Bispecific antibody and use thereof

Abstract

A bispecific antibody targeting CD3 and a tumor target and a use of the bispecific antibody. The bispecific antibody comprises a first antigen binding region, a second antigen binding region, and an Fc portion; the Fc portion comprises a first Fc peptide fragment and a second Fc peptide fragment; the first antigen binding region comprises an scFv fragment and a binding protein of a first molecule or a fragment thereof, the binding protein of the first molecule or the fragment thereof is linked to the N-terminal of the scFv fragment, and the C-terminal of the scFv fragment is linked to the N-terminal of the first Fc peptide fragment; the second antigen binding region comprises a binding protein of a second molecule or a fragment thereof, and the binding protein of the second molecule or the fragment thereof is linked to the N-terminal of the second Fc peptide fragment; and the first molecule and the second molecule are the same, and the scFv fragment has CD3 binding activity. The bispecific antibody has weak binding to T-cells, strong binding to tumor cells, good safety, high anticancer activity, and significant value for clinical use and drug development.

Description

Bispecific antibodies and their applications 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 and weakening their binding to T cells. This allows the CD3 bispecific antibody to be more effectively distributed to the tumor, increasing local tumor drug concentration, reducing peripheral drug concentration, and minimizing off-target toxicity. Therefore, clinically, there is an urgent need to develop CD3 bispecific antibodies with improved safety and greater clinical application value.

[0005] Summary of the Invention

[0006] 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 a tumor target. This bispecific antibody can bind to both CD3 and the tumor target, has low CD3 binding activity, weak T cell binding, and weak ability to bridge T cells and tumor cells. It can also effectively promote the activity of PBMCs in killing tumor cells, has improved T cell activation performance, and can promote drug distribution to the tumor rather than to the periphery or lymphoid tissues. As a result, the bispecific antibody of the present invention has good safety, high anti-cancer activity, and high clinical application and drug development value.

[0007] The present invention is based on the inventor's discovery and understanding of the following facts and problems:

[0008] To improve the safety of CD3 and MICA bispecific antibodies, the inventors, through extensive screening and experimental verification, have developed a novel configuration of CD3 and MICA bispecific antibodies. Further experimental results demonstrate that this CD3 and MICA bispecific antibody exhibits weak T cell binding, strong tumor binding, and high cytotoxicity, effectively promoting PBMCs to kill tumor cells and demonstrating excellent anti-cancer activity. Furthermore, this bispecific antibody exhibits reduced T cell binding activity, high T cell activation, and enhanced safety, demonstrating promising clinical application and drug development value.

[0009] Therefore, in a 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, a second antigen-binding region, and an Fc portion;

[0010] The Fc portion comprises a first Fc peptide segment and a second Fc peptide segment;

[0011] The first antigen binding region comprises an scFv fragment and a first molecule of a binding protein or a fragment thereof, wherein the first molecule of the binding protein or the fragment thereof is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment;

[0012] The second antigen binding region comprises a second molecule of binding protein or a fragment thereof, and the second molecule of binding protein or a fragment thereof is connected to the N-terminus of the second Fc peptide segment;

[0013] The first molecule and the second molecule are identical, and the scFv fragment has CD3 binding activity.

[0014] The bispecific antibodies according to the embodiments of the present invention can bind to CD3 and tumor targets. The bispecific antibodies can bind to CD3 and tumor targets, and have low CD3 binding activity, weak binding to T cells, and weak ability to bridge T cells and tumor cells. At the same time, they can effectively promote the activity of PBMCs in killing tumor cells, have improved T cell activation performance, and can promote the distribution of drugs to the local tumor rather than the periphery or lymphoid tissues. Therefore, the bispecific antibodies of the present invention have good safety, high anti-cancer activity, and high clinical application and drug development value.

[0015] 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. The nucleic acid according to an embodiment of the present invention may encode a bispecific antibody that can simultaneously target CD3 and a tumor target.

[0016] 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.

[0017] 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. The cell according to embodiments of the present invention is obtained by transfecting or transforming the aforementioned vector or transformant, and the cell can efficiently express the aforementioned bispecific antibody under appropriate conditions.

[0018] In its 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 PBMCs to kill tumor cells, demonstrating high anti-cancer activity; the bispecific antibody has low CD3 binding activity and good safety. The resulting drug can be further used to prevent and / or treat diseases mediated by CD3 and / or tumor targets.

[0019] 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, 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 tumor targets, and can effectively identify CD3 protein and / or tumor targets.

[0020] Those skilled in the art will appreciate that the features and advantages described above for antibodies, nucleic acids, vectors or transformants, cells or pharmaceutical compositions are also applicable to the kit and will not be described in detail here.

[0021] In the seventh aspect of the present invention, the invention proposes the use of the aforementioned bispecific antibody, the aforementioned nucleic acid, the aforementioned vector or transformant or the aforementioned cell in preparing a kit for detecting CD3 and / or tumor targets.

[0022] Those skilled in the art will appreciate that the features and advantages described above for antibodies, nucleic acids, vectors or transformants, cells or pharmaceutical compositions are also applicable to the use in the preparation kit and will not be described in detail here.

[0023] In the eighth aspect of the present invention, the present invention proposes the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, the aforementioned cell or the aforementioned pharmaceutical composition in the preparation of a drug, which is used to prevent and / or treat related diseases mediated by CD3 and / or tumor targets.

[0024] Those skilled in the art will appreciate that the features and advantages described above for antibodies, nucleic acid molecules, vectors or transformants, cells and pharmaceutical compositions are also applicable to this use and will not be elaborated here.

[0025] 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

[0026] 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:

[0027] FIG1 is a schematic diagram of a CD3×MICA bispecific antibody according to a specific embodiment of the present invention;

[0028] FIG2 is a graph showing the ELISA results of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies to MICA protein according to a specific embodiment of the present invention;

[0029] FIG3 is a graph showing the ELISA results of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies to CD3E&D proteins according to a specific embodiment of the present invention;

[0030] FIG4 is a graph showing the ELISA results of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies bridging CD3E&D and MICA protein according to a specific embodiment of the present invention;

[0031] FIG5 is a flow cytometry result diagram of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies to CD8+ T cells according to a specific embodiment of the present invention;

[0032] FIG6 is a flow cytometry result diagram of the binding of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies to HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0033] 7 is a graph showing the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting PBMC to kill HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0034] FIG8 is a graph showing the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting PBMC to kill NCI-H1299 lung cancer cells according to a specific embodiment of the present invention;

[0035] FIG9 is a graph showing the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting PBMC to kill PANC-1 pancreatic cancer cells according to a specific embodiment of the present invention;

[0036] FIG10 is a graph showing the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting PBMC to kill A498 renal cancer cells according to a specific embodiment of the present invention;

[0037] FIG11 is a graph showing the results of Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoting PBMC to kill PC-3 prostate cancer cells according to a specific embodiment of the present invention;

[0038] FIG12 is a graph showing the results of Cross3×h5A1002 1:1 and Cross3×h5A1002 2:1 antibodies promoting the secretion of immune-activating cytokines IL-2 and IFN-γ after co-incubation of PBMC with HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0039] FIG13 is a graph showing the results of Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1, and SP34×h5A1002 2:1 antibodies promoting PBMC to kill HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0040] FIG14 is a graph showing the results of Cross3×h5A1002 2:1, Cross3×1D5 1:1, and Cross3×1D5 2:1 antibodies promoting PBMC to kill HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0041] FIG15 is a graph showing the results of Cross3×h5A1002 2:1, Cross3×13A9 1:1, and Cross3×13A9 2:1 antibodies promoting PBMC to kill HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0042] FIG16 is a graph showing the results of Cross3×h5A1002 2:1, Cross3×619 1:1, and Cross3×619 2:1 antibodies promoting PBMC to kill HCT-15 colorectal cancer cells according to a specific embodiment of the present invention;

[0043] FIG17 is a graph showing the in vivo efficacy of the Cross3×h5A1002 2:1 antibody according to a specific embodiment of the present invention in an immune reconstitution mouse A-375 human melanoma model;

[0044] FIG18 is a graph showing the in vivo efficacy of the Cross3×h5A1002 2:1 antibody according to a specific embodiment of the present invention in the immune reconstitution mouse NCI-H1299 human lung cancer model;

[0045] FIG19 is a graph showing the in vivo efficacy of the Cross3×h5A1002 2:1 antibody in promoting immune reconstitution in PC-3 human prostate cancer models in mice according to a specific embodiment of the present invention. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Terms and Definitions

[0050] 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.

[0051] 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, bispecific antibodies, heavy chain antibodies only, three-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, as long as they exhibit the desired biological activity. 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 an immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof that contains 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.

[0052] 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, it generally includes: 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; 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.

[0053] 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."

[0054] 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. Examples include, but are not limited to, Fv, scFv, Fab, Fab', Fab'-SH, scFab (single chain Fab fragment, scFab), F(ab')2, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that 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.

[0055] 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).

[0056] 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.

[0057] 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 2357768-A.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] As used herein, the term "pharmaceutical composition" generally refers to a 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 a carrier which constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and thoroughly combining the active bispecific antibody with a liquid carrier, a finely divided solid carrier, or both.

[0062] As used herein, the term "pharmaceutically acceptable ingredients" refers to substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), ie, substances with a reasonable benefit / risk ratio.

[0063] As used herein, the term "pharmaceutically acceptable excipient" includes any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the bispecific antibodies of the present invention, for example, by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0064] In this article, the term "administration" refers to introducing a predetermined amount of material into a patient by a certain suitable manner. The antibody or antigen-binding fragment, recombinant protein, bispecific antibody, conjugate or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the expected 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.

[0065] As used herein, the term "treatment" refers to a method for obtaining 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 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 an individual who is susceptible to the disease but has 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 symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or bispecific antibody to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing the bispecific antibody described herein to an individual in need.

[0066] 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.

[0067] The present invention provides a bispecific antibody, nucleic acid molecule, vector or transformant, cell, pharmaceutical composition, kit and application thereof that targets CD3 and tumor targets, which are described in detail below.

[0068] Bispecific antibodies

[0069] The present invention provides a bispecific antibody. The bispecific antibody comprises: a first antigen-binding region, a second antigen-binding region, and an Fc portion; the Fc portion comprises a first Fc peptide segment and a second Fc peptide segment; the first antigen-binding region comprises an scFv fragment and a first molecule of a binding protein or a fragment thereof, the first molecule of the binding protein or a fragment thereof is linked to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is linked to the N-terminus of the first Fc peptide segment; the second antigen-binding region comprises a second molecule of a binding protein or a fragment thereof, the second molecule of the binding protein or a fragment thereof is linked to the N-terminus of the second Fc peptide segment; the first and second molecules are identical, and the scFv fragment has CD3 binding activity.

[0070] The bispecific antibodies according to the embodiments of the present invention can bind to CD3 and tumor targets. The bispecific antibodies can bind to CD3 and tumor targets, and have low CD3 binding activity and weak ability to bridge T cells and tumor cells. At the same time, they can effectively promote the activity of PBMCs to kill tumor cells, have improved T cell activation performance, and can promote the distribution of drugs to the local tumor rather than the periphery or lymphoid tissues. Therefore, the bispecific antibodies of the present invention have good safety, high anti-cancer activity, and high clinical application and drug development value.

[0071] According to an embodiment of the present invention, the first molecule and the second molecule are selected from MICA, PDL1, CD47, TIGIT, CD73, CD33, CEACAM1, CEACAM5, CEACAM6, STING, WNT, Beta catenin, B7H3, VISITA, CD19, BCMA, CD22, CD20, CD123, CD38, CEA, CD25, CD46, CD138, PSCA, PSMA, PSA, MUC1, MUC16, NY-ESO-1, GD2, WT1, Mesothelin, MAGE-A3, GPC3, PRAME, Globo H, AFP, Trop2, FOLR1, SP, Sca-1, CD133 or EPCAM.

[0072] According to an embodiment of the present invention, both the first molecule and the second molecule are MICA. The bispecific antibody of the present invention can bind to CD3 and MICA, and has low CD3 binding activity and weak ability to bridge T cells and tumor cells. As a result, the bispecific antibody has low T cell binding activity and high tumor binding activity, can effectively promote PBMC to kill tumor cells, and can promote drug distribution to the local tumor rather than the periphery or lymphoid tissue. As a result, the bispecific antibody has good safety and high anti-cancer activity.

[0073] According to an embodiment of the present invention, the binding protein or fragment thereof is selected from at least one of an antibody or an antigen-binding fragment thereof, a receptor, and a ligand.

[0074] According to an embodiment of the present invention, the binding protein or fragment thereof of the first molecule is a first Fab fragment, and / or the binding protein or fragment thereof of the second molecule is a second Fab fragment.

[0075] It should be noted that the amino acid sequences involved in the present invention are all shown from N-terminus to C-terminus.

[0076] According to an embodiment of the present invention, the C-terminus of the CH1 fragment of the first Fab fragment is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment; and / or the C-terminus of the CH1 fragment of the second Fab fragment is connected to the N-terminus of the second Fc peptide segment.

[0077] In some embodiments of the present invention, the first molecule of binding protein or fragment thereof is a first scFab fragment, and / or the second molecule of binding protein or fragment thereof is a second scFab fragment.

[0078] In some embodiments of the present invention, the C-terminus of the CH1 fragment of the first scFab fragment is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment; and / or the C-terminus of the CH1 fragment of the second scFab fragment is connected to the N-terminus of the second Fc peptide segment.

[0079] In the bispecific antibodies of the embodiments of the present invention, the scFv fragment is successfully masked by the first Fab fragment or the first scFab fragment. This reduces the bispecific antibody's CD3 binding activity and its ability to bridge T cells and tumor cells, resulting in improved safety. Contrary to the inventors' expectations, the bispecific antibody with this configuration exhibits significantly enhanced tumor cell binding activity.

[0080] According to embodiments of the present invention, the first and second Fab fragments have MICA binding activity. In bispecific antibodies according to embodiments of the present invention, the CD3 antigen-binding portion of the bispecific antibody has little effect on the affinity of the MICA antigen-binding portion. Therefore, to obtain bispecific antibodies with excellent safety and high anti-cancer activity, the MICA antigen-binding portion is preferably an antigen-binding fragment of an anti-MICA antibody with strong MICA binding ability. For example, the MICA antigen-binding region is selected from the antigen-binding fragment Fab of the h5A1002 antibody.

[0081] In some embodiments of the invention, the first Fab fragment and the second Fab fragment are not identical.

[0082] In some embodiments of the invention, the first Fab fragment and the second Fab fragment are identical.

[0083] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment both comprise: a second heavy chain variable region CDR1-3 and a second light chain variable region CDR1-3; wherein,

[0084] The second heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 7 or a conservative modification thereof,

[0085] The second heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 8 or a conservative modification thereof,

[0086] The second heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 9 or a conservative modification thereof,

[0087] The second light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 10 or a conservatively modified form thereof,

[0088] The second light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 11 or a conservatively modified form thereof,

[0089] The second light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 12 or a conservative modification thereof.

[0090] According to the embodiments of the present invention, the bispecific antibody of the present invention not only has low CD3 binding activity and weak ability to bridge T cells and tumor cells, but also has high tumor cell binding activity, and also significantly enhances the activity of promoting PBMC to kill tumor cells.

[0091] It should be noted that one or more amino acid residues in the heavy chain variable region CDR sequence or light chain variable region CDR sequence of the present invention 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 assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0092] According to an embodiment of the present invention, the first Fab fragment and / or the second Fab fragment comprises a second heavy chain framework region and / or a second light chain framework region.

[0093] According to an embodiment of the present invention, at least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a cow 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, preferably at least one of a murine antibody, a human antibody and a primate antibody.

[0094] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment each independently contain: a second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 29 or a conservative modification thereof, and a second light chain variable region having an amino acid sequence as shown in SEQ ID NO: 30 or a conservative modification thereof.

[0095] According to an embodiment of the present invention, the first Fab fragment and / or the second Fab fragment further comprises a CH1 fragment and a CL fragment; 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 second light chain variable region is connected to the N-terminus of the CL fragment, and the CH1 fragment and the CL fragment are connected via an interchain disulfide bond.

[0096] According to an embodiment of the present invention, the first Fab fragment and the second Fab fragment each independently contain: the second heavy chain variable region and CH1 fragment having the amino acid sequence shown in SEQ ID NO: 31, and the second light chain variable region and CL fragment having the amino acid sequence shown in SEQ ID NO: 32.

[0097] According to an embodiment of the present invention, the scFv fragment comprises: a first heavy chain variable region CDR1-3 and a first light chain variable region CDR1-3; wherein,

[0098] The first heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 1,

[0099] The first heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2,

[0100] The first heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3,

[0101] The first light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 4,

[0102] The first light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 5,

[0103] The first light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO:6.

[0104] According to an embodiment of the present invention, when the scFv fragment of the bispecific antibody of the present invention has the above-mentioned CDRs amino acid sequence, it has significantly reduced CD3 binding activity and significantly enhanced tumor cell binding ability, and the bispecific antibody further improves the activity of promoting PBMC to kill tumor cells, thereby further improving the anti-cancer efficacy and safety.

[0105] According to an embodiment of the present invention, the scFv fragment comprises a first heavy chain framework region and / or a first light chain framework region.

[0106] According to an embodiment of the present invention, at least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from at least one of a murine 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, preferably at least one of a murine antibody, a human antibody and a primate antibody.

[0107] 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.

[0108] According to an embodiment of the present invention, the scFv fragment comprises: a first heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 33 or a conservatively modified form thereof, and a first light chain variable region having an amino acid sequence as shown in SEQ ID NO: 34 or a conservatively modified form thereof.

[0109] It should be noted that one or more amino acid residues in the heavy chain variable region or light chain variable region of the present invention 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 assay method described herein. Preferably, the number of conservative modifications does not exceed 1 or 2.

[0110] According to an embodiment of the present invention, the scFv fragment further comprises: a connecting peptide 1; wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the first heavy chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the first light chain variable region; or the N-terminus of the connecting peptide 1 is connected to the C-terminus of the first light chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the first heavy chain variable region.

[0111] The connecting peptide 1 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.

[0112] In some embodiments of the present invention, the connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO:11.

[0113] According to an embodiment of the present invention, the scFv fragment has an amino acid sequence as shown in SEQ ID NO: 36 or 37.

[0114] According to an embodiment of the present invention, the first antigen binding region further includes a connecting peptide 2; wherein the C-terminus of CH1 of the first Fab fragment is connected to the N-terminus of the connecting peptide 2, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the scFv fragment.

[0115] According to an embodiment of the present invention, the connecting peptide 2 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.

[0116] According to an embodiment of the present invention, the connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO: 36 or 37.

[0117] 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.

[0118] According to an embodiment of the present invention, the first Fc peptide segment and the second Fc peptide segment are both human Fc peptide segments.

[0119] According to an embodiment of the present invention, the human Fc peptide segment is a human IgG1 Fc peptide segment.

[0120] According to an embodiment of the present invention, the first Fc peptide segment has an amino acid sequence as shown in SEQ ID NO: 38, and the second Fc peptide segment has an amino acid sequence as shown in SEQ ID NO: 39 or 40.

[0121] According to an embodiment of the present invention, the bispecific antibody comprises:

[0122] a first polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 13, 18, 19, 22, 25, or 28, or an amino acid sequence at least 80% identical thereto;

[0123] a second polypeptide chain having an amino acid sequence as set forth in SEQ ID NO: 14, 20, 23, or 26, or an amino acid sequence at least 80% identical thereto;

[0124] a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, 21, 24 or 27, or an amino acid sequence at least 80% identical thereto;

[0125] A fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, 21, 24 or 27, or an amino acid sequence at least 80% identical thereto.

[0126] It should be noted that, without substantially affecting the CD3 and MICA binding activity of the multispecific 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 multispecific 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.

[0127] According to an embodiment of the present invention, the bispecific antibody comprises:

[0128] a first polypeptide chain having the amino acid sequence of SEQ ID NO: 13, a second polypeptide chain having the amino acid sequence of SEQ ID NO: 14, a third polypeptide chain having the amino acid sequence of SEQ ID NO: 15, and a fourth polypeptide chain having the amino acid sequence of SEQ ID NO: 15; or

[0129] a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 18, a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 14, a third polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 15, and a fourth polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 15; or

[0130] a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 19, a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 14, a third polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 15, and a fourth polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 15; or

[0131] a first polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 22, a second polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 20, a third polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 21, and a fourth polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 21; or

[0132] a first polypeptide chain having the amino acid sequence of SEQ ID NO: 25, a second polypeptide chain having the amino acid sequence of SEQ ID NO: 23, a third polypeptide chain having the amino acid sequence of SEQ ID NO: 24, and a fourth polypeptide chain having the amino acid sequence of SEQ ID NO: 24; or

[0133] A first polypeptide chain having the amino acid sequence shown in SEQ ID NO: 28, a second polypeptide chain having the amino acid sequence shown in SEQ ID NO: 26, a third polypeptide chain having the amino acid sequence shown in SEQ ID NO: 27, and a fourth polypeptide chain having the amino acid sequence shown in SEQ ID NO: 27.

[0134] Nucleic Acids

[0135] 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 a tumor target.

[0136] 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.

[0137] Vector or transformant

[0138] The present invention provides a vector or transformant. The vector or transformant contains the aforementioned nucleic acid. The vector or transformant according to an embodiment of the present invention may include optional control sequences, such as, for example, control sequences operably linked to the nucleic acid molecule. The control sequences are one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The vector or transformant thus constructed can effectively express the aforementioned bispecific antibody.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] According to an embodiment of the present invention, the vector is a eukaryotic vector or a prokaryotic vector.

[0143] 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.

[0144] cell

[0145] 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. The cell according to an embodiment of the present invention is obtained by transfecting or transforming the aforementioned vector or transformant, and the cell can efficiently express the aforementioned bispecific antibody under appropriate conditions.

[0146] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

[0147] According to an embodiment of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0148] According to an embodiment of the present invention, the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0149] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0150] 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.

[0151] According to an embodiment of the present invention, the cell is a mammalian cell.

[0152] According to an embodiment of the present invention, the cell is a BHK cell, a CHO cell, a COS cell or a NSO cell.

[0153] 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.

[0154] Pharmaceutical composition

[0155] 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. The bispecific antibody according to the embodiments of the present invention can effectively promote PBMCs to kill tumor cells, exhibiting high anti-cancer activity; it also has low CD3 binding activity and good safety. The resulting drug can be further used to prevent and / or treat diseases mediated by CD3 and / or tumor targets.

[0156] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0157] According to an embodiment of the present invention, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0158] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] Reagent test kit

[0164] 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. The kit according to an embodiment of the present invention can bind to CD3 protein and / or tumor targets, and can effectively identify CD3 protein and / or tumor targets.

[0165] Those skilled in the art will appreciate that the features and advantages described above for antibodies, nucleic acids, vectors or transformants, cells or pharmaceutical compositions are also applicable to the kit and will not be described in detail here.

[0166] In some embodiments of the present invention, the tumor target is MICA. As previously mentioned, the bispecific antibodies according to the embodiments of the present invention can specifically bind to CD3 and MICA. Kits developed utilizing this property for CD3 and / or MICA proteins can be used in CD3 and / or MICA protein-related research, such as for detecting and / or enriching and / or isolating and purifying CD3 and / or MICA proteins from humans or other mammals. These kits can effectively detect, enrich, isolate, and purify CD3 and / or MICA proteins from biological samples, and can be further used in scientific research, such as for qualitative or quantitative detection of CD3 and / or MICA protein molecules in biological samples. More specifically, these kits can be used in immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of CD3 and / or MICA proteins to 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 serum or blood of the subject can be tested to detect CD3 and / or MICA-mediated diseases.

[0167] Use in the preparation of kits

[0168] 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 tumor targets.

[0169] In some embodiments of the present invention, the tumor target is MICA.

[0170] Those skilled in the art will appreciate that the features and advantages described above for antibodies, nucleic acids, vectors or transformants, cells or pharmaceutical compositions are also applicable to the use in the preparation kit and will not be described in detail here.

[0171] Use in preparing medicines

[0172] The present invention provides use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned vector or transformant, the aforementioned cell, or the aforementioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating CD3 and / or tumor target-mediated diseases.

[0173] Those skilled in the art will appreciate that the features and advantages described above for antibodies, nucleic acid molecules, vectors or transformants, cells and pharmaceutical compositions are also applicable to this use and will not be elaborated here.

[0174] According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.

[0175] 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.

[0176] According to an embodiment of the present invention, the tumor target is MICA. The bispecific antibody, nucleic acid, vector, transformant, or pharmaceutical composition according to an embodiment of the present invention can be further prepared into a drug, which can be clinically used to prevent or treat diseases mediated by CD3 and / or MICA.

[0177] 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.

[0178] 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.

[0179] Disease treatment methods

[0180] 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.

[0181] 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.

[0182] The effective amount of the antibody or antigen-binding fragment thereof, 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). Such 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, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0183] In some embodiments, the CD3-mediated related disease comprises an autoimmune disease.

[0184] 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.

[0185] In some embodiments, the tumor target is MICA.

[0186] In some embodiments, the MICA-mediated disease is cancer, disease caused by transplant rejection, autoimmune disease, or infectious disease.

[0187] 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.

[0188] The amino acid sequences involved in the present invention are detailed in Table 1.

[0189] Table 1 Amino acid sequence description

[0190] 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.

[0191] 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 indicated, the technology or conditions described in the literature in this area or the product instructions are used.If the manufacturer of the reagents or instruments is not indicated, they are all conventional products that can be purchased commercially.

[0192] Example 1: Production of antibodies

[0193] 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 bispecific antibody is of A configuration (Figure 1), the pcDNA3.4 vector containing CD3 antibody, MICA antibody heavy chain, and MICA antibody light chain (commissioned by Nanjing GenScript for synthesis) is added to 2mL OptiSFM culture medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a; or, when the bispecific antibody is of B configuration (Figure 1), the pcDNA3.4 vector containing MICA heavy chain-CD3 antibody, MICA antibody heavy chain, and MICA antibody light chain (commissioned by Nanjing GenScript for synthesis) is added to 2mL OptiSFM culture medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a. (3) 160μL ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) is added to 2mL OptiSFM culture 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, 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, 5% CO2 for 9 days. The culture supernatant was harvested, with 8 mL of feed 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).

[0194] In this example, 11 CD3 and MICA bispecific antibodies were prepared to investigate the various properties of the bispecific antibodies of the present invention. The configurations and amino acid sequences of each antibody are described in Figure 1, Table 1, and Table 2.

[0195] Table 2 Structure of bispecific antibody and corresponding amino acid sequence in Example 1

[0196] Note: The configurations of class A and class B antibodies are shown in Figure 1. Specifically,

[0197] The amino acid sequence of the CD3 single-chain antibody of Cross3×h5A1002 1:1 is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15.

[0198] The amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody of Cross3×h5A1002 2:1 is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15.

[0199] The amino acid sequence of the UCHT1×h5A1002 1:1 CD3 single-chain antibody is shown in SEQ ID NO: 17, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15.

[0200] The amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody of UCHT1×h5A1002 2:1 is shown in SEQ ID NO:18, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:15.

[0201] The amino acid sequence of the SP34×h5A1002 2:1 MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 19, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15.

[0202] The amino acid sequence of the Cross3×1D5 1:1 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: 20, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 21.

[0203] The amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody of Cross3×1D5 2:1 is shown in SEQ ID NO:22, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO:20, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO:21.

[0204] The amino acid sequence of the Cross3×13A9 1:1 CD3 single-chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 24.

[0205] The amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody of Cross3×13A9 2:1 is shown in SEQ ID NO: 25, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 24.

[0206] The amino acid sequence of the Cross3×619 1:1 CD3 single-chain antibody is shown in SEQ ID NO: 16, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 26, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 27.

[0207] The amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody of Cross3×619 2:1 is shown in SEQ ID NO: 28, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 26, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 27.

[0208] Example 2: ELISA binding experiment of the antibody of the present invention

[0209] 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.

[0210] (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 six times with PBST. Then, 100 μl / well of a serial dilution of the bispecific antibodies to be tested (Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1), and control hIgG1LALA (purchased from Bio-Bio) diluted in PBST / 0.05% BSA (maximum concentration 20 μg / ml, eight steps, 5-fold dilution) was added and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. Then, 100 μl / well of an HRP-conjugated anti-human IgG secondary antibody (purchased from Jackson Lab) diluted in PBST / 0.05% BSA was added and incubated at 37°C for 1 hour. 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.

[0211] The results are shown in Figure 2. Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies were all able to bind to MICA protein with comparable binding abilities.

[0212] (2) CD3 E&D protein (purchased from Acro) was diluted to 2 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μl / well. The plate was incubated at 4°C overnight. The PBS buffer in the 96-well plate was aspirated and the plate was washed six times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer. 200 μl / well of PBS / 10% BSA was added and the plate was incubated at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μl / well of a serial dilution of the bispecific antibodies to be tested (Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1), and the control hIgG1LALA (purchased from Bio-Bio) diluted in PBST / 0.05% BSA (maximum concentration 20 μg / ml, eight steps, 5-fold dilutions) was added and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. Then, 100 μl / well of an HRP (horseradish peroxidase)-conjugated anti-human IgG secondary antibody (purchased from Southern Biotech) diluted in PBST / 0.05% BSA was added and incubated at 37°C for 1 hour. 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.

[0213] The results are shown in Figure 3. Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies all bound to CD3E&D proteins; Cross3×h5A1002 2:1 had weaker binding to CD3E&D than Cross3×h5A1002 1:1, and Cross3×h5A1002 2:1 had the weakest CD3 binding activity.

[0214] The above experimental results show that after adjusting the structure of the bispecific antibody (Figure 1, configuration B), the antigen-binding fragment of the anti-CD3 antibody was successfully masked in the configuration, and its ability to bind to CD3 was weakened.

[0215] Example 3: Antibody ELISA bridging experiment

[0216] 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.

[0217] MICA-His protein (purchased from Acro) was diluted to 2 μg / ml in PBS buffer and added to a 96-well plate at a volume of 100 μl / well. The plate was incubated at 4°C overnight. The PBS buffer was aspirated from the 96-well plate, and the plate was washed six times with PBST (PBS pH 7.2 containing 0.1% Tween 20). Then, 200 μl / well of PBS / 10% BSA was added and the plate was incubated at 37°C for 2 h for blocking. The blocking solution was removed, and the plate was washed six times with PBST. Then, 100 μl / well of a series of PBST / 0.05% BSA dilutions (maximum concentration 20 μg / ml, eight steps, 5-fold dilutions) containing the bispecific antibodies to be tested (Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1), and control hIgG1LALA (purchased from Bio-Bio) were added and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. Then, MICA*002α3-Fc-Biotin diluted to the appropriate concentration was added and incubated at 37°C for 1 hour. 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.

[0218] The results are shown in Figure 4. Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies were able to bridge CD3E&D and MICA proteins; the bridging ability of Cross3×h5A1002 2:1 antibody was weaker than that of Cross3×h5A1002 1:1, and Cross3×h5A1002 2:1 antibody had the weakest bridging ability.

[0219] According to the results analysis of Figures 2 and 3, the weak bridging ability of the Cross3×h5A1002 2:1 antibody is due to the low affinity of the Cross3×h5A1002 2:1 antibody to CD3.

[0220] Example 4: Antibody Flow Cytometry Binding Experiment

[0221] 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.

[0222] (1) Dilute PBMC to 2×106 / ml, add 100μl / tube to a 1.5ml EP tube, add 10μl / tube of goat serum, and block at 4°C for 30 min. Add serial dilutions of the bispecific antibody Cross3×h5A1002 (1:1), Cross3×h5A1002 (2:1), UCHT1×h5A1002 (1:1), UCHT1×h5A1002 (2:1), and control hIgG1LALA (purchased from Bio-Bio), and incubate at 4°C for 30 min. Add 1ml of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash once more with PBS. After centrifugation, discard the supernatant and resuspend the cells in 100 μl / tube of PBS. Add 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. Incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μl / tube of PBS and analyze by flow cytometry.

[0223] The results are shown in Figure 5. Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies were able to bind to CD8 T cells, and the T cell binding ability of the 2:1 configuration (configuration B) bispecific antibody was weaker than that of the 1:1 configuration (configuration A) bispecific antibody. Among them, the Cross3×h5A1002 2:1 bispecific antibody had the weakest T cell binding activity.

[0224] (2) HCT-15 tumor cells were diluted to 2×10 6 / ml, added to a 100μl / tube volume in a 1.5ml EP tube. 10μl / tube of goat serum was added and the cells were blocked at 4°C for 30 min. A serial dilution of the bispecific antibodies Cross3×h5A1002 (1:1), Cross3×h5A1002 (2:1), UCHT1×h5A1002 (2:1), and control hIgG1LALA (purchased from Bio-Bio) was added and incubated at 4°C for 30 min. 1ml 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 cells were washed once with PBS. After centrifugation, the supernatant was discarded and the cells were resuspended in 100μl / tube of PBS. 1μl / tube of Alexa-647-labeled goat anti-human IgG secondary antibody (purchased from Jackson Labs) was added and the cells were 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.

[0225] The results are shown in Figure 6. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, and UCHT1×h5A1002 2:1 antibodies were able to bind to tumor cells. (2) Contrary to the inventors' expectations, the 2:1 configuration (configuration B) bispecific antibody bound to the EC of HCT-15 tumor cells. 50 The bispecific antibodies with a configuration lower than 1:1 (configuration A) have a stronger ability to bind to tumor cells, among which the Cross3×h5A1002 2:1 and UCHT1×h5A1002 2:1 antibodies have similar tumor binding activities.

[0226] Example 5: Experiment on Bispecific Antibodies Promoting PBMC to Kill Tumor Cells

[0227] The ability of bispecific antibodies to promote PBMC to kill HCT-15 colorectal cancer cells, NCI-H1299 lung cancer cells, PANC-1 pancreatic cancer cells, A-498 renal cancer cells, and PC-3 prostate cancer cells was tested.

[0228] (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.

[0229] (b) Tumor cells were diluted to 2×10 5 / mL, and added 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;

[0230] (c) Add the serially diluted bispecific antibodies Cross3×h5A1002 (1:1), Cross3×h5A1002 (2:1), UCHT1×h5A1002 (1:1), UCHT1×h5A1002 (2:1), and control hIgG1LALA (purchased from Bio-Tech) (maximum concentration 0.2 μg / ml, 8 serial dilutions, 5-fold dilutions) to the RTCA plate obtained in step (2) using complete RPMI-1640 medium at a volume of 20 μl / well.

[0231] (4) PBMC (purchased from Saili Bio) were diluted to 1.25×10 6 pcs / ml, and added to the RTCA plate obtained in step (3) at a volume of 80 μl / well;

[0232] (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.

[0233] The results are shown in Figure 7. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoted PBMC killing of HCT-15 colorectal cancer cells. (2) Contrary to the inventors' expectations, the 2:1 configuration (Configuration B) bispecific antibody had a stronger killing ability than the 1:1 configuration (Configuration A) bispecific antibody, and Cross3×h5A1002 2:1 had the highest killing activity.

[0234] The results are shown in Figure 8. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoted PBMC killing of NCI-H1299 lung cancer cells. (2) Contrary to the inventors' expectations, the 2:1 configuration (Configuration B) bispecific antibody exhibited stronger killing activity than the 1:1 configuration (Configuration A) bispecific antibody, and Cross3×h5A1002 2:1 had the highest killing activity.

[0235] The results are shown in Figure 9. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoted PBMC killing of PANC-1 pancreatic cancer cells. (2) Contrary to the inventors' expectations, the 2:1 configuration (Configuration B) bispecific antibody exhibited stronger killing activity than the 1:1 configuration (Configuration A) bispecific antibody, and Cross3×h5A1002 2:1 had the highest killing activity.

[0236] The results are shown in Figure 10. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoted PBMC killing of A498 renal cancer cells. The killing ability of the 2:1 configuration (configuration B) dual antibody was stronger than that of the 1:1 configuration (configuration A) dual antibody, and Cross3×h5A1002 2:1 had the highest killing activity.

[0237] The results are shown in Figure 11. (1) Cross3×h5A1002 1:1, Cross3×h5A1002 2:1, UCHT1×h5A1002 1:1, and UCHT1×h5A1002 2:1 antibodies promoted PBMC killing of PC-3 prostate cancer cells. (2) Contrary to the inventors' expectations, the 2:1 configuration (Configuration B) bispecific antibody exhibited stronger killing activity than the 1:1 configuration (Configuration A) bispecific antibody, and Cross3×h5A1002 2:1 had the highest killing activity.

[0238] The above results show that the 2:1 configuration (Configuration B) of the present invention significantly enhances the ability of PBMCs to kill HCT-15 colorectal cancer cells, NCI-H1299 lung cancer cells, PANC-1 pancreatic cancer cells, A498 renal cancer cells, and PC-3 prostate cancer cells. Specifically, when the CD3 antigen-binding region is the antigen-binding fragment of the Cross3 antibody, the 2:1 configuration (Configuration B) of the present invention exhibits the strongest activity in promoting PBMCs to kill tumor cells.

[0239] Example 6: Experiment on Bispecific Antibodies Promoting Cytokine Secretion by PBMC

[0240] 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.

[0241] (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;

[0242] (2) The bispecific antibodies Cross3×h5A1002 (1:1), Cross3×h5A1002 (2:1), and control hIgG1LALA (purchased from Bio-Bio) were serially diluted using complete RPMI 1640 medium and added to a 96-well plate at 20 μl / well.

[0243] (3) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 / ml, added to 96-well plate, 80 μl / well;

[0244] (4) Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 48 h;

[0245] (5) Centrifuge at 300 g for 10 min at room temperature and collect the cell culture supernatant;

[0246] (6) The cytokine content in the supernatant was detected using a CBA kit (purchased from BD).

[0247] The results are shown in Figure 12. (1) Cross3×h5A1002 1:1 and Cross3×h5A1002 2:1 antibodies promoted the secretion of immune-activating cytokines IL-2 and IFN-γ by PBMCs. (2) Cross3×h5A1002 2:1 antibody was more effective in promoting the secretion of immune-activating cytokines by PBMCs.

[0248] The above results show that the 2:1 configuration (Configuration B) bispecific antibody of the present invention has an enhanced ability to promote the secretion of immune-activating cytokines by PBMCs. Considering that the 2:1 configuration (Configuration B) bispecific antibody binds weakly to T cells and strongly to tumor cells, after administration, more drug can be distributed to the tumor, enhancing anti-cancer effectiveness.

[0249] Example 7: Experiment on the promotion of PBMC killing tumor cells by bispecific antibodies at different effector-target ratios

[0250] The ability of the bispecific antibody of the present invention to promote PBMC to kill HCT-15 colorectal cancer cells was tested under different effector-target ratios (PBMC:tumor).

[0251] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0252] (2) Dilute the tumor cells to 2×10 5 / mL, and added 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;

[0253] (3) Add the gradient dilutions of bispecific antibodies Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1, SP34×h5A1002 2:1, and control hIgG1LALA (purchased from Bio-Bio) (maximum concentration 0.2 μg / ml, 8 gradients, 5-fold dilution) to the RTCA plate obtained in step (2) using complete RPMI-1640 medium, with an addition volume of 20 μl / well;

[0254] (4) PBMC (purchased from Saili Bio) were diluted to 1.25×10 6 / ml (efficacy-target ratio 10:1), 0.625×10 6 / ml (efficacy-target ratio 5:1), 0.3125×10 6pcs / ml (target ratio 2.5:1), added to the RTCA plate obtained in step (3), with an addition volume of 80 μl / well;

[0255] (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.

[0256] The results are shown in Figure 13. Under different effector-target ratios, Cross3×h5A1002 2:1, UCHT1×h5A1002 2:1, and SP34×h5A1002 2:1 antibodies were able to promote PBMCs to kill HCT-15 colorectal cancer cells, and Cross3×h5A1002 2:1 had the highest killing activity.

[0257] Example 8: Experiments on promoting PBMC to kill tumor cells with other MICA dual antibodies

[0258] The ability of the 2:1 configuration (Configuration B) bispecific antibodies of the present invention, formed based on other MICA antibodies, to promote PBMC to kill HCT-15 colorectal cancer cells was tested to evaluate whether the ability of the 2:1 configuration (Configuration B) bispecific antibodies of the present invention to promote PBMC to kill tumor cells is generally better than the 1:1 configuration (Configuration A). Other MICA antibodies are selected from: 1D5 (Genentech monoclonal antibody, US20200055939A1), 13A9 (Genentech monoclonal antibody, US20200055939A1), and 619 (Cullinan monoclonal antibody, US20210253711A1).

[0259] 1. Expression of 2:1 configuration (configuration B) antibody: the same method as Example 1.

[0260] 2. Investigation of the ability of PBMC to kill tumor cells: Referring to Example 7, the following steps were performed.

[0261] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0262] (2) Dilute the tumor cells to 2×10 5 / mL, and added 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;

[0263] (3) Add the serially diluted bispecific antibodies Cross3×h5A1002 2:1, Cross3×1D5 1:1, Cross3×1D5 2:1, Cross3×13A9 1:1, Cross3×13A9 2:1, Cross3×619 1:1, Cross3×619 2:1, and control hIgG1LALA (purchased from Bio-Tech) (maximum concentration 0.2 μg / ml, 8 serial dilutions, 5-fold dilutions) to the RTCA plate obtained in step (2) using complete RPMI-1640 medium at a volume of 20 μl / well;

[0264] (4) PBMC (purchased from Saili Biotechnology) were diluted to 1.25×10 6 pcs / ml, and added to the RTCA plate obtained in step (3) at a volume of 80 μl / well;

[0265] (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.

[0266] The results are shown in Figure 14. (1) The killing ability of the 2:1 bispecific antibody based on 1D5 was weaker than that of the 1:1 bispecific antibody. (2) In addition, the killing activity of Cross3×h5A1002 2:1 was higher than that of the 1D5 bispecific antibody.

[0267] The results are shown in Figure 15. (1) The 2:1 bispecific antibody based on 13A9 had weaker killing ability than the 1:1 bispecific antibody. (2) Furthermore, the Cross3×h5A1002 2:1 bispecific antibody had higher killing activity than the 13A9 bispecific antibody.

[0268] The results are shown in Figure 16. (1) The killing ability of the 2:1 bispecific antibody based on 619 was weaker than that of the 1:1 bispecific antibody. (2) In addition, the killing activity of Cross3×h5A1002 2:1 was higher than that of the 619 bispecific antibody.

[0269] The above results show that only when the MICA antibody is selected from the h5A1002 antibody, the 2:1 configuration (configuration B) bispecific antibody of the present invention exhibits an enhanced ability to promote PBMC to kill tumor cells.

[0270] Example 9: Effect of CD3×MICA Antibody on Anti-cancer in Mice

[0271] The in vivo efficacy experiment was used to detect the anti-cancer function of the Cross3×h5A1002 2:1 bispecific antibody of the present invention in promoting immune reconstitution in mice.

[0272] (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;

[0273] (2) On day 10, NSG mice were subcutaneously injected with 2×10 6 A-375 human melanoma cells, 2×10 6 NCI-H1299 human lung cancer cells, or 5 × 10 6 PC3 human prostate cancer cells;

[0274] (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;

[0275] (4) On days 0, 4, 7, and 11, mice were injected with Cross3×h5A1002 2:1 (the amino acid sequence of the MICA antibody heavy chain-CD3 single-chain antibody is shown in SEQ ID NO: 13, the amino acid sequence of the MICA antibody heavy chain is shown in SEQ ID NO: 14, and the amino acid sequence of the MICA antibody light chain is shown in SEQ ID NO: 15) (0.1 mg / kg) and the solvent control PBS, 250 μl per mouse;

[0276] (4) After injection of the above antibodies, the tumor volume was measured and the mice were weighed twice a week.

[0277] The results are shown in Figures 17, 19 and 20. The Cross3×h5A1002 2:1 antibody of the present invention can effectively promote anti-cancer in immune reconstructed mice and can be used to treat tumor-bearing mice, resulting in complete regression of tumors.

[0278] 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 low-affinity binding to CD3, with higher anti-cancer activity and better safety. In summary, the bispecific antibody of the present invention can promote immune cell anti-cancer, has good anti-cancer activity, and higher safety, and has high clinical application value and drug development value.

[0279] 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.

[0280] 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.

[0281] 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: Comprising: a first antigen binding region, a second antigen binding region and an Fc portion; The Fc portion comprises a first Fc peptide segment and a second Fc peptide segment; The first antigen binding region comprises an scFv fragment and a first molecule of a binding protein or a fragment thereof, the first molecule of a binding protein or a fragment thereof is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment; The second antigen binding region comprises a second molecule of binding protein or a fragment thereof, and the second molecule of binding protein or a fragment thereof is connected to the N-terminus of the second Fc peptide segment; The first molecule and the second molecule are the same, and the scFv fragment has CD3 binding activity.

2. The bispecific antibody according to claim 1, characterized in that The first molecule and the second molecule are selected from MICA, PDL1, CD47, TIGIT, CD73, CD33, CEACAM1, CEACAM5, CEACAM6, STING, WNT, Beta catenin, B7H3, VISITA, CD19, BCMA, CD22, CD20, CD123, CD38, CEA, CD25, CD46, CD138, PSCA, PSMA, PSA, MUC1, MUC16, NY-ESO-1, GD2, WT1, Mesothelin, MAGE-A3, GPC3, PRAME, Globo H, AFP, Trop2, FOLR1, SP, Sca-1, CD133 or EPCAM.

3. The bispecific antibody according to claim 2, characterized in that The first molecule and the second molecule are both MICA.

4. The bispecific antibody according to claim 1, characterized in that The binding protein or fragment thereof is selected from at least one of an antibody or an antigen-binding fragment thereof, a receptor, and a ligand.

5. The bispecific antibody according to claim 1, characterized in that The binding protein or fragment thereof of the first molecule and / or the second molecule is independently selected from one of a Fab fragment and a scFab fragment.

6. The bispecific antibody according to claim 1, characterized in that The binding protein or fragment thereof of the first molecule is a first Fab fragment, and / or the binding protein or fragment thereof of the second molecule is a second Fab fragment.

7. The bispecific antibody according to claim 6, characterized in that The C-terminus of the CH1 fragment of the first Fab fragment is connected to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is connected to the N-terminus of the first Fc peptide segment; and / or The C-terminus of the CH1 fragment of the second Fab fragment is connected to the N-terminus of the second Fc peptide segment.

8. The bispecific antibody according to claim 6, characterized in that The first Fab fragment and the second Fab fragment have MICA binding activity.

9. The bispecific antibody according to claim 6, characterized in that The first Fab fragment and the second Fab fragment both comprise: a second heavy chain variable region CDR1-3 and a second light chain variable region CDR1-3; wherein, The second heavy chain variable region CDR1 has an amino acid sequence as shown in SEQ ID NO: 7 or a conservatively modified form thereof, The second heavy chain variable region CDR2 has an amino acid sequence as shown in SEQ ID NO: 8 or a conservatively modified form thereof, The second heavy chain variable region CDR3 has an amino acid sequence as shown in SEQ ID NO: 9 or a conservatively modified form thereof, The second light chain variable region CDR1 has an amino acid sequence as shown in SEQ ID NO: 10 or a conservatively modified form thereof, The second light chain variable region CDR2 has an amino acid sequence as shown in SEQ ID NO: 11 or a conservatively modified form thereof, The second light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 12 or a conservatively modified version thereof.

10. The bispecific antibody according to claim 6, characterized in that The first Fab fragment and / or the second Fab fragment comprises a second heavy chain framework region and / or a second light chain framework region.

11. The bispecific antibody according to claim 10, characterized in that: At least a portion of the second heavy chain framework region and / or the second light chain framework region is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow 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, preferably at least one of a mouse antibody, a human antibody and a primate antibody.

12. The bispecific antibody according to claim 6, characterized in that The first Fab fragment and the second Fab fragment each independently comprise: a second heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 29 or a conservatively modified form thereof, and a second light chain variable region having an amino acid sequence as shown in SEQ ID NO: 30 or a conservatively modified form thereof.

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

14. The bispecific antibody according to claim 6, characterized in that The first Fab fragment and the second Fab fragment each independently comprise: The second heavy chain variable region and CH1 fragment have the amino acid sequence shown in SEQ ID NO:31, and the second light chain variable region and CL fragment have the amino acid sequence shown in SEQ ID NO:

32.

15. The bispecific antibody according to any one of claims 1 to 14, characterized in that: The scFv fragment comprises: a first heavy chain variable region CDR1-3 and a first light chain variable region CDR1-3; wherein, The first heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 1, The first heavy chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 2, The first heavy chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO: 3, The first light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 4, The first light chain variable region CDR2 has the amino acid sequence shown in SEQ ID NO: 5, The first light chain variable region CDR3 has the amino acid sequence shown in SEQ ID NO:

6.

16. The bispecific antibody according to any one of claims 1 to 14, characterized in that: The scFv fragment comprises a first heavy chain framework region and / or a first light chain framework region.

17. The bispecific antibody according to claim 16, characterized in that At least a portion of the first heavy chain framework region and / or the first light chain framework region is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow 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, preferably at least one of a mouse antibody, a human antibody and a primate antibody.

18. The bispecific antibody according to any one of claims 1 to 14, characterized in that: The scFv fragment comprises: A first heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 33 or a conservative modification thereof, and a first light chain variable region having an amino acid sequence as shown in SEQ ID NO: 34 or a conservative modification thereof.

19. The bispecific antibody according to claim 18, characterized in that The scFv fragment further comprises: a connecting peptide 1; wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the first heavy chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the first light chain variable region; or The N-terminus of the connecting peptide 1 is connected to the C-terminus of the first light chain variable region, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the first heavy chain variable region.

20. The bispecific antibody according to claim 19, characterized in that The connecting peptide 1 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.

21. The bispecific antibody according to claim 19, characterized in that The scFv fragment has the amino acid sequence shown in SEQ ID NO:

35.

22. The bispecific antibody according to claim 19, characterized in that The first antigen binding region further comprises a connecting peptide 2; The C-terminus of CH1 of the first Fab fragment is connected to the N-terminus of the connecting peptide 2, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the scFv fragment.

23. The bispecific antibody according to claim 22, characterized in that The connecting peptide 2 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.

24. The bispecific antibody according to claim 22, characterized in that The connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO: 36 or 37.

25. The bispecific antibody according to any one of claims 1 to 7, characterized in that The first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure.

26. The bispecific antibody according to any one of claims 1 to 7, characterized in that: The first Fc peptide segment and the second Fc peptide segment are both human Fc peptide segments.

27. The bispecific antibody according to claim 26, characterized in that The human Fc peptide segment is a human IgG1 Fc peptide segment.

28. The bispecific antibody according to claim 26, characterized in that The first Fc peptide segment has an amino acid sequence as shown in SEQ ID NO:38, and the second Fc peptide segment has an amino acid sequence as shown in SEQ ID NO:39 or 40.

29. The bispecific antibody according to any one of claims 2 to 7, characterized in that The bispecific antibody comprises: A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 13, 18, 19, 22, 25 or 28, or an amino acid sequence having at least 80% identity thereto; a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 14, 20, 23 or 26, or an amino acid sequence having at least 80% identity thereto; a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, 21, 24 or 27, or an amino acid sequence having at least 80% identity thereto; A fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, 21, 24 or 27, or an amino acid sequence having at least 80% identity thereto.

30. The bispecific antibody according to claim 29, characterized in that The bispecific antibody comprises: A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 13, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 14, a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15; or A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 18, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 14, a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15; or A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 19, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 14, a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15; or A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 22, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 20, a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 21, and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 21; or A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 25, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 23, a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 24, and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 24; or A first polypeptide chain having an amino acid sequence as shown in SEQ ID NO:28, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO:26, a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO:27, and a fourth polypeptide chain having an amino acid sequence as shown in SEQ ID NO:

27.

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

32. A vector or transformant, characterized in that: Containing the nucleic acid according to claim 31.

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

34. The vector or transformant according to claim 32, 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.

35. A cell, characterized in that Carrying the nucleic acid of claim 31, the vector or transformant of claims 32 to 34; or Expressing the multispecific antibody according to any one of claims 1 to 30.

36. The cell according to claim 35, characterized in that The cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

37. A pharmaceutical composition, characterized in that The method comprises: the multispecific antibody according to any one of claims 1 to 30, the nucleic acid according to claim 31, the vector or transformant according to any one of claims 32 to 34, or the cell according to claim 35 or 36.

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

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

40. The pharmaceutical composition according to claim 37, characterized in that The pharmaceutical composition is an injection.

41. A kit, characterized in that The method comprises: the multispecific antibody according to any one of claims 1 to 30, the nucleic acid according to claim 31, the vector or transformant according to claims 32 to 34, or the cell according to claim 35 or 36.

42. Use of the multispecific antibody according to any one of claims 1 to 30, the nucleic acid according to claim 31, the vector or transformant according to claims 32 to 34, or the cell according to claim 35 or 36 in the preparation of a kit for detecting CD3 and / or tumor targets.

43. Use of the multispecific antibody of any one of claims 1 to 30, the nucleic acid of claim 31, the vector or transformant of claims 32 to 34, the cell of claim 35 or 36, or the pharmaceutical composition of claims 37 to 40 in the preparation of a drug for preventing and / or treating CD3 and / or tumor target-mediated related diseases.

44. The use according to claim 43, characterized in that The CD3-mediated related diseases include autoimmune diseases.

45. The use according to claim 44, characterized in that The autoimmune diseases include 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.

46. ​​The use according to claim 43, characterized in that The tumor target is MICA.

47. The use according to claim 46, characterized in that The MICA-mediated diseases include cancer or transplant rejection, autoimmune diseases and infectious diseases.

48. The use according to claim 47, characterized in that The cancer includes 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.

49. A method for preventing and / or treating a disease mediated by CD3 and / or MICA, characterized in that: include: A pharmaceutically acceptable amount of the bispecific antibody of claims 1 to 30, the nucleic acid of claim 31, the vector or transformant of claims 32 to 34, the cell of claim 35 or 36, or the pharmaceutical composition of claims 37 to 40 is administered to a subject.

50. The method according to claim 49, characterized in that The CD3-mediated related diseases include autoimmune diseases.

51. The method according to claim 50, characterized in that 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.

52. The method according to claim 51, characterized in that The tumor target is MICA.

53. The method according to claim 52, characterized in that The MICA-mediated diseases include cancer or transplant rejection, autoimmune diseases and infectious diseases.

54. The method according to claim 53, characterized in that The cancer includes 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.

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