plate obtained in step (2), with an addition volume of 20 μl / well;

[0242] (4) PBMC (purchased from Miaoshun Biotechnology) were diluted to 1.25×10 6pcs / ml, and added to the RTCA plate obtained in step (3) at a volume of 80 μl / well;

[0243] (5) The reaction system obtained in step (4) was incubated at 37° C. and 5% CO 2 for 24 h to detect the cell coefficient using an xCELLigence RTCA MP device.

[0244] The results are as follows Figure 9 As shown, Cross3×h5A1 and Cross3×h5A1002 antibodies promoted PBMC to kill HCT-15 colorectal cancer cells, and the killing ability of Cross3×h5A1002 antibody was stronger than that of Cross3×h5A1.

[0245] The results are as follows Figure 13 As shown, Cross3×h5A1002, TR66×h5A1002, and UCHT1×h5A1002 promoted PBMC to kill A-375 melanoma cells, and the killing ability of Cross3×h5A1002 and UCHT1×h5A1002 antibodies was stronger than that of TR66×h5A1002.

[0246] The results are as follows Figure 20 As shown, Cross3×h5A1002 antibody and Cross313×h5A1002 and Cross325×h5A1002 of the present invention promoted 100% killing of HCT-15 melanoma cells by PBMC, and the EC50 of Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention for promoting killing was higher than that of Cross3×h5A1002.

[0247] As shown in the figure, the Cross313×h5A1002 of the present invention promoted PBMC to kill NCI-H1299 lung cancer cells, HCC827 lung cancer cells, A-498 renal cancer cells, PANC-1 pancreatic cancer cells, and PC-3 prostate cancer cells by 100%.

[0248] Example 6: Experiment on Bispecific Antibodies Promoting Cytokine Secretion by PBMC

[0249] The bispecific antibody was added to the PBMC and tumor cell co-incubation system. After 48 hours of culture, the culture supernatant was collected and the cytokine content in the supernatant was detected to determine the characteristics of the bispecific antibody in inducing cytokine release.

[0250] (1) Dilute the tumor to 1×10 5 / ml, added to 96-well plates, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0251] (2) The bispecific antibodies Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross313×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 15, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), and Cross325×h5A1002 (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: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18) were cultured in complete RPMI 1640 medium. NO: 18), control hIgG1LALA (purchased from Bio-Tech), were serially diluted and added to a 96-well plate at 20 μl / well;

[0252] (3) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / ml, added to 96-well plate, 80 μl / well;

[0253] (4) Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 48 h;

[0254] (5) Centrifuge at 300 g for 10 min at room temperature and collect the cell culture supernatant;

[0255] (6) The cytokine content in the supernatant was detected using a CBA kit (purchased from BD).

[0256] The results are as follows Figure 21 As shown, Cross3×h5A1002 antibody and Cross313×h5A1002 and Cross325×h5A1002 of the present invention promote the secretion of pro-inflammatory cytokine TNFα by PBMC, and Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention promote TNFα secretion less than Cross3×h5A1002.

[0257] The results are as follows Figure 22As shown, Cross3×h5A1002 antibody and Cross313×h5A1002 and Cross325×h5A1002 of the present invention promoted PBMC to secrete the proinflammatory cytokine IL-6, and Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention promoted IL-6 secretion less than Cross3×h5A1002.

[0258] Example 7: Bispecific Antibody Promotes T Cell Activation Experiment

[0259] Bispecific antibodies were added to the PBMC and tumor cell co-incubation system. After 48 hours of culture, the culture supernatant was collected and the expression of CD8 T cell surface activation markers CD69 and CD25 was detected to determine the characteristics of bispecific antibodies in inducing T cell activation.

[0260] (1) Dilute the tumor to 1×10 5 / ml, added to 96-well plates, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0261] (2) The bispecific antibodies Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross313×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 15, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), and Cross325×h5A1002 (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: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18) were cultured in complete RPMI 1640 medium. NO: 18), control hIgG1LALA (purchased from Bio-Tech), were serially diluted and added to a 96-well plate at 20 μl / well;

[0262] (3) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / ml, added to 96-well plate, 80 μl / well;

[0263] (4) Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 48 h;

[0264] (5) Centrifuge at 300g for 10 min at room temperature, discard the supernatant, resuspend the cells in 90 μl / well PBS, add 10 μl / well goat serum, and block at 4°C for 30 min. Add PerCP-Cy5.5-labeled CD8 antibody, and PE-labeled CD25 or BV421-labeled CD69 antibody, and incubate at 4°C in the dark for 30 min.

[0265] (6) Wash twice with PBS and discard the supernatant after centrifugation.

[0266] (7) Resuspend the cells in 200 μl / tube of PBS and analyze them using a flow cytometer.

[0267] The results are as follows Figure 23 As shown, Cross3×h5A1002 antibody and Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention promote T cells to upregulate the expression of the activation marker CD25, and the ability of Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention to promote CD25 expression is lower than that of Cross3×h5A1002 antibody.

[0268] The results are as follows Figure 24 As shown, Cross3×h5A1002 antibody and Cross313×h5A1002 and Cross325×h5A1002 of the present invention promote T cells to upregulate the expression of activation marker CD69, and the ability of Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention to promote CD69 expression is lower than that of Cross3×h5A1002.

[0269] Example 8: Effect of CD3×MICA Antibody on Anti-cancer in Mice

[0270] In vivo efficacy experiments were used to detect the anti-cancer function of Cross3×h5A1002 and the Cross313×h5A1002 and Cross325×h5A1002 bispecific antibodies of the present invention in promoting immune reconstitution in mice.

[0271] (1) On day -14, human PBMC (purchased from Saili Biotechnology) were transfused into NSG mice (purchased from Southern Model) via the tail vein at an injection volume of 5×10 6 / Only;

[0272] (2) On day 10, NSG mice were subcutaneously injected with 2×10 6 A-375 human melanoma cells;

[0273] (3) On day 0, the mice were weighed and the tumor volume was measured. The mice were divided into 6 groups based on the weighing results and tumor volume;

[0274] (4) On days 0, 4, 7, and 11, the mice were injected with the Cross3×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 21, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross313×h5A1002 (the amino acid sequence of the CD3 single-chain antibody is shown in SEQ ID NO: 15, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18), Cross325×h5A1002 (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: 17, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 18) antibodies and the solvent control PBS into the tail vein, 250 μl per mouse;

[0275] (4) After injection of the above antibodies, the tumor volume was measured and the mice were weighed twice a week.

[0276] The results are shown in the figure. It can be seen that Cross3×h5A1002 antibody and Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention can effectively promote PBMC anti-cancer activity, and the anti-cancer function of Cross313×h5A1002 and Cross325×h5A1002 antibodies of the present invention is better than that of Cross3×h5A1002.

[0277] The above experimental results show that the bispecific antibody of the present invention can bind to CD3 and MICA, thereby promoting T cell activation and cytokine secretion, effectively promoting PBMC to kill tumor cells, and has good anti-cancer activity; the bispecific antibody of the present invention can achieve high-affinity binding to MICA and reduce the performance of promoting pro-inflammatory cytokine secretion and T cell activation, and has higher safety. In summary, the bispecific antibody of the present invention can promote immune cells to fight cancer, has good anti-cancer activity, and has higher safety, and has good clinical application value and drug development value.

[0278] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

[0279] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0280] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A bispecific antibody, characterized in that: Include: a first antigen-binding region comprising an anti-CD3 antibody or an antigen-binding fragment thereof; a second antigen-binding region, wherein the second antigen-binding region has MICA binding activity, Wherein, the anti-CD3 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region; The first heavy chain variable region has heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 3, and the first light chain variable region has light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 6; or The first heavy chain variable region has heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1, 2 and 7, and the first light chain variable region has light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 4, 5 and 8; The second antigen-binding region comprises a first anti-MICA antibody or an antigen-binding fragment thereof, wherein the anti-MICA antibody or the antigen-binding fragment thereof is a first Fab fragment, a first scFab fragment or a first scFv fragment of anti-MICA; The first anti-MICA antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region; The second heavy chain variable region has heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 9, 10 and 11, and the second light chain variable region has light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 12, 13 and 14; The first antigen binding region comprises a first Fc fragment; The second antigen binding region comprises a second Fc fragment; The anti-CD3 antibody or antigen-binding fragment thereof comprises a CL fragment and a CH1 fragment; The second antigen-binding region comprises a second anti-MICA antibody or an antigen-binding fragment thereof, and the second anti-MICA antibody or an antigen-binding fragment thereof is a second scFab fragment or a second Fab fragment of anti-MICA; The second anti-MICA antibody or antigen-binding fragment thereof comprises a third heavy chain variable region and a third light chain variable region; The third heavy chain variable region has heavy chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 9, 10 and 11, and the third light chain variable region has light chain variable region CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 12, 13 and 14; The bispecific antibody is in one of the following configurations: (a) configuration A; (b) configuration B; (c) configuration C; (d) configuration D; (e) configuration E; Wherein, the (a) configuration A comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises a first scFv fragment and a first Fc fragment, and the C-terminus of the first scFv fragment is connected to the N-terminus of the first Fc fragment; The second polypeptide chain comprises a second Fab fragment and a second Fc fragment, and the C-terminus of the CH1 domain of the second Fab fragment is connected to the N-terminus of the second Fc fragment; The first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure; The (b) configuration B comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first scFv fragment and a first Fc fragment, and the C-terminus of the first scFv fragment is connected to the N-terminus of the first Fc fragment; The second polypeptide chain comprises a second scFab fragment and a second Fc fragment, and the C-terminus of the second scFab fragment is connected to the N-terminus of the second Fc fragment; The first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure; Said (c) configuration C comprises a first polypeptide chain and a second polypeptide chain, The first polypeptide chain comprises a first scFab fragment and a first Fc fragment, wherein the C-terminus of the first scFab fragment is connected to the N-terminus of the first Fc fragment; The second polypeptide chain comprises a second Fab fragment and a second Fc fragment, and the C-terminus of the CH1 fragment of the second Fab fragment is connected to the N-terminus of the second Fc fragment; The first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure; The (d) configuration D comprises a first polypeptide chain and a second polypeptide chain, The first polypeptide chain comprises a first scFab fragment and a first Fc fragment, wherein the C-terminus of the first scFab fragment is connected to the N-terminus of the first Fc fragment; The second polypeptide chain comprises a second scFab fragment and a second Fc fragment, and the C-terminus of the second scFab fragment is connected to the N-terminus of the second Fc fragment; The first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure; The (e) configuration E comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, The first polypeptide chain comprises a second heavy chain variable region, a CH1 domain, a third connecting peptide, a first scFv fragment and a first Fc fragment, wherein the C-terminus of the second heavy chain variable region is connected to the N-terminus of the CH1 fragment, the C-terminus of the CH1 fragment is connected to the N-terminus of the third connecting peptide, the C-terminus of the third connecting peptide is connected to the N-terminus of the first scFv fragment, and the C-terminus of the first scFv fragment is connected to the N-terminus of the first Fc fragment; The second polypeptide chain comprises a second light chain variable region and a CL fragment; The third polypeptide chain comprises a third heavy chain variable region, a CH1 fragment and a second Fc fragment, wherein the C-terminus of the third heavy chain variable region is connected to the N-terminus of the CH1 fragment, and the C-terminus of the CH1 fragment is connected to the N-terminus of the second Fc fragment; The first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure; Among them, in configuration E, the second heavy chain variable region and the third heavy chain variable region are both the heavy chain variable regions of the second antigen binding region and have the same sequence, the second light chain variable region is the light chain variable region of the second antigen binding region, and the first scFv fragment includes the heavy chain variable region and light chain variable region of the first antigen binding region.

2. The bispecific antibody according to claim 1, characterized in that The first heavy chain variable region further comprises a heavy chain framework region, and / or the first light chain variable region further comprises a light chain framework region.

3. The bispecific antibody according to claim 2, characterized in that At least a portion of the heavy chain framework region and / or light chain framework region is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy bovine antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

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

5. The bispecific antibody according to claim 1, wherein The first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 31 or 34 or a conservative modification thereof, and / or the first light chain variable region has an amino acid sequence as shown in SEQ ID NO: 32 or 35 or a conservative modification thereof.

6. The bispecific antibody according to claim 1, characterized in that The first heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 31, and the first light chain variable region has the amino acid sequence shown in SEQ ID NO: 32; or The first heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 34, and the first light chain variable region has the amino acid sequence shown in SEQ ID NO:

35.

7. The bispecific antibody according to claim 1, characterized in that The N-terminus of the first light chain variable region is connected to the C-terminus of the first heavy chain variable region, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region.

8. The bispecific antibody according to claim 1, wherein The first antigen binding region further includes a first connecting peptide, the N-terminus of the first light chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first heavy chain variable region, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first light chain variable region.

9. The bispecific antibody according to claim 8, characterized in that The N-terminus of the first light chain variable region is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first light chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or The N-terminus of the first light chain variable region is connected to the C-terminus of the first connecting peptide, the N-terminus of the first connecting peptide is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or, the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first connecting peptide, the N-terminus of the first connecting peptide is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first light chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment.

10. The bispecific antibody according to claim 9, characterized in that The C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 segment, and the C-terminus of the first light chain variable region is connected to the N-terminus of the CL segment.

11. The bispecific antibody according to claim 10, characterized in that The anti-CD3 antibody or antigen-binding fragment thereof further includes a second connecting peptide, the C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment, the C-terminus of the CL fragment is connected to the N-terminus of the second connecting peptide, the C-terminus of the second connecting peptide is connected to the N-terminus of the first heavy chain variable region, and the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment.

12. The bispecific antibody according to claim 11, characterized in that The N-terminus of the first light chain variable region is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first Fc fragment is connected to the C-terminus of the first light chain variable region, or the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first Fc fragment is connected to the C-terminus of the first heavy chain variable region; or The N-terminus of the first Fc fragment is connected to the C-terminus of the first light chain variable region, the N-terminus of the first light chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first heavy chain variable region; or the N-terminus of the first Fc fragment is connected to the C-terminus of the first heavy chain variable region, the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first connecting peptide, and the N-terminus of the first connecting peptide is connected to the C-terminus of the first light chain variable region; or The N-terminus of the first Fc fragment is connected to the C-terminus of the first light chain variable region, the N-terminus of the first light chain variable region is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or, the N-terminus of the first Fc fragment is connected to the C-terminus of the first heavy chain variable region, the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first light chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or The N-terminus of the first Fc fragment is connected to the C-terminus of the first light chain variable region, the N-terminus of the first light chain variable region is connected to the C-terminus of the first connecting peptide, the N-terminus of the first connecting peptide is connected to the C-terminus of the first heavy chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or, the N-terminus of the first Fc fragment is connected to the C-terminus of the first heavy chain variable region, the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first connecting peptide, the N-terminus of the first connecting peptide is connected to the C-terminus of the first light chain variable region, and the N-terminus of the first light chain variable region is connected to the C-terminus of the CH1 fragment of the first Fab fragment or the first scFab fragment; or The N-terminus of the first Fc fragment is connected to the C-terminus of the CH1 fragment, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment, and the C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment; or The C-terminus of the first light chain variable region is connected to the N-terminus of the CL fragment, the C-terminus of the CL fragment is connected to the N-terminus of the second connecting peptide, the C-terminus of the second connecting peptide is connected to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 fragment, and the N-terminus of the first Fc fragment is connected to the C-terminus of the CH1 fragment.

13. The bispecific antibody according to claim 1, characterized in that The first Fc fragment is selected from human Fc peptide fragments.

14. The bispecific antibody according to claim 1, characterized in that The first Fc fragment is a human IgG1 Fc peptide segment.

15. The bispecific antibody according to claim 1, characterized in that The first Fc fragment has the amino acid sequence shown in SEQ ID NO: 51 or 53.

16. The bispecific antibody according to claim 8, characterized in that The first connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1.

17. The bispecific antibody according to claim 8, characterized in that The first connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

18. The bispecific antibody according to claim 11, characterized in that The second connecting peptide has the amino acid sequence shown in SEQ ID NO:

50.

19. The bispecific antibody according to claim 1, characterized in that The second heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 37, and the second light chain variable region has the amino acid sequence shown in SEQ ID NO:

38.

20. The bispecific antibody according to claim 1, wherein The first scFab fragment has the amino acid sequence shown in SEQ ID NO: 44; or The first Fab fragment has the amino acid sequence shown in SEQ ID NOs: 42 and 43.

21. The bispecific antibody according to claim 1, characterized in that The first antigen-binding region has an amino acid sequence as shown in SEQ ID NO: 15 or 16; or The first antigen-binding region has the amino acid sequence shown in SEQ ID NO: 46 and SEQ ID NO: 53, or has the amino acid sequence shown in SEQ ID NO: 46 and SEQ ID NO:

54.

22. The bispecific antibody according to claim 1, characterized in that The third heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 37, and the third light chain variable region has the amino acid sequence shown in SEQ ID NO:

38.

23. The bispecific antibody according to claim 1, characterized in that The second scFab fragment has the amino acid sequence shown in SEQ ID NO: 44; or The second Fab fragment has the amino acid sequence shown in SEQ ID NOs: 42 and 43.

24. The bispecific antibody according to claim 1, characterized in that The second Fc fragment is selected from human Fc peptide fragments.

25. The bispecific antibody according to claim 1, characterized in that The second Fc fragment is a human IgG1 Fc peptide segment.

26. The bispecific antibody according to claim 1, characterized in that The second Fc fragment has the amino acid sequence shown in SEQ ID NO: 52 or 54.

27. The bispecific antibody according to claim 1, characterized in that The second scFab fragment has the amino acid sequence shown in SEQ ID NO: 44; or The second Fab fragment has the amino acid sequence shown in SEQ ID NOs: 42 and 43.

28. The bispecific antibody according to claim 1, characterized in that The second antigen-binding region has the amino acid sequence shown in SEQ ID NO: 23; or The second antigen-binding region has the amino acid sequences shown in SEQ ID NO: 17 and SEQ ID NO:

18.

29. The bispecific antibody according to claim 1, characterized in that The bispecific antibody comprises: A first polypeptide chain having an amino acid sequence as shown in one of SEQ ID NOs: 15, 16, 39 or 40, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 44; or A first polypeptide chain having an amino acid sequence as shown in one of SEQ ID NOs: 15, 16, 39 or 40, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 17, and a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 18; 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 either SEQ ID NO: 47 or SEQ ID NO: 48, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 18, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO:

17.

30. A nucleic acid, characterized in that Encodes the bispecific antibody according to any one of claims 1 to 29.

31. A vector or transformant, characterized in that: Comprising the nucleic acid of claim 30.

32. The carrier according to claim 31, characterized in that The vector is a eukaryotic vector or a prokaryotic vector.

33. The carrier according to claim 31, characterized in that The vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

34. A cell, characterized in that Carrying the nucleic acid according to claim 30, the vector or transformant according to any one of claims 31 to 33; or Expressing the bispecific antibody according to any one of claims 1 to 29.

35. The cell according to claim 34, characterized in that The cells are prokaryotic cells or eukaryotic cells.

36. A pharmaceutical composition, characterized in that Include: The bispecific antibody according to any one of claims 1 to 29, the nucleic acid according to claim 30, the vector or transformant according to any one of claims 31 to 33, or the cell according to claim 34 or 35.

37. The pharmaceutical composition according to claim 36, characterized in that Further includes pharmaceutically acceptable excipients.

38. The pharmaceutical composition according to claim 37, characterized in that The excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

39. The pharmaceutical composition according to claim 36, characterized in that The pharmaceutical composition is an injection.

40. A kit, characterized in that include: The bispecific antibody according to any one of claims 1 to 29, the nucleic acid molecule according to claim 30, the vector or transformant according to any one of claims 31 to 33, or the cell according to claim 34 or 35.

41. Use of the bispecific antibody according to any one of claims 1 to 29, the nucleic acid molecule according to claim 30, the vector or transformant according to any one of claims 31 to 33, or the cell according to claim 34 or 35 in preparing a kit for detecting CD3 and / or MICA.

42. Use of the bispecific antibody of any one of claims 1 to 29, the nucleic acid of claim 30, the vector or transformant of any one of claims 31 to 33, the cell of claim 34 or 35, or the pharmaceutical composition of any one of claims 36 to 39 in the preparation of a medicament for treating at least one of the following cancers: melanoma, lung cancer, colon cancer, pancreatic cancer, renal cancer, and prostate cancer.

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